Joseph C. Wu, MD, PhD is Director of the Stanford Cardiovascular Institute (http://cvi.stanford.edu/) and Professor in the Department of Medicine (Cardiology) and Department of Radiology (Molecular Imaging Program) at the Stanford University School of Medicine. Dr. Wu received his medical degree from Yale. He completed his medicine internship, residency and cardiology fellowship training at UCLA followed by a PhD (Molecular & Medical Pharmacology) at UCLA. Dr. Wu has received several awards, including the Burroughs Wellcome Foundation Career Award in Medical Sciences, Baxter Foundation Faculty Scholar Award, AHA Innovative Research Award, AHA Established Investigator Award, NIH Director’s New Innovator Award, NIH Roadmap Transformative Award, and Presidential Early Career Award for Scientists and Engineers given out by President Obama. He is on the editorial board of Journal Clinical Investigation, Circulation Research, Circulation Cardiovascular Imaging, JACC Imaging, Human Gene Therapy, Molecular Therapy, Stem Cell Research, and Journal of Nuclear Cardiology. He is a Council Member for the American Society for Clinical Investigation and a Scientific Advisory Board Member for the Keystone Symposia. His clinical activities involve adult congenital heart disease and cardiovascular imaging. His lab research focuses on stem cells, drug discovery, and molecular imaging (http://wulab.stanford.edu/).
- Congenital Heart Disease (Adult)
- Cardiovascular Disease
- Cardiovascular Imaging
Director, Stanford Cardiovascular Institute (2013 - Present)
Co-Director, Stanford Cardiovascular Institute (2012 - 2013)
Editorial Board, Scientific Report (2015 - Present)
Editorial Board, Journal Clinical Investigation (2012 - Present)
Editorial Board, Circulation Research (2011 - Present)
Editorial Board, Stem Cell Research (2011 - Present)
Editorial Board, Molecular Therapy (2011 - Present)
Editorial Board, Human Gene Therapy (2009 - Present)
Editorial Board, JACC: Cardiovascular Imaging (2008 - Present)
Editorial Board, Circulation: Cardiovascular Imaging (2008 - Present)
Editorial Board, Journal of Nuclear Cardiology (2009 - Present)
Board Member, SNM Molecular Imaging Center of Excellence (2006 - 2009)
Honors & Awards
Elected, Association of American Physicians (AAP) (2015)
Scientific Advisory Board Member, Keystone Symposia (2014-2017)
Council Member, American Society Clinical Investigation (2014-2017)
Distinguished Investigator Award, Academy of Radiology Research (2014)
Established Investigator Award, American Heart Association (2013-2017)
Best Manuscript Award, Circulation Research (2013)
Elected, Association of University Cardiologists (AUC) (2013)
Presidential Early Career Award for Scientists and Engineers (PECASE), White House Office of Technology (2010)
NIH Roadmap Transformative Award, National Institutes of Health (2009-2014)
NIH Director's New Innovator Award, National Institutes of Health (2008-2013)
Elected, American Society Clinical Investigation (ASCI) (2012)
Bernard and Joan Marshall Research Excellence Prize, British Society for Cardiovascular Research (2011)
Fellow American Heart Association, FAHA (2010)
National Innovative Research Grant, American Heart Association (2009)
Douglas P. Zipes Distinguished Young Scientist Award, American College of Cardiology (2009)
William Parmley Prize, Journal American College of Cardiology (2009)
Baxter Faculty Scholar Award, Baxter Foundation (2008)
Division of Cardiology Teaching Award (Innovation), Stanford School of Medicine (2008)
Edward Mallinckrodt Jr. Foundation, Junior Faculty Award (2008)
Career Award for Medical Scientists, Burroughs Wellcome Foundation (2007-2012)
Best Basic Science Paper, Circulation (2007)
Fellow American College of Cardiology, FACC (2006)
Career Award in Cardiovascular Imaging, American College Cardiology/General Electrics (2006-2008)
Best Drug Development Young Investigator Award, Academy of Molecular Imaging (2005)
K08 Mentored Clinician Scientist Award, National Institutes of Health (2005-2008)
GSK Cardiovascular Young Investigator Award, GlaxoSmithKline (2003-2005)
Best Basic Science Abstract, Bristol Myers Squibb Cardiology Fellows Forum (2003)
Cardiovascular Young Investigator Award (First Place), Society of Nuclear Medicine (2002)
MD, Yale University School of Medicine, Medicine (1997)
Medical Education:Yale University School of Medicine (1997) CT
Residency:UCLA Medical Center (1999) CA
Fellowship:UCLA Medical Center (2004) CA
Board Certification: Echocardiography, National Board of Echocardiography (2004)
Board Certification: Cardiovascular Disease, American Board of Internal Medicine (2004)
PhD, UCLA, Molecular & Medical Pharmacology (2004)
Current Research and Scholarly Interests
My lab works on biological mechanisms of adult stem cells, embryonic stem cells, and induced pluripotent stem cells. We use a combination of gene profiling, tissue engineering, physiological testing, and molecular imaging technologies to better understand stem cell biology in vitro and in vivo. For adult stem cells, we are interested in monitoring stem cell survival, proliferation, and differentiation. For ESC, we are currently studying their tumorigenicity, immunogenicity, and differentiation. For iPSC, we are working on novel derivation techniques. We also work on development of novel vectors and therapeutic genes for cardiovascular gene therapy applications.
Defining the Role of Insulin Resistance in 'Idiopathic' Dilated Cardiomyopathy
This study will investigate the effects of rosiglitazone, a medicine commonly used to treat type 2 diabetes, on the utilization of glucose by the heart in patients with heart failure which is not due to heart attacks. The primary purpose of the study is to determine whether treatment with an insulin-sensitizing medication will improve the heart's ability to metabolize glucose (sugar).
Stanford is currently not accepting patients for this trial. For more information, please contact Michael B Fowler, MB, 650-723-7846.
An Efficacy, Safety and Tolerability Study of Ixmyelocel-T Administered Via Transendocardial Catheter-based Injections to Subjects With Heart Failure Due to Ischemic Dilated Cardiomyopathy (IDCM)
This study is designed to assess the efficacy, safety and tolerability of ixmyelocel-T compared to placebo (vehicle control) when administered via transendocardial catheter-based injections to patients with end stage heart failure due to IDCM, who have no reasonable revascularization options (either surgical or percutaneous interventional) likely to provide clinical benefit.
NBS10 (Also Known as AMR-001) Versus Placebo Post ST Segment Elevation Myocardial Infarction
This study will assess the safety and efficacy of intracoronary artery administered autologous bone marrow derived stem cells in subjects post ST segment elevation myocardial infarction (STEMI). This will be assessed by evaluating and comparing the autologous stem cell treatment group to the control group in terms of the occurrence of AE's, SAE's and Major Adverse Cardiac Events (MACE), by the change in myocardial perfusion (RTSS) measured quantitatively by gated single photon emission computed tomography myocardial perfusion imaging (gated SPECT MPI), and other secondary endpoints such as LVEF measured by cardiac MRI in addition to other endpoints.
Independent Studies (25)
- Bioengineering Problems and Experimental Investigation
BIOE 191 (Win, Spr, Sum)
- Directed Investigation
BIOE 392 (Win, Spr, Sum)
- Directed Reading in Immunology
IMMUNOL 299 (Aut, Win, Spr, Sum)
- Directed Reading in Medicine
MED 299 (Aut, Win, Spr, Sum)
- Directed Reading in Radiology
RAD 299 (Aut, Win, Spr, Sum)
- Directed Reading in Stem Cell Biology and Regenerative Medicine
STEMREM 299 (Aut, Win, Spr)
- Directed Study
BIOE 391 (Aut, Win, Spr, Sum)
- Early Clinical Experience in Immunology
IMMUNOL 280 (Aut, Win, Spr, Sum)
- Early Clinical Experience in Medicine
MED 280 (Aut, Win, Spr, Sum)
- Early Clinical Experience in Radiology
RAD 280 (Aut, Win, Spr, Sum)
- Graduate Research
IMMUNOL 399 (Aut, Win, Spr, Sum)
- Graduate Research
MED 399 (Aut, Win, Spr, Sum)
- Graduate Research
RAD 399 (Aut, Win, Spr, Sum)
- Graduate Research
STEMREM 399 (Aut, Win, Spr)
- Medical Scholars Research
MED 370 (Aut, Win, Spr, Sum)
- Medical Scholars Research
RAD 370 (Aut, Win, Spr, Sum)
- Medical Scholars Research
STEMREM 370 (Aut, Win, Spr)
- Out-of-Department Advanced Research Laboratory in Bioengineering
BIOE 191X (Aut, Win, Spr)
- Out-of-Department Advanced Research Laboratory in Experimental Biology
BIO 199X (Aut, Win, Spr, Sum)
- Out-of-Department Graduate Research
BIO 300X (Win)
- Teaching in Immunology
IMMUNOL 290 (Aut, Win, Spr, Sum)
- Undergraduate Research
IMMUNOL 199 (Aut, Win, Spr, Sum)
- Undergraduate Research
MED 199 (Aut, Win, Spr, Sum)
- Undergraduate Research
RAD 199 (Aut, Win, Spr, Sum)
- Undergraduate Research
STEMREM 199 (Aut, Win, Spr)
- Bioengineering Problems and Experimental Investigation
- Induced pluripotent stem cells. JAMA 2015; 313 (16): 1613-4
- Characterization of the molecular mechanisms underlying increased ischemic damage in the aldehyde dehydrogenase 2 genetic polymorphism using a human induced pluripotent stem cell model system SCIENCE TRANSLATIONAL MEDICINE 2014; 6 (255)
Chemically defined generation of human cardiomyocytes.
2014; 11 (8): 855-860
Existing methods for human induced pluripotent stem cell (hiPSC) cardiac differentiation are efficient but require complex, undefined medium constituents that hinder further elucidation of the molecular mechanisms of cardiomyogenesis. Using hiPSCs derived under chemically defined conditions on synthetic matrices, we systematically developed an optimized cardiac differentiation strategy, using a chemically defined medium consisting of just three components: the basal medium RPMI 1640, L-ascorbic acid 2-phosphate and rice-derived recombinant human albumin. Along with small molecule-based induction of differentiation, this protocol produced contractile sheets of up to 95% TNNT2(+) cardiomyocytes at a yield of up to 100 cardiomyocytes for every input pluripotent cell and was effective in 11 hiPSC lines tested. This chemically defined platform for cardiac specification of hiPSCs will allow the elucidation of cardiomyocyte macromolecular and metabolic requirements and will provide a minimal system for the study of maturation and subtype specification.
View details for DOI 10.1038/nmeth.2999
View details for PubMedID 24930130
Stem Cell Imaging: From Bench to Bedside
CELL STEM CELL
2014; 14 (4): 431-444
Although cellular therapies hold great promise for the treatment of human disease, results from several initial clinical trials have not shown a level of efficacy required for their use as a first line therapy. Here we discuss how in vivo molecular imaging has helped identify barriers to clinical translation and potential strategies that may contribute to successful transplantation and improved outcomes, with a focus on cardiovascular and neurological diseases. We conclude with a perspective on the future role of molecular imaging in defining safety and efficacy for clinical implementation of stem cell therapies.
View details for DOI 10.1016/j.stem.2014.03.009
View details for Web of Science ID 000334766400008
Tumorigenicity as a clinical hurdle for pluripotent stem cell therapies
2013; 19 (8): 998-1004
Human pluripotent stem cells (PSCs) are a leading candidate for cell-based therapies because of their capacity for unlimited self renewal and pluripotent differentiation. These advances have recently culminated in the first-in-human PSC clinical trials by Geron, Advanced Cell Technology and the Kobe Center for Developmental Biology for the treatment of spinal cord injury and macular degeneration. Despite their therapeutic promise, a crucial hurdle for the clinical implementation of human PSCs is their potential to form tumors in vivo. In this Perspective, we present an overview of the mechanisms underlying the tumorigenic risk of human PSC-based therapies and discuss current advances in addressing these challenges.
View details for DOI 10.1038/nm.3267
View details for Web of Science ID 000322779600025
Global Epigenomic Reconfiguration During Mammalian Brain Development.
Science (New York, N.Y.)
DNA methylation is implicated in mammalian brain development and plasticity underlying learning and memory. We report the genome-wide composition, patterning, cell specificity, and dynamics of DNA methylation at single-base resolution in human and mouse frontal cortex throughout their lifespan. Widespread methylome reconfiguration occurs during fetal to young adult development, coincident with synaptogenesis. During this period, highly conserved non-CG methylation (mCH) accumulates in neurons, but not glia, to become the dominant form of methylation in the human neuronal genome. Moreover, we found an mCH signature that identifies genes escaping X-chromosome inactivation. Finally, whole-genome single-base resolution 5-hydroxymethylcytosine (hmC) maps revealed that hmC marks fetal brain cell genomes at putative regulatory regions that are CG-demethylated and activated in the adult brain, and that CG demethylation at these hmC-poised loci depends on Tet2 activity.
View details for PubMedID 23828890
Patient-Specific Induced Pluripotent Stem Cells as a Model for Familial Dilated Cardiomyopathy
SCIENCE TRANSLATIONAL MEDICINE
2012; 4 (130)
Characterized by ventricular dilatation, systolic dysfunction, and progressive heart failure, dilated cardiomyopathy (DCM) is the most common form of cardiomyopathy in patients. DCM is the most common diagnosis leading to heart transplantation and places a significant burden on healthcare worldwide. The advent of induced pluripotent stem cells (iPSCs) offers an exceptional opportunity for creating disease-specific cellular models, investigating underlying mechanisms, and optimizing therapy. Here, we generated cardiomyocytes from iPSCs derived from patients in a DCM family carrying a point mutation (R173W) in the gene encoding sarcomeric protein cardiac troponin T. Compared to control healthy individuals in the same family cohort, cardiomyocytes derived from iPSCs from DCM patients exhibited altered regulation of calcium ion (Ca(2+)), decreased contractility, and abnormal distribution of sarcomeric ?-actinin. When stimulated with a ?-adrenergic agonist, DCM iPSC-derived cardiomyocytes showed characteristics of cellular stress such as reduced beating rates, compromised contraction, and a greater number of cells with abnormal sarcomeric ?-actinin distribution. Treatment with ?-adrenergic blockers or overexpression of sarcoplasmic reticulum Ca(2+) adenosine triphosphatase (Serca2a) improved the function of iPSC-derived cardiomyocytes from DCM patients. Thus, iPSC-derived cardiomyocytes from DCM patients recapitulate to some extent the morphological and functional phenotypes of DCM and may serve as a useful platform for exploring disease mechanisms and for drug screening.
View details for DOI 10.1126/scitranslmed.3003552
View details for Web of Science ID 000303045900004
View details for PubMedID 22517884
Short-Term Immunosuppression Promotes Engraftment of Embryonic and Induced Pluripotent Stem Cells
CELL STEM CELL
2011; 8 (3): 309-317
Embryonic stem cells (ESCs) are an attractive source for tissue regeneration and repair therapies because they can be differentiated into virtually any cell type in the adult body. However, for this approach to succeed, the transplanted ESCs must survive long enough to generate a therapeutic benefit. A major obstacle facing the engraftment of ESCs is transplant rejection by the immune system. Here we show that blocking leukocyte costimulatory molecules permits ESC engraftment. We demonstrate the success of this immunosuppressive therapy for mouse ESCs, human ESCs, mouse induced pluripotent stem cells (iPSCs), human induced pluripotent stem cells, and more differentiated ESC/(iPSCs) derivatives. Additionally, we provide evidence describing the mechanism by which inhibition of costimulatory molecules suppresses T cell activation. This report describes a short-term immunosuppressive approach capable of inducing engraftment of transplanted ESCs and iPSCs, providing a significant improvement in our mechanistic understanding of the critical role costimulatory molecules play in leukocyte activation.
View details for DOI 10.1016/j.stem.2011.01.012
View details for Web of Science ID 000288404400012
View details for PubMedID 21362570
- Identification of a New Modulator of the Intercalated Disc in a Zebrafish Model of Arrhythmogenic Cardiomyopathy SCIENCE TRANSLATIONAL MEDICINE 2014; 6 (240)
- Human Stem Cells for Modeling Heart Disease and for Drug Discovery SCIENCE TRANSLATIONAL MEDICINE 2014; 6 (239)
- Transplanted terminally differentiated induced pluripotent stem cells are accepted by immune mechanisms similar to self-tolerance NATURE COMMUNICATIONS 2014; 5
Human-induced pluripotent stem cell models of inherited cardiomyopathies
CURRENT OPINION IN CARDIOLOGY
2014; 29 (3): 214-219
This article provides an overview of the latest advances in in-vitro modeling of inherited cardiomyopathies using human-induced pluripotent stem cells (iPSCs).Inherited cardiomyopathies have been recently modeled by generating iPSCs from patients harboring mutations in genes associated with the pathogenesis of hypertrophic cardiomyopathy, dilated cardiomyopathy, and arrhythmogenic right ventricular cardiomyopathy/dysplasia.Patient-specific iPSCs and their differentiated cardiomyocytes (induced pluripotent stem cell-derived cardiomyocytes) now provide a novel model to study the underlying molecular mechanism of the pathogenesis of familial cardiomyopathies as well as for in-vitro drug screening and drug discovery.
View details for DOI 10.1097/HCO.0000000000000049
View details for Web of Science ID 000335060500003
View details for PubMedID 24576884
- Rapid and Efficient Conversion of Integration-Free Human Induced Pluripotent Stem Cells to GMP-Grade Culture Conditions PLOS ONE 2014; 9 (4)
Relationship between Echocardiographic and Magnetic Resonance Derived Measures of Right Ventricular Size and Function in Patients with Pulmonary Hypertension
JOURNAL OF THE AMERICAN SOCIETY OF ECHOCARDIOGRAPHY
2014; 27 (4): 405-412
Transthoracic echocardiographic (TTE) imaging is the mainstay of clinical practice for evaluating right ventricular (RV) size and function, but its accuracy in patients with pulmonary hypertension has not been well validated.Magnetic resonance imaging (MRI) and TTE images were retrospectively reviewed in 40 consecutive patients with pulmonary hypertension. RV and left ventricular volumes and ejection fractions were calculated using MRI. TTE areas and indices of RV ejection fraction (RVEF) were compared.The average age was 42 ± 12 years, with a majority of women (85%). There was a wide range of mean pulmonary arterial pressures (27-81 mm Hg) and RV end-diastolic volumes (111-576 mL), RVEFs (8%-67 %), and left ventricular ejection fractions (26%-72%) by MRI. There was a strong association between TTE and MRI-derived parameters: RV end-diastolic area (by TTE imaging) and RV end-diastolic volume (by MRI), R(2) = 0.78 (P < .001); RV fractional area change by TTE imaging and RVEF by MRI, R(2) = 0.76 (P < .001); and tricuspid annular plane systolic excursion by TTE imaging and RVEF by MRI, R(2) = 0.64 (P < .001). By receiver operating characteristic curve analysis, an RV fractional area change < 25% provided excellent discrimination of moderate systolic dysfunction (RVEF < 35%), with an area under the curve of 0.97 (P < .001). An RV end-diastolic area index of 18 cm(2)/m(2) provided excellent discrimination for moderate RV enlargement (area under the curve, 0.89; P < .001).Echocardiographic estimates of RV volume (by RV end-diastolic area) and function (by RV fractional area change and tricuspid annular plane systolic excursion) offer good approximations of RV size and function in patients with pulmonary hypertension and allow the accurate discrimination of normal from abnormal.
View details for DOI 10.1016/j.echo.2013.12.011
View details for Web of Science ID 000334315700006
View details for PubMedID 24444659
- Stem cells and cardiovascular drug development--reply. JAMA : the journal of the American Medical Association 2014; 311 (10): 1070-1071
Construction and Validation of Nano Gold Tripods for Molecular Imaging of Living Subjects
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2014; 136 (9): 3560-3571
Anisotropic colloidal hybrid nanoparticles exhibit superior optical and physical properties compared to their counterparts with regular architectures. We herein developed a controlled, stepwise strategy to build novel, anisotropic, branched, gold nanoarchitectures (Au-tripods) with predetermined composition and morphology for bioimaging. The resultant Au-tripods with size less than 20 nm showed great promise as contrast agents for in vivo photoacoustic imaging (PAI). We further identified Au-tripods with two possible configurations as high-absorbance nanomaterials from various gold multipods using a numerical simulation analysis. The PAI signals were linearly correlated with their concentrations after subcutaneous injection. The in vivo biodistribution of Au-tripods favorable for molecular imaging was confirmed using small animal positron emission tomography (PET). Intravenous administration of cyclic Arg-Gly-Asp-d-Phe-Cys (RGDfC) peptide conjugated Au-tripods (RGD-Au-tripods) to U87MG tumor-bearing mice showed PAI contrasts in tumors almost 3-fold higher than for the blocking group. PAI results correlated well with the corresponding PET images. Quantitative biodistribution data revealed that 7.9% ID/g of RGD-Au-tripods had accumulated in the U87MG tumor after 24 h post-injection. A pilot mouse toxicology study confirmed that no evidence of significant acute or systemic toxicity was observed in histopathological examination. Our study suggests that Au-tripods can be reliably synthesized through stringently controlled chemical synthesis and could serve as a new generation of platform with high selectivity and sensitivity for multimodality molecular imaging.
View details for DOI 10.1021/ja412001e
View details for Web of Science ID 000332684700036
View details for PubMedID 24495038
- Tracking gene and cell fate for therapeutic gain NATURE MATERIALS 2014; 13 (2): 106-109
- Predicting the Future With Stem Cells CIRCULATION 2014; 129 (2): 136-138
Cardiac stem cell biology: glimpse of the past, present, and future.
2014; 114 (1): 21-27
Cardiac regeneration strategies and de novo generation of cardiomyocytes have long been significant areas of research interest in cardiovascular medicine. In this review, we outline a variety of common cell sources and methods used to regenerate cardiomyocytes and highlight the important role that key Circulation Research articles have played in this flourishing field.
View details for DOI 10.1161/CIRCRESAHA.113.302895
View details for PubMedID 24385505
Short Hairpin RNA Gene Silencing of Prolyl Hydroxylase-2 with a Minicircle Vector Improves Neovascularization of Hindlimb Ischemia
HUMAN GENE THERAPY
2014; 25 (1): 41-49
Abstract In this study, we target the hypoxia inducible factor-1 alpha (HIF-1-alpha) pathway by short hairpin RNA interference therapy targeting prolyl hydroxylase-2 (shPHD2). We use the minicircle (MC) vector technology as an alternative for conventional nonviral plasmid (PL) vectors in order to improve neovascularization after unilateral hindlimb ischemia in a murine model. Gene expression and transfection efficiency of MC and PL, both in vitro and in vivo, were assessed using bioluminescence imaging (BLI) and firefly luciferase (Luc) reporter gene. C57Bl6 mice underwent unilateral electrocoagulation of the femoral artery and gastrocnemic muscle injection with MC-shPHD2, PL-shPHD2, or phosphate-buffered saline (PBS) as control. Blood flow recovery was monitored using laser Doppler perfusion imaging, and collaterals were visualized by immunohistochemistry and angiography. MC-Luc showed a 4.6-fold higher in vitro BLI signal compared with PL-Luc. BLI signals in vivo were 4.3×10(5)±3.3×10(5) (MC-Luc) versus 0.4×10(5)±0.3×10(5) (PL-Luc) at day 28 (p=0.016). Compared with PL-shPHD2 or PBS, MC-shPHD2 significantly improved blood flow recovery, up to 50% from day 3 until day 14 after ischemia induction. MC-shPHD2 significantly increased collateral density and capillary density, as monitored by alpha-smooth muscle actin expression and CD31(+) expression, respectively. Angiography data confirmed the histological findings. Significant downregulation of PHD2 mRNA levels by MC-shPHD2 was confirmed by quantitative polymerase chain reaction. Finally, Western blot analysis confirmed significantly higher levels of HIF-1-alpha protein by MC-shPHD2, compared with PL-shPHD2 and PBS. This study provides initial evidence of a new potential therapeutic approach for peripheral artery disease. The combination of HIF-1-alpha pathway targeting by shPHD2 with the robust nonviral MC plasmid improved postischemic neovascularization, making this approach a promising potential treatment option for critical limb ischemia.
View details for DOI 10.1089/hum.2013.110
View details for Web of Science ID 000329848600007
View details for PubMedID 24090375
VEGF-C and aortic cardiomyocytes guide coronary artery stem development.
The Journal of clinical investigation
Coronary arteries (CAs) stem from the aorta at 2 highly stereotyped locations, deviations from which can cause myocardial ischemia and death. CA stems form during embryogenesis when peritruncal blood vessels encircle the cardiac outflow tract and invade the aorta, but the underlying patterning mechanisms are poorly understood. Here, using murine models, we demonstrated that VEGF-C-deficient hearts have severely hypoplastic peritruncal vessels, resulting in delayed and abnormally positioned CA stems. We observed that VEGF-C is widely expressed in the outflow tract, while cardiomyocytes develop specifically within the aorta at stem sites where they surround maturing CAs in both mouse and human hearts. Mice heterozygous for islet 1 (Isl1) exhibited decreased aortic cardiomyocytes and abnormally low CA stems. In hearts with outflow tract rotation defects, misplaced stems were associated with shifted aortic cardiomyocytes, and myocardium induced ectopic connections with the pulmonary artery in culture. These data support a model in which CA stem development first requires VEGF-C to stimulate vessel growth around the outflow tract. Then, aortic cardiomyocytes facilitate interactions between peritruncal vessels and the aorta. Derangement of either step can lead to mispatterned CA stems. Studying this niche for cardiomyocyte development, and its relationship with CAs, has the potential to identify methods for stimulating vascular regrowth as a treatment for cardiovascular disease.
View details for DOI 10.1172/JCI77483
View details for PubMedID 25271623
Second Generation Codon Optimized Minicircle (CoMiC) for Nonviral Reprogramming of Human Adult Fibroblasts.
Methods in molecular biology (Clifton, N.J.)
2014; 1181: 1-13
The ability to induce pluripotency in somatic cells is one of the most important scientific achievements in the fields of stem cell research and regenerative medicine. This technique allows researchers to obtain pluripotent stem cells without the controversial use of embryos, providing a novel and powerful tool for disease modeling and drug screening approaches. However, using viruses for the delivery of reprogramming genes and transcription factors may result in integration into the host genome and cause random mutations within the target cell, thus limiting the use of these cells for downstream applications. To overcome this limitation, various non-integrating techniques, including Sendai virus, mRNA, minicircle, and plasmid-based methods, have recently been developed. Utilizing a newly developed codon optimized 4-in-1 minicircle (CoMiC), we were able to reprogram human adult fibroblasts using chemically defined media and without the need for feeder cells.
View details for DOI 10.1007/978-1-4939-1047-2_1
View details for PubMedID 25070322
Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes as an In Vitro Model for Coxsackievirus B3-Induced Myocarditis and Antiviral Drug Screening Platform.
Rationale: Viral myocarditis is a life-threatening illness that may lead to heart failure or cardiac arrhythmias. A major causative agent for viral myocarditis is the B3 strain of coxsackievirus, a positive-sense RNA enterovirus. However, human cardiac tissues are difficult to procure in sufficient enough quantities for studying the mechanisms of cardiac-specific viral infection. Objective: This study examined whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) could be used to model the pathogenic processes of coxsackievirus-induced viral myocarditis and to screen antiviral therapeutics for efficacy. Methods and Results: Human iPSC-CMs were infected with a luciferase-expressing coxsackievirus B3 strain (CVB3-Luc). Brightfield microscopy, immunofluorescence, and calcium imaging were utilized to characterize virally-infected hiPSC-CMs for alterations in cellular morphology and calcium handling. Viral proliferation in hiPSC-CMs was quantified using bioluminescence imaging. Antiviral compounds including interferon beta 1 (IFNβ1), ribavirin, pyrrolidine dithiocarbamate, and fluoxetine were tested for their capacity to abrogate CVB3-Luc proliferation in hiPSC-CMs in vitro. The ability of these compounds to reduce CVB3-Luc proliferation in hiPSC-CMs was consistent with reported drug effects in previous studies. Mechanistic analyses via gene expression profiling of hiPSC-CMs infected with CVB3-Luc revealed an activation of viral RNA and protein clearance pathways after IFNβ1 treatment. Conclusions: This study demonstrates that hiPSC-CMs express the coxsackievirus and adenovirus receptor, are susceptible to coxsackievirus infection, and can be used to predict antiviral drug efficacy. Our results suggest that the hiPSC-CM/CVB3-Luc assay is a sensitive platform that can screen novel antiviral therapeutics for their effectiveness in a high-throughput fashion.
View details for DOI 10.1161/CIRCRESAHA.115.303810
View details for PubMedID 25015077
- On-line visualization of ischemic burden during repetitive ischemia/reperfusion. JACC. Cardiovascular imaging 2014; 7 (9): 956-8
Cardiac tissue slice transplantation as a model to assess tissue-engineered graft thickness, survival, and function.
2014; 130 (11 Suppl 1): S77-86
Cell therapies offer the potential to improve cardiac function after myocardial infarction. Although injection of single-cell suspensions has proven safe, cell retention and survival rates are low. Tissue-engineered grafts allow cell delivery with minimal initial cell loss and mechanical support to the heart. However, graft performance cannot be easily compared, and optimal construct thickness, vascularization, and survival kinetics are unknown.Cardiac tissue slices (CTS) were generated by sectioning mouse hearts (n=40) expressing firefly luciferase and green fluorescent protein into slices of defined size and thickness using a vibrating blade microtome. Bioluminescence imaging of CTS transplanted onto hearts of immunodeficient mice demonstrated survival of ≤30% of transplanted cells. Cardiac slice perfusion was re-established within 3 days, likely through anastomosis of pre-existing vessels with the host vasculature and invasion of vessels from the host. Immunofluorescence showed a peak in cell death 3 days after transplantation and a gradual decline thereafter. MRI revealed preservation of contractile function and an improved ejection fraction 1 month after transplantation of CTS (28±2% CTS versus 22±2% control; P=0.05). Importantly, this effect was specific to CTS because transplantation of skeletal muscle tissue slices led to faster dilative remodeling and higher animal mortality.In summary, this is the first study to use CTS as a benchmark to validate and model tissue-engineered graft studies. CTS transplantation improved cell survival, established reperfusion, and enhanced cardiac function after myocardial infarction. These findings also confirm that dilative remodeling can be attenuated by topical transplantation of CTS but not skeletal muscle tissue grafts.
View details for DOI 10.1161/CIRCULATIONAHA.113.007920
View details for PubMedID 25200059
Transplanted terminally differentiated induced pluripotent stem cells are accepted by immune mechanisms similar to self-tolerance.
2014; 5: 3903-?
The exact nature of the immune response elicited by autologous-induced pluripotent stem cell (iPSC) progeny is still not well understood. Here we show in murine models that autologous iPSC-derived endothelial cells (iECs) elicit an immune response that resembles the one against a comparable somatic cell, the aortic endothelial cell (AEC). These cells exhibit long-term survival in vivo and prompt a tolerogenic immune response characterized by elevated IL-10 expression. In contrast, undifferentiated iPSCs elicit a very different immune response with high lymphocytic infiltration and elevated IFN-γ, granzyme-B and perforin intragraft. Furthermore, the clonal structure of infiltrating T cells from iEC grafts is statistically indistinguishable from that of AECs, but is different from that of undifferentiated iPSC grafts. Taken together, our results indicate that the differentiation of iPSCs results in a loss of immunogenicity and leads to the induction of tolerance, despite expected antigen expression differences between iPSC-derived versus original somatic cells.
View details for DOI 10.1038/ncomms4903
View details for PubMedID 24875164
Rapid and efficient conversion of integration-free human induced pluripotent stem cells to GMP-grade culture conditions.
2014; 9 (4)
Data suggest that clinical applications of human induced pluripotent stem cells (hiPSCs) will be realized. Nonetheless, clinical applications will require hiPSCs that are free of exogenous DNA and that can be manufactured through Good Manufacturing Practice (GMP). Optimally, derivation of hiPSCs should be rapid and efficient in order to minimize manipulations, reduce potential for accumulation of mutations and minimize financial costs. Previous studies reported the use of modified synthetic mRNAs to reprogram fibroblasts to a pluripotent state. Here, we provide an optimized, fully chemically defined and feeder-free protocol for the derivation of hiPSCs using synthetic mRNAs. The protocol results in derivation of fully reprogrammed hiPSC lines from adult dermal fibroblasts in less than two weeks. The hiPSC lines were successfully tested for their identity, purity, stability and safety at a GMP facility and cryopreserved. To our knowledge, as a proof of principle, these are the first integration-free iPSCs lines that were reproducibly generated through synthetic mRNA reprogramming that could be putatively used for clinical purposes.
View details for DOI 10.1371/journal.pone.0094231
View details for PubMedID 24718618
Cross Talk of Combined Gene and Cell Therapy in Ischemic Heart Disease: Role of Exosomal MicroRNA Transfer.
2014; 130 (11 Suppl 1): S60-9
Despite the promise shown by stem cells for restoration of cardiac function after myocardial infarction, the poor survival of transplanted cells has been a major issue. Hypoxia-inducible factor-1 (HIF1) is a transcription factor that mediates adaptive responses to ischemia. Here, we hypothesize that codelivery of cardiac progenitor cells (CPCs) with a nonviral minicircle plasmid carrying HIF1 (MC-HIF1) into the ischemic myocardium can improve the survival of transplanted CPCs.After myocardial infarction, CPCs were codelivered intramyocardially into adult NOD/SCID mice with saline, MC-green fluorescent protein, or MC-HIF1 versus MC-HIF1 alone (n=10 per group). Bioluminescence imaging demonstrated better survival when CPCs were codelivered with MC-HIF1. Importantly, echocardiography showed mice injected with CPCs+MC-HIF1 had the highest ejection fraction 6 weeks after myocardial infarction (57.1±2.6%; P=0.002) followed by MC-HIF1 alone (48.5±2.6%; P=0.04), with no significant protection for CPCs+MC-green fluorescent protein (44.8±3.3%; P=NS) when compared with saline control (38.7±3.2%). In vitro mechanistic studies confirmed that cardiac endothelial cells produced exosomes that were actively internalized by recipient CPCs. Exosomes purified from endothelial cells overexpressing HIF1 had higher contents of miR-126 and miR-210. These microRNAs activated prosurvival kinases and induced a glycolytic switch in recipient CPCs, giving them increased tolerance when subjected to in vitro hypoxic stress. Inhibiting both of these miRs blocked the protective effects of the exosomes.In summary, HIF1 can be used to modulate the host microenvironment for improving survival of transplanted cells. The exosomal transfer of miRs from host cells to transplanted cells represents a unique mechanism that can be potentially targeted for improving survival of transplanted cells.
View details for DOI 10.1161/CIRCULATIONAHA.113.007917
View details for PubMedID 25200057
High efficiency differentiation of human pluripotent stem cells to cardiomyocytes and characterization by flow cytometry.
Journal of visualized experiments : JoVE
There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle "in a dish" for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.
View details for DOI 10.3791/52010
View details for PubMedID 25286293
Modeling inherited cardiac disorders.
2014; 78 (4): 784-794
Advances in the understanding and treatment of cardiac disorders have been thwarted by the inability to study beating human cardiac cells in vitro. Induced pluripotent stem cells (iPSCs) bypass this hurdle by enabling the creation of patient-specific iPSC-derived cardiomyocytes (iPSC-CMs). These cells provide a unique platform to study cardiac diseases in vitro, especially hereditary cardiac conditions. To date, iPSC-CMs have been used to successfully model arrhythmic disorders, showing excellent recapitulation of cardiac channel function and electrophysiologic features of long QT syndrome types 1, 2, 3, and 8, and catecholaminergic polymorphic ventricular tachycardia (CPVT). Similarly, iPSC-CM models of dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) have shown robust correlation of predicted morphologic, contractile, and electrical phenotypes. In addition, iPSC-CMs have shown some features of the respective phenotypes for arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C), LEOPARD syndrome, Pompe's disease, and Friedriech's ataxia. In this review, we examine the progress of utilizing iPSC-CMs as a model for cardiac conditions and analyze the potential for the platform in furthering the biology and treatment of cardiac disorders.
View details for PubMedID 24632794
- Patient-specific stem cells and cardiovascular drug discovery. JAMA-the journal of the American Medical Association 2013; 310 (19): 2039-2040
Costimulation-Adhesion Blockade Is Superior to Cyclosporine A and Prednisone Immunosuppressive Therapy for Preventing Rejection of Differentiated Human Embryonic Stem Cells Following Transplantation
2013; 31 (11): 2354-2363
Rationale: Human embryonic stem cell (hESC) derivatives are attractive candidates for therapeutic use. The engraftment and survival of hESC derivatives as xenografts or allografts require effective immunosuppression to prevent immune cell infiltration and graft destruction. Objective: To test the hypothesis that a short-course, dual-agent regimen of two costimulation-adhesion blockade agents can induce better engraftment of hESC derivatives compared to current immunosuppressive agents. Methods and Results: We transduced hESCs with a double fusion reporter gene construct expressing firefly luciferase (Fluc) and enhanced green fluorescent protein, and differentiated these cells to endothelial cells (hESC-ECs). Reporter gene expression enabled longitudinal assessment of cell engraftment by bioluminescence imaging. Costimulation-adhesion therapy resulted in superior hESC-EC and mouse EC engraftment compared to cyclosporine therapy in a hind limb model. Costimulation-adhesion therapy also promoted robust hESC-EC and hESC-derived cardiomyocyte survival in an ischemic myocardial injury model. Improved hESC-EC engraftment had a cardioprotective effect after myocardial injury, as assessed by magnetic resonance imaging. Mechanistically, costimulation-adhesion therapy is associated with systemic and intragraft upregulation of T-cell immunoglobulin and mucin domain 3 (TIM3) and a reduced proinflammatory cytokine profile. Conclusions: Costimulation-adhesion therapy is a superior alternative to current clinical immunosuppressive strategies for preventing the post-transplant rejection of hESC derivatives. By extending the window for cellular engraftment, costimulation-adhesion therapy enhances functional preservation following ischemic injury. This regimen may function through a TIM3-dependent mechanism. Stem Cells 2013;31:2354-2363.
View details for DOI 10.1002/stem.1501
View details for Web of Science ID 000327025600007
- Noninvasive Imaging of Hypoxia-Inducible Factor-1 alpha Gene Therapy for Myocardial Ischemia HUMAN GENE THERAPY METHODS 2013; 24 (5): 279-288
Imaging cardiac stem cell transplantation using radionuclide labeling techniques: clinical applications and future directions.
Methodist DeBakey cardiovascular journal
2013; 9 (4): 218-222
Stem cell therapy is emerging as a potential new therapy for patients with advanced heart failure. In recent years, advances in molecular imaging have allowed monitoring of stem cell homing and survival. In this review article, we will discuss the clinical application and future directions of stem cell imaging in advanced heart failure.
View details for PubMedID 24298314
CD34(+) Stem Cell Therapy in Nonischemic Dilated Cardiomyopathy Patients.
Clinical pharmacology & therapeutics
2013; 94 (4): 452-458
Recent trends indicate that patients with nonischemic dilated cardiomyopathy represent the largest subpopulation of heart failure patients with a significant need for alternative treatment modalities. Similar to patients with ischemic cardiomyopathy, patients with nonischemic dilated cardiomyopathy have been found to have myocardial regions with flow abnormalities, which may represent targets for neoangiogenic therapies. CD34(+) stem cells might contribute to the formation of new blood vessels from existing vascular structures in ischemic tissues by the direct incorporation of injected cells into the newly developing vasculature or by the production and secretion of angiogenic cytokines. This review summarizes the long-term clinical effects and potential underlying mechanisms of CD34(+) cell therapy in patients with nonischemic dilated cardiomyopathy.Clinical Pharmacology & Therapeutics (2013); 94 4, 452-458. doi:10.1038/clpt.2013.134.
View details for DOI 10.1038/clpt.2013.134
View details for PubMedID 23903668
Noninvasive imaging of hypoxia-inducible factor-1a gene therapy for myocardial ischemia.
Human gene therapy methods
2013; 24 (5): 279-288
Abstract Hypoxia-inducible factor-1 alpha (HIF-1α) gene therapy holds great promise for the treatment of myocardial ischemia. Both preclinical and clinical evaluations of this therapy are underway and can benefit from a vector strategy that allows noninvasive assessment of HIF-1α expression as an objective measure of gene delivery. We have developed a novel bidirectional plasmid vector (pcTnT-HIF-1α-VP2-TSTA-fluc), which employs the cardiac troponin T (cTnT) promoter in conjunction with a two-step transcriptional amplification (TSTA) system to drive the linked expression of a recombinant HIF-1α gene (HIF-1α-VP2) and the firefly luciferase gene (fluc). The firefly luciferase (FLuc) activity serves as a surrogate for HIF-1α-VP2 expression, and can be noninvasively assessed in mice using bioluminescence imaging after vector delivery. Transfection of cultured HL-1 cardiomyocytes with pcTnT-HIF-1α-VP2-TSTA-fluc led to a strong correlation between FLuc and HIF-1α-dependent vascular endothelial growth factor expression (r(2)=0.88). Intramyocardial delivery of pcTnT-HIF-1α-VP2-TSTA-fluc into infarcted mouse myocardium led to persistent HIF-1α-VP2 expression for 4 weeks, even though it improved neither CD31+ microvessel density nor echocardiographically determined left ventricular systolic function. These results lend support to recent findings of suboptimal efficacy associated with plasmid-mediated HIF-1α therapy. The imaging techniques developed herein should be useful for further optimizing HIF-1α-VP2 therapy in preclinical models of myocardial ischemia.
View details for DOI 10.1089/hgtb.2013.028
View details for PubMedID 23937265
Screening drug-induced arrhythmia [corrected] using human induced pluripotent stem cell-derived cardiomyocytes and low-impedance microelectrode arrays.
2013; 128 (11): S3-13
Drug-induced arrhythmia is one of the most common causes of drug development failure and withdrawal from market. This study tested whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) combined with a low-impedance microelectrode array (MEA) system could improve on industry-standard preclinical cardiotoxicity screening methods, identify the effects of well-characterized drugs, and elucidate underlying risk factors for drug-induced arrhythmia. hiPSC-CMs may be advantageous over immortalized cell lines because they possess similar functional characteristics as primary human cardiomyocytes and can be generated in unlimited quantities.Pharmacological responses of beating embryoid bodies exposed to a comprehensive panel of drugs at 65 to 95 days postinduction were determined. Responses of hiPSC-CMs to drugs were qualitatively and quantitatively consistent with the reported drug effects in literature. Torsadogenic hERG blockers, such as sotalol and quinidine, produced statistically and physiologically significant effects, consistent with patch-clamp studies, on human embryonic stem cell-derived cardiomyocytes hESC-CMs. False-negative and false-positive hERG blockers were identified accurately. Consistent with published studies using animal models, early afterdepolarizations and ectopic beats were observed in 33% and 40% of embryoid bodies treated with sotalol and quinidine, respectively, compared with negligible early afterdepolarizations and ectopic beats in untreated controls.We found that drug-induced arrhythmias can be recapitulated in hiPSC-CMs and documented with low impedance MEA. Our data indicate that the MEA/hiPSC-CM assay is a sensitive, robust, and efficient platform for testing drug effectiveness and for arrhythmia screening. This system may hold great potential for reducing drug development costs and may provide significant advantages over current industry standard assays that use immortalized cell lines or animal models.
View details for DOI 10.1161/CIRCULATIONAHA.112.000570
View details for PubMedID 24030418
Comparison of Transendocardial and Intracoronary CD34(+) Cell Transplantation in Patients With Nonischemic Dilated Cardiomyopathy
2013; 128 (11): S42-S49
In an open-label blinded study, we compared intracoronary and transendocardial CD34(+) cell transplantation in patients with nonischemic dilated cardiomyopathy.Of the 40 patients with dilated cardiomyopathy, 20 were randomized to receive intracoronary injection and 20 received transendocardial CD34(+) cell delivery. In both groups, CD34(+) cells were mobilized by filgrastim, collected via apheresis, and labeled with technetium-99m radioisotope for single-photon emission computed tomographic imaging. In the intracoronary group, cells were injected intracoronarily in the artery supplying segments of greater perfusion defect on myocardial perfusion scintigraphy. In the transendocardial group, electroanatomic mapping was used to identify viable but dysfunctional myocardium, and transendocardial cell injections were performed. Nuclear single-photon emission computed tomographic imaging for quantification of myocardial retention was performed 18 hours thereafter. At baseline, groups did not differ in age, sex, left ventricular ejection fraction, or N-terminal pro-brain natriuretic peptide levels. The number of CD34(+) cells was also comparable (105 ± 31 × 10(6) in the transendocardial group versus 103 ± 27 × 10(6) in the intracoronary group, P=0.62). At 18 hours after procedure, myocardial retention was higher in the transendocardial group (19.2 ± 4.8%) than in the intracoronary group (4.4 ± 1.2%, P<0.01). At 6 months, left ventricular ejection fraction improved more in the transendocardial group (+8.1 ± 4.3%) than in the intracoronary group (+4.2 ± 2.3%, P=0.03). The same pattern was observed for the 6-minute walk test distance (+125 ± 33 m in the transendocardial group versus +86 ± 13 m in the intracoronary group, P=0.03) and N-terminal pro-brain natriuretic peptide (-628 ± 211 versus -315 ± 133 pg/mL, P=0.04).In patients with dilated cardiomyopathy, transendocardial CD34(+) cell transplantation is associated with higher myocardial retention rates and greater improvement in ventricular function, N-terminal pro-brain natriuretic peptide, and exercise capacity compared with intracoronary route.http://www.clinicaltrials.gov. Unique identifier: NCT01350310.
View details for DOI 10.1161/CIRCULATIONAHA.112.000230
View details for Web of Science ID 000330535500005
View details for PubMedID 24030420
A Transgenic Tri-Modality Reporter Mouse
2013; 8 (8)
Transgenic mouse with a stably integrated reporter gene(s) can be a valuable resource for obtaining uniformly labeled stem cells, tissues, and organs for various applications. We have generated a transgenic mouse model that ubiquitously expresses a tri-fusion reporter gene (fluc2-tdTomato-ttk) driven by a constitutive chicken β-actin promoter. This "Tri-Modality Reporter Mouse" system allows one to isolate most cells from this donor mouse and image them for bioluminescent (fluc2), fluorescent (tdTomato), and positron emission tomography (PET) (ttk) modalities. Transgenic colonies with different levels of tri-fusion reporter gene expression showed a linear correlation between all three-reporter proteins (R(2)=0.89 for TdTomato vs Fluc, R(2)=0.94 for Fluc vs TTK, R(2)=0.89 for TdTomato vs TTK) in vitro from tissue lysates and in vivo by optical and PET imaging. Mesenchymal stem cells (MSCs) isolated from this transgenics showed high level of reporter gene expression, which linearly correlated with the cell numbers (R(2)=0.99 for bioluminescence imaging (BLI)). Both BLI (R(2)=0.93) and micro-PET (R(2)=0.94) imaging of the subcutaneous implants of Tri-Modality Reporter Mouse derived MSCs in nude mice showed linear correlation with the cell numbers and across different imaging modalities (R(2)=0.97). Serial imaging of MSCs transplanted to mice with acute myocardial infarction (MI) by intramyocardial injection exhibited significantly higher signals in MI heart at days 2, 3, 4, and 7 (p<0.01). MSCs transplanted to the ischemic hindlimb of nude mice showed significantly higher BLI and PET signals in the first 2 weeks that dropped by 4(th) week due to poor cell survival. However, laser Doppler perfusion imaging revealed that blood circulation in the ischemic limb was significantly improved in the MSCs transplantation group compared with the control group. In summary, this mouse can be used as a source of donor cells and organs in various research areas such as stem cell research, tissue engineering research, and organ transplantation.
View details for DOI 10.1371/journal.pone.0073580
View details for Web of Science ID 000326473200094
View details for PubMedID 23951359
Molecular imaging: The key to advancing cardiac stem cell therapy
TRENDS IN CARDIOVASCULAR MEDICINE
2013; 23 (6): 201-210
Cardiac stem cell therapy continues to hold promise for the treatment of ischemic heart disease despite the fact that early promising pre-clinical findings have yet to be translated into consistent clinical success. The latest human studies have collectively identified a pressing need to better understand stem cell behavior in humans and called for more incorporation of noninvasive imaging techniques into the design and evaluation of human stem cell therapy trials. This review discusses the various molecular imaging techniques validated to date for studying stem cells in living subjects, with a particular emphasis on their utilities in assessing the acute retention and the long-term survival of transplanted stem cells. These imaging techniques will be essential for advancing cardiac stem cell therapy by providing the means to both guide ongoing optimization and predict treatment response in humans.
View details for DOI 10.1016/j.tcm.2012.12.003
View details for Web of Science ID 000322147600003
View details for PubMedID 23561794
Development of Poly(beta-amino ester)-Based Biodegradable Nanoparticles for Nonviral Delivery of Minicircle DNA
2013; 7 (8): 7241-7250
Gene therapy provides a powerful tool for regulating cellular processes and tissue repair. Minicircle (MC) DNA are supercoiled DNA molecules free of bacterial plasmid backbone elements and have been reported to enhance prolonged gene expression compared to conventional plasmids. Despite the great promise of MC DNA for gene therapy, methods for safe and efficient MC DNA delivery remain lacking. To overcome this bottleneck, here we report the development of a poly(β-amino ester) (PBAE)-based, biodegradable nanoparticulate platform for efficient delivery of MC DNA driven by a Ubc promoter in vitro and in vivo. By synthesizing and screening a small library of 18 PBAE polymers with different backbone and end-group chemistry, we identified lead cationic PBAE structures that can complex with minicircle DNA to form nanoparticles, and delivery efficiency can be further modulated by tuning PBAE chemistry. Using human embryonic kidney 293 cells and mouse embryonic fibroblasts as model cell types, we identified a few PBAE polymers that allow efficient MC delivery at levels that are comparable or even surpassing Lipofectamine 2000. The biodegradable nature of PBAE-based nanoparticles facilitates in vivo applications and clinical translation. When injected via intraperitoneal route in vivo, MC alone resulted in high transgene expression, and a lead PBAE/MC nanoparticle formulation achieved a further 2-fold increase in protein expression compared to MC alone. Together, our results highlight the promise of PBAE-based nanoparticles as promising nonviral gene carriers for MC delivery, which may provide a valuable tool for broad applications of MC DNA-based gene therapy.
View details for DOI 10.1021/nn402657d
View details for Web of Science ID 000323810600089
View details for PubMedID 23837668
Clonal precursor of bone, cartilage, and hematopoietic niche stromal cells
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2013; 110 (31): 12643-12648
Organs are composites of tissue types with diverse developmental origins, and they rely on distinct stem and progenitor cells to meet physiological demands for cellular production and homeostasis. How diverse stem cell activity is coordinated within organs is not well understood. Here we describe a lineage-restricted, self-renewing common skeletal progenitor (bone, cartilage, stromal progenitor; BCSP) isolated from limb bones and bone marrow tissue of fetal, neonatal, and adult mice. The BCSP clonally produces chondrocytes (cartilage-forming) and osteogenic (bone-forming) cells and at least three subsets of stromal cells that exhibit differential expression of cell surface markers, including CD105 (or endoglin), Thy1 [or CD90 (cluster of differentiation 90)], and 6C3 [ENPEP glutamyl aminopeptidase (aminopeptidase A)]. These three stromal subsets exhibit differential capacities to support hematopoietic (blood-forming) stem and progenitor cells. Although the 6C3-expressing subset demonstrates functional stem cell niche activity by maintaining primitive hematopoietic stem cell (HSC) renewal in vitro, the other stromal populations promote HSC differentiation to more committed lines of hematopoiesis, such as the B-cell lineage. Gene expression analysis and microscopic studies further reveal a microenvironment in which CD105-, Thy1-, and 6C3-expressing marrow stroma collaborate to provide cytokine signaling to HSCs and more committed hematopoietic progenitors. As a result, within the context of bone as a blood-forming organ, the BCSP plays a critical role in supporting hematopoiesis through its generation of diverse osteogenic and hematopoietic-promoting stroma, including HSC supportive 6C3(+) niche cells.
View details for DOI 10.1073/pnas.1310212110
View details for Web of Science ID 000322441500042
View details for PubMedID 23858471
The Role of SIRT6 Protein in Aging and Reprogramming of Human Induced Pluripotent Stem Cells
JOURNAL OF BIOLOGICAL CHEMISTRY
2013; 288 (25): 18439-18447
Aging is known to be the single most important risk factor for multiple diseases. Sirtuin-6, or SIRT6, has recently been identified as a critical regulator of transcription, genome stability, telomere integrity, DNA repair, and metabolic homeostasis. A knockout mouse model of SIRT6 has displayed dramatic phenotypes of accelerated aging. In keeping with its role in aging, we demonstrated that human dermal fibroblasts (HDFs) from older subjects were more resistant to reprogramming by classic Yamanaka factors than those from young subjects, but the addition of SIRT6 during reprogramming substantially improved such efficiency in older HDFs. Despite the importance of SIRT6, little is known about the molecular mechanism of its regulation. We show for the first time post-transcriptional regulation of SIRT6 by miR-766 and inverse correlation in the expression of this microRNA in HDFs from different age groups. Our results suggest that SIRT6 regulates miR-766 transcription via a feedback regulatory loop, which has implications for the modulation of SIRT6 expression in reprogramming of aging cells.
View details for DOI 10.1074/jbc.M112.405928
View details for Web of Science ID 000320721900046
View details for PubMedID 23653361
Overview of High Throughput Sequencing Technologies to Elucidate Molecular Pathways in Cardiovascular Diseases
2013; 112 (12): 1613-1623
High throughput sequencing technologies have become essential in studies on genomics, epigenomics, and transcriptomics. Although sequencing information has traditionally been elucidated using a low throughput technique called Sanger sequencing, high throughput sequencing technologies are capable of sequencing multiple DNA molecules in parallel, enabling hundreds of millions of DNA molecules to be sequenced at a time. This advantage allows high throughput sequencing to be used to create large data sets, generating more comprehensive insights into the cellular genomic and transcriptomic signatures of various diseases and developmental stages. Within high throughput sequencing technologies, whole exome sequencing can be used to identify novel variants and other mutations that may underlie many genetic cardiac disorders, whereas RNA sequencing can be used to analyze how the transcriptome changes. Chromatin immunoprecipitation sequencing and methylation sequencing can be used to identify epigenetic changes, whereas ribosome sequencing can be used to determine which mRNA transcripts are actively being translated. In this review, we will outline the differences in various sequencing modalities and examine the main sequencing platforms on the market in terms of their relative read depths, speeds, and costs. Finally, we will discuss the development of future sequencing platforms and how these new technologies may improve on current sequencing platforms. Ultimately, these sequencing technologies will be instrumental in further delineating how the cardiovascular system develops and how perturbations in DNA and RNA can lead to cardiovascular disease.
View details for DOI 10.1161/CIRCRESAHA.113.300939
View details for Web of Science ID 000330350900018
- Stem cell isolation: Differential stickiness. Nature materials 2013; 12 (6): 474-476
Dissecting the molecular relationship among various cardiogenic progenitor cells.
2013; 112 (9): 1253-1262
Rationale: Multiple progenitors derived from the heart and bone marrow (BM) have been used for cardiac repair. Despite this, not much is known about the molecular identity and relationship among these progenitors. To develop a robust stem cell therapy for the heart, it is critical to understand the molecular identity of the multiple cardiogenic progenitor cells. Objective: This study is the first report of high-throughput transcriptional profiling of cardiogenic progenitor cells carried out on an identical platform. Method and Results: Microarray-based transcriptional profiling was carried out for 3 cardiac (ckit(+), Sca1(+), and side population) and 2 BM (ckit(+) and mesenchymal stem cell) progenitors, obtained from age- and sex-matched wild-type C57BL/6 mice. Analysis indicated that cardiac-derived ckit(+) population was very distinct from Sca1(+) and side population cells in the downregulation of genes encoding for cell-cell and cell-matrix adhesion proteins, and in the upregulation of developmental genes. Significant enrichment of transcripts involved in DNA replication and repair was observed in BM-derived progenitors. The BM ckit(+) cells seemed to have the least correlation with the other progenitors, with enrichment of immature neutrophil-specific molecules. Conclusions: Our study indicates that cardiac ckit(+) cells represent the most primitive population in the rodent heart. Primitive cells of cardiac versus BM origin differ significantly with respect to stemness and cardiac lineage-specific genes, and molecules involved in DNA replication and repair. The detailed molecular profile of progenitors reported here will serve as a useful reference to determine the molecular identity of progenitors used in future preclinical and clinical studies.
View details for DOI 10.1161/CIRCRESAHA.112.300779
View details for PubMedID 23463815
Drug screening using a library of human induced pluripotent stem cell-derived cardiomyocytes reveals disease-specific patterns of cardiotoxicity.
2013; 127 (16): 1677-1691
Cardiotoxicity is a leading cause for drug attrition during pharmaceutical development and has resulted in numerous preventable patient deaths. Incidents of adverse cardiac drug reactions are more common in patients with preexisting heart disease than the general population. Here we generated a library of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from patients with various hereditary cardiac disorders to model differences in cardiac drug toxicity susceptibility for patients of different genetic backgrounds.Action potential duration and drug-induced arrhythmia were measured at the single cell level in hiPSC-CMs derived from healthy subjects and patients with hereditary long QT syndrome, familial hypertrophic cardiomyopathy, and familial dilated cardiomyopathy. Disease phenotypes were verified in long QT syndrome, hypertrophic cardiomyopathy, and dilated cardiomyopathy hiPSC-CMs by immunostaining and single cell patch clamp. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and the human ether-a-go-go-related gene expressing human embryonic kidney cells were used as controls. Single cell PCR confirmed expression of all cardiac ion channels in patient-specific hiPSC-CMs as well as hESC-CMs, but not in human embryonic kidney cells. Disease-specific hiPSC-CMs demonstrated increased susceptibility to known cardiotoxic drugs as measured by action potential duration and quantification of drug-induced arrhythmias such as early afterdepolarizations and delayed afterdepolarizations.We have recapitulated drug-induced cardiotoxicity profiles for healthy subjects, long QT syndrome, hypertrophic cardiomyopathy, and dilated cardiomyopathy patients at the single cell level for the first time. Our data indicate that healthy and diseased individuals exhibit different susceptibilities to cardiotoxic drugs and that use of disease-specific hiPSC-CMs may predict adverse drug responses more accurately than the standard human ether-a-go-go-related gene test or healthy control hiPSC-CM/hESC-CM screening assays.
View details for DOI 10.1161/CIRCULATIONAHA.113.001883
View details for PubMedID 23519760
- Enhanced A beta(1-40) Production in Endothelial Cells Stimulated with Fibrillar A beta(1-42) PLOS ONE 2013; 8 (3)
Effective Delivery of Stem Cells Using an Extracellular Matrix Patch Results in Increased Cell Survival and Proliferation and Reduced Scarring in Skin Wound Healing
TISSUE ENGINEERING PART A
2013; 19 (5-6): 738-747
Wound healing is one of the most complex biological processes and occurs in all tissues and organs of the body. In humans, fibrotic tissue, or scar, hinders function and is aesthetically unappealing. Stem cell therapy offers a promising new technique for aiding in wound healing; however, current findings show that stem cells typically die and/or migrate from the wound site, greatly decreasing efficacy of the treatment. Here, we demonstrate effectiveness of a stem cell therapy for improving wound healing in the skin and reducing scarring by introducing stem cells using a natural patch material. Adipose-derived stromal cells were introduced to excisional wounds created in mice using a nonimmunogenic extracellular matrix (ECM) patch material derived from porcine small-intestine submucosa (SIS). The SIS served as an attractive delivery vehicle because of its natural ECM components, including its collagen fiber network, providing the stem cells with a familiar structure. Experimental groups consisted of wounds with stem cell-seeded patches removed at different time points after wounding to determine an optimal treatment protocol. Stem cells delivered alone to skin wounds did not survive post-transplantation as evidenced by bioluminescence in vivo imaging. In contrast, delivery with the patch enabled a significant increase in stem cell proliferation and survival. Wound healing rates were moderately improved by treatment with stem cells on the patch; however, areas of fibrosis, indicating scarring, were significantly reduced in wounds treated with the stem cells on the patch compared to untreated wounds.
View details for DOI 10.1089/ten.tea.2012.0480
View details for Web of Science ID 000314581100015
View details for PubMedID 23072446
Sacrificial layer technique for axial force post assay of immature cardiomyocytes
2013; 15 (1): 171-181
Immature primary and stem cell-derived cardiomyocytes provide useful models for fundamental studies of heart development and cardiac disease, and offer potential for patient specific drug testing and differentiation protocols aimed at cardiac grafts. To assess their potential for augmenting heart function, and to gain insight into cardiac growth and disease, tissue engineers must quantify the contractile forces of these single cells. Currently, axial contractile forces of isolated adult heart cells can only be measured by two-point methods such as carbon fiber techniques, which cannot be applied to neonatal and stem cell-derived heart cells because they are more difficult to handle and lack a persistent shape. Here we present a novel axial technique for measuring the contractile forces of isolated immature cardiomyocytes. We overcome cell manipulation and patterning challenges by using a thermoresponsive sacrificial support layer in conjunction with arrays of widely separated elastomeric microposts. Our approach has the potential to be high-throughput, is functionally analogous to current gold-standard axial force assays for adult heart cells, and prescribes elongated cell shapes without protein patterning. Finally, we calibrate these force posts with piezoresistive cantilevers to dramatically reduce measurement error typical for soft polymer-based force assays. We report quantitative measurements of peak contractile forces up to 146 nN with post stiffness standard error (26 nN) far better than that based on geometry and stiffness estimates alone. The addition of sacrificial layers to future 2D and 3D cell culture platforms will enable improved cell placement and the complex suspension of cells across 3D constructs.
View details for DOI 10.1007/s10544-012-9710-3
View details for Web of Science ID 000313517800018
Advances in nanotechnology for the management of coronary artery disease.
Trends in cardiovascular medicine
2013; 23 (2): 39-45
Nanotechnology holds tremendous potential to advance the current treatment of coronary artery disease. Nanotechnology may assist medical therapies by providing a safe and efficacious delivery platform for a variety of drugs aimed at modulating lipid disorders, decreasing inflammation and angiogenesis within atherosclerotic plaques, and preventing plaque thrombosis. Nanotechnology may improve coronary stent applications by promoting endothelial recovery on a stent surface utilizing bio-mimetic nanofibrous scaffolds, and also by preventing in-stent restenosis using nanoparticle-based delivery of drugs that are decoupled from stents. Additionally, nanotechnology may enhance tissue-engineered graft materials for application in coronary artery bypass grafting by facilitating cellular infiltration and remodeling of a graft matrix.
View details for DOI 10.1016/j.tcm.2012.08.009
View details for PubMedID 23245913
Immunogenicity of Pluripotent Stem Cells and Their Derivatives
2013; 112 (3): 549-561
The ability of pluripotent stem cells to self-renew and differentiate into all somatic cell types brings great prospects to regenerative medicine and human health. However, before clinical applications, much translational research is necessary to ensure that their therapeutic progenies are functional and nontumorigenic, that they are stable and do not dedifferentiate, and that they do not elicit immune responses that could threaten their survival in vivo. For this, an in-depth understanding of their biology, genetic, and epigenetic make-up and of their antigenic repertoire is critical for predicting their immunogenicity and for developing strategies needed to assure successful long-term engraftment. Recently, the expectation that reprogrammed somatic cells would provide an autologous cell therapy for personalized medicine has been questioned. Induced pluripotent stem cells display several genetic and epigenetic abnormalities that could promote tumorigenicity and immunogenicity in vivo. Understanding the persistence and effects of these abnormalities in induced pluripotent stem cell derivatives is critical to allow clinicians to predict graft fate after transplantation, and to take requisite measures to prevent immune rejection. With clinical trials of pluripotent stem cell therapy on the horizon, the importance of understanding immunologic barriers and devising safe, effective strategies to bypass them is further underscored. This approach to overcome immunologic barriers to stem cell therapy can take advantage of the validated knowledge acquired from decades of hematopoietic stem cell transplantation.
View details for DOI 10.1161/CIRCRESAHA.111.249243
View details for Web of Science ID 000314356700022
View details for PubMedID 23371903
A Review of Human Pluripotent Stem Cell-Derived Cardiomyocytes for High-Throughput Drug Discovery, Cardiotoxicity Screening, and Publication Standards
JOURNAL OF CARDIOVASCULAR TRANSLATIONAL RESEARCH
2013; 6 (1): 22-30
Drug attrition rates have increased in past years, resulting in growing costs for the pharmaceutical industry and consumers. The reasons for this include the lack of in vitro models that correlate with clinical results and poor preclinical toxicity screening assays. The in vitro production of human cardiac progenitor cells and cardiomyocytes from human pluripotent stem cells provides an amenable source of cells for applications in drug discovery, disease modeling, regenerative medicine, and cardiotoxicity screening. In addition, the ability to derive human-induced pluripotent stem cells from somatic tissues, combined with current high-throughput screening and pharmacogenomics, may help realize the use of these cells to fulfill the potential of personalized medicine. In this review, we discuss the use of pluripotent stem cell-derived cardiomyocytes for drug discovery and cardiotoxicity screening, as well as current hurdles that must be overcome for wider clinical applications of this promising approach.
View details for DOI 10.1007/s12265-012-9423-2
View details for Web of Science ID 000313657700003
View details for PubMedID 23229562
MicroRNA-302 Increases Reprogramming Efficiency via Repression of NR2F2
2013; 31 (2): 259-268
MicroRNAs (miRNAs) have emerged as critical regulators of gene expression through translational inhibition and RNA decay and have been implicated in the regulation of cellular differentiation, proliferation, angiogenesis, and apoptosis. In this study, we analyzed global miRNA and mRNA microarrays to predict novel miRNA-mRNA interactions in human embryonic stem cells and induced pluripotent stem cells (iPSCs). In particular, we demonstrate a regulatory feedback loop between the miR-302 cluster and two transcription factors, NR2F2 and OCT4. Our data show high expression of miR-302 and OCT4 in pluripotent cells, while NR2F2 is expressed exclusively in differentiated cells. Target analysis predicts that NR2F2 is a direct target of miR-302, which we experimentally confirm by reporter luciferase assays and real-time polymerase chain reaction. We also demonstrate that NR2F2 directly inhibits the activity of the OCT4 promoter and thus diminishes the positive feedback loop between OCT4 and miR-302. Importantly, higher reprogramming efficiencies were obtained when we reprogrammed human adipose-derived stem cells into iPSCs using four factors (KLF4, C-MYC, OCT4, and SOX2) plus miR-302 (this reprogramming cocktail is hereafter referred to as "KMOS3") when compared to using four factors ("KMOS"). Furthermore, shRNA knockdown of NR2F2 mimics the over-expression of miR-302 by also enhancing reprogramming efficiency. Interestingly, we were unable to generate iPSCs from miR-302a/b/c/d alone, which is in contrast to previous publications that have reported that miR-302 by itself can reprogram human skin cancer cells and human hair follicle cells. Taken together, these findings demonstrate that miR-302 inhibits NR2F2 and promotes pluripotency through indirect positive regulation of OCT4. This feedback loop represents an important new mechanism for understanding and inducing pluripotency in somatic cells.
View details for DOI 10.1002/stem.1278
View details for Web of Science ID 000314873000006
Effects of Intracoronary CD34(+) Stem Cell Transplantation in Nonischemic Dilated Cardiomyopathy Patients 5-Year Follow-Up
2013; 112 (1): 165-173
CD34+ transplantation in dilated cardiomyopathy was associated with short-term improvement in left ventricular ejection fraction and exercise tolerance.We investigated long-term effects of intracoronary CD34+ cell transplantation in dilated cardiomyopathy and the relationship between intramyocardial cell homing and clinical response.Of 110 dilated cardiomyopathy patients, 55 were randomized to receive CD34+ stem cell transplantation (SC group) and 55 received no cell therapy (controls). In the SC group, CD34+ cells were mobilized by granulocyte colony-stimulating factor and collected via apheresis. Patients underwent myocardial scintigraphy and cells were injected in the artery supplying segments with the greatest perfusion defect. At baseline, 2 groups did not differ in age, sex, left ventricular ejection fraction, or N-terminal B-type natriuretic peptide levels. At 5 years, stem cell therapy was associated with increased left ventricular ejection fraction (from 24.3 ± 6.5% to 30.0 ± 5.1%; P=0.02), increased 6-minute walk distance (from 344 ± 90 m to 477 ± 130 m; P<0.001), and decreased N-terminal B-type natriuretic peptide (from 2322 ± 1234 pg/mL to 1011 ± 893 pg/mL; P<0.01). Left ventricular ejection fraction improvement was more significant in patients with higher myocardial homing of injected cells. During follow-up, 27 (25%) patients died and 9 (8%) underwent heart transplantation. Of the 27 deaths, 13 were attributed to pump failure and 14 were attributed to sudden cardiac death. Total mortality was lower in the SC group (14%) than in controls (35%; P=0.01). The same was true of pump failure (5% vs. 18%; P=0.03), but not of sudden cardiac death (9% vs. 16%; P=0.39).Intracoronary stem cell transplantation may be associated with improved ventricular function, exercise tolerance, and long-term survival in patients with dilated cardiomyopathy. Higher intramyocardial homing is associated with better stem cell therapy response.
View details for DOI 10.1161/CIRCRESAHA.112.276519
View details for Web of Science ID 000313053000021
View details for PubMedID 23065358
Abnormal Calcium Handling Properties Underlie Familial Hypertrophic Cardiomyopathy Pathology in Patient-Specific Induced Pluripotent Stem Cells
CELL STEM CELL
2013; 12 (1): 101-113
Familial hypertrophic cardiomyopathy (HCM) is a prevalent hereditary cardiac disorder linked to arrhythmia and sudden cardiac death. While the causes of HCM have been identified as genetic mutations in the cardiac sarcomere, the pathways by which sarcomeric mutations engender myocyte hypertrophy and electrophysiological abnormalities are not understood. To elucidate the mechanisms underlying HCM development, we generated patient-specific induced pluripotent stem cell cardiomyocytes (iPSC-CMs) from a ten-member family cohort carrying a hereditary HCM missense mutation (Arg663His) in the MYH7 gene. Diseased iPSC-CMs recapitulated numerous aspects of the HCM phenotype including cellular enlargement and contractile arrhythmia at the single-cell level. Calcium (Ca(2+)) imaging indicated dysregulation of Ca(2+) cycling and elevation in intracellular Ca(2+) ([Ca(2+)](i)) are central mechanisms for disease pathogenesis. Pharmacological restoration of Ca(2+) homeostasis prevented development of hypertrophy and electrophysiological irregularities. We anticipate that these findings will help elucidate the mechanisms underlying HCM development and identify novel therapies for the disease.
View details for DOI 10.1016/j.stem.2012.10.010
View details for Web of Science ID 000313839500014
Induced pluripotency of human prostatic epithelial cells.
2013; 8 (5)
Induced pluripotent stem (iPS) cells are a valuable resource for discovery of epigenetic changes critical to cell type-specific differentiation. Although iPS cells have been generated from other terminally differentiated cells, the reprogramming of normal adult human basal prostatic epithelial (E-PZ) cells to a pluripotent state has not been reported. Here, we attempted to reprogram E-PZ cells by forced expression of Oct4, Sox2, c-Myc, and Klf4 using lentiviral vectors and obtained embryonic stem cell (ESC)-like colonies at a frequency of 0.01%. These E-PZ-iPS-like cells with normal karyotype gained expression of pluripotent genes typical of iPS cells (Tra-1-81, SSEA-3, Nanog, Sox2, and Oct4) and lost gene expression characteristic of basal prostatic epithelial cells (CK5, CK14, and p63). E-PZ-iPS-like cells demonstrated pluripotency by differentiating into ectodermal, mesodermal, and endodermal cells in vitro, although lack of teratoma formation in vivo and incomplete demethylation of pluripotency genes suggested only partial reprogramming. Importantly, E-PZ-iPS-like cells re-expressed basal epithelial cell markers (CD44, p63, MAO-A) in response to prostate-specific medium in spheroid culture. Androgen induced expression of androgen receptor (AR), and co-culture with rat urogenital sinus further induced expression of prostate-specific antigen (PSA), a hallmark of secretory cells, suggesting that E-PZ-iPS-like cells have the capacity to differentiate into prostatic basal and secretory epithelial cells. Finally, when injected into mice, E-PZ-iPS-like cells expressed basal epithelial cell markers including CD44 and p63. When co-injected with rat urogenital mesenchyme, E-PZ-iPS-like cells expressed AR and expression of p63 and CD44 was repressed. DNA methylation profiling identified epigenetic changes in key pathways and genes involved in prostatic differentiation as E-PZ-iPS-like cells converted to differentiated AR- and PSA-expressing cells. Our results suggest that iPS-like cells derived from prostatic epithelial cells are pluripotent and capable of prostatic differentiation; therefore, provide a novel model for investigating epigenetic changes involved in prostate cell lineage specification.
View details for DOI 10.1371/journal.pone.0064503
View details for PubMedID 23717621
- Cardiovascular molecular imaging as a tool to study biology. Theranostics 2013; 3 (11): 914-915
- Imaging Neural Stem Cell Graft-Induced Structural Repair in Stroke CELL TRANSPLANTATION 2013; 22 (5): 881-892
Generation of Human iPSCs from Human Peripheral Blood Mononuclear Cells Using Non-integrative Sendai Virus in Chemically Defined Conditions.
Methods in molecular biology (Clifton, N.J.)
2013; 1036: 81-88
Human-induced pluripotent stem cells (hiPSCs) have received enormous attention because of their ability to differentiate into multiple cell types that demonstrate the patient's original phenotype. The use of hiPSCs is particularly valuable to the study of cardiac biology, as human cardiomyocytes are difficult to isolate and culture and have a limited proliferative potential. By deriving iPSCs from patients with heart disease and subsequently differentiating these hiPSCs to cardiomyocytes, it is feasible to study cardiac biology in vitro and model cardiac diseases. While there are many different methods for deriving hiPSCs, clinical use of these hiPSCs will require derivation by methods that do not involve modification of the original genome (non-integrative) or incorporate xeno-derived products (such as bovine serum albumin) which may contain xeno-agents. Ideally, this derivation would be carried out under chemically defined conditions to prevent lot-to-lot variability and enhance reproducibility. Additionally, derivation from cell types such as fibroblasts requires extended culture (4-6 weeks), greatly increasing the time required to progress from biopsy to hiPSC. Herein, we outline a method of culturing peripheral blood mononuclear cells (PBMCs) and reprogramming PBMCs into hiPSCs using a non-integrative Sendai virus.
View details for DOI 10.1007/978-1-62703-511-8_7
View details for PubMedID 23807788
MicroRNA expression profiling of human-induced pluripotent and embryonic stem cells.
Methods in molecular biology (Clifton, N.J.)
2013; 936: 247-256
Clinical implications of induced pluripotent stem (iPS) cell technology are enormous for personalized medicine. However, extensive use of viral approach for ectopic expression of reprogramming factors is a major hurdle in realization of its true potential. Non-viral methods for making iPS cells, although plausible, are impractical because of high cost. MicroRNAs are important cellular modulators that have been shown to rival transcription factors and are important players in embryonic development. We have generated distinct "microRNA-omes" signature of iPS cells that remain in a near embryonic stem (ES) cell state and distinct from differentiated cells. Recent advances in the microRNA field and experimentally validated microRNAs warrant a review in experimental protocols for microRNA expression profile.
View details for PubMedID 23007513
American Journal of Nuclear Medicine and Molecular Imaging: Editorial Board (2014) e-Century Publishing Corporation.
American journal of nuclear medicine and molecular imaging
2013; 4 (1): 89-95
View details for PubMedID 24380049
Enhanced Aß(1-40) production in endothelial cells stimulated with fibrillar Aß(1-42).
2013; 8 (3)
Amyloid accumulation in the brain of Alzheimer's patients results from altered processing of the 39- to 43-amino acid amyloid ? protein (A?). The mechanisms for the elevated amyloid (A?(1-42)) are considered to be over-expression of the amyloid precursor protein (APP), enhanced cleavage of APP to A?, and decreased clearance of A? from the central nervous system (CNS). We report herein studies of A? stimulated effects on endothelial cells. We observe an interesting and as yet unprecedented feedback effect involving A?(1-42) fibril-induced synthesis of APP by Western blot analysis in the endothelial cell line Hep-1. We further observe an increase in the expression of A?(1-40) by flow cytometry and fluorescence microscopy. This phenomenon is reproducible for cultures grown both in the presence and absence of serum. In the former case, flow cytometry reveals that A?(1-40) accumulation is less pronounced than under serum-free conditions. Immunofluorescence staining further corroborates these observations. Cellular responses to fibrillar A?(1-42) treatment involving eNOS upregulation and increased autophagy are also reported.
View details for DOI 10.1371/journal.pone.0058194
View details for PubMedID 23505467
Pluripotent Stem Cells: Immune to the Immune System?
SCIENCE TRANSLATIONAL MEDICINE
2012; 4 (164)
Human embryonic stem cells (hESCs), initially thought to be immune privileged cells, are now known to be susceptible to immune recognition. Human induced pluripotent stem cells (iPSCs) have been proposed as a potential source of autologous stem cells for therapy, but even these autologous stem cells may be targets of immune rejection. With clinical trials on the horizon, it is imperative that the immunogenicity of hESCs and iPSCs be definitively understood.
View details for DOI 10.1126/scitranslmed.3005090
View details for Web of Science ID 000312393900002
View details for PubMedID 23241742
In vivo directed differentiation of pluripotent stem cells for skeletal regeneration
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2012; 109 (50): 20379-20384
Pluripotent cells represent a powerful tool for tissue regeneration, but their clinical utility is limited by their propensity to form teratomas. Little is known about their interaction with the surrounding niche following implantation and how this may be applied to promote survival and functional engraftment. In this study, we evaluated the ability of an osteogenic microniche consisting of a hydroxyapatite-coated, bone morphogenetic protein-2-releasing poly-L-lactic acid scaffold placed within the context of a macroenvironmental skeletal defect to guide in vivo differentiation of both embryonic and induced pluripotent stem cells. In this setting, we found de novo bone formation and participation by implanted cells in skeletal regeneration without the formation of a teratoma. This finding suggests that local cues from both the implanted scaffold/cell micro- and surrounding macroniche may act in concert to promote cellular survival and the in vivo acquisition of a terminal cell fate, thereby allowing for functional engraftment of pluripotent cells into regenerating tissue.
View details for DOI 10.1073/pnas.1218052109
View details for Web of Science ID 000312605600055
View details for PubMedID 23169671
Genome Editing of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells With Zinc Finger Nucleases for Cellular Imaging
2012; 111 (12): 1494-?
Molecular imaging has proven to be a vital tool in the characterization of stem cell behavior in vivo. However, the integration of reporter genes has typically relied on random integration, a method that is associated with unwanted insertional mutagenesis and positional effects on transgene expression.To address this barrier, we used genome editing with zinc finger nuclease (ZFN) technology to integrate reporter genes into a safe harbor gene locus (PPP1R12C, also known as AAVS1) in the genome of human embryonic stem cells and human induced pluripotent stem cells for molecular imaging.We used ZFN technology to integrate a construct containing monomeric red fluorescent protein, firefly luciferase, and herpes simplex virus thymidine kinase reporter genes driven by a constitutive ubiquitin promoter into a safe harbor locus for fluorescence imaging, bioluminescence imaging, and positron emission tomography imaging, respectively. High efficiency of ZFN-mediated targeted integration was achieved in both human embryonic stem cells and induced pluripotent stem cells. ZFN-edited cells maintained both pluripotency and long-term reporter gene expression. Functionally, we successfully tracked the survival of ZFN-edited human embryonic stem cells and their differentiated cardiomyocytes and endothelial cells in murine models, demonstrating the use of ZFN-edited cells for preclinical studies in regenerative medicine.Our study demonstrates a novel application of ZFN technology to the targeted genetic engineering of human pluripotent stem cells and their progeny for molecular imaging in vitro and in vivo.
View details for DOI 10.1161/CIRCRESAHA.112.274969
View details for Web of Science ID 000311994700042
View details for PubMedID 22967807
Transient, Inducible, Placenta-Specific Gene Expression in Mice
2012; 153 (11): 5637-5644
Molecular understanding of placental functions and pregnancy disorders is limited by the absence of methods for placenta-specific gene manipulation. Although persistent placenta-specific gene expression has been achieved by lentivirus-based gene delivery methods, developmentally and physiologically important placental genes have highly stage-specific functions, requiring controllable, transient expression systems for functional analysis. Here, we describe an inducible, placenta-specific gene expression system that enables high-level, transient transgene expression and monitoring of gene expression by live bioluminescence imaging in mouse placenta at different stages of pregnancy. We used the third generation tetracycline-responsive tranactivator protein Tet-On 3G, with 10- to 100-fold increased sensitivity to doxycycline (Dox) compared with previous versions, enabling unusually sensitive on-off control of gene expression in vivo. Transgenic mice expressing Tet-On 3G were created using a new integrase-based, site-specific approach, yielding high-level transgene expression driven by a ubiquitous promoter. Blastocysts from these mice were transduced with the Tet-On 3G-response element promoter-driving firefly luciferase using lentivirus-mediated placenta-specific gene delivery and transferred into wild-type pseudopregnant recipients for placenta-specific, Dox-inducible gene expression. Systemic Dox administration at various time points during pregnancy led to transient, placenta-specific firefly luciferase expression as early as d 5 of pregnancy in a Dox dose-dependent manner. This system enables, for the first time, reliable pregnancy stage-specific induction of gene expression in the placenta and live monitoring of gene expression during pregnancy. It will be widely applicable to studies of both placental development and pregnancy, and the site-specific Tet-On G3 mouse will be valuable for studies in a broad range of tissues.
View details for DOI 10.1210/en.2012-1556
View details for Web of Science ID 000310359300049
View details for PubMedID 23011919
- Returns of the Living Dead Therapeutic Action of Irradiated and Mitotically Inactivated Embryonic Stem Cells CIRCULATION RESEARCH 2012; 111 (10): 1250-1252
Induced Pluripotent Stem Cells as a Disease Modeling and Drug Screening Platform
JOURNAL OF CARDIOVASCULAR PHARMACOLOGY
2012; 60 (4): 408-416
Induced pluripotent stem cells (iPSCs) hold great hopes for therapeutic application in various diseases. Although ongoing research is dedicated to achieving clinical translation of iPSCs, further understanding of the mechanisms that underlie complex pathogenic conditions is required. Compared with other classical models for studying diseases, iPSCs provide considerable advantages. A newly emerging application of iPSCs is in vitro disease modeling, which can significantly improve the never-ending search for new pharmacological cures. Here, we will discuss current efforts to create iPSC-dependent patient-specific disease models. Furthermore, we will review the use of iPSCs for development and testing of new therapeutic agents and the implications for high-throughput drug screening.
View details for DOI 10.1097/FJC.0b013e318247f642
View details for Web of Science ID 000309977900012
View details for PubMedID 22240913
- Pushing the Reset Button: Chemical-Induced Conversion of Amniotic Fluid Stem Cells Into a Pluripotent State MOLECULAR THERAPY 2012; 20 (10): 1839-1841
Microfluidic Single-Cell Analysis Shows That Porcine Induced Pluripotent Stem Cell-Derived Endothelial Cells Improve Myocardial Function by Paracrine Activation
2012; 111 (7): 882-893
Induced pluripotent stem cells (iPSCs) hold great promise for the development of patient-specific therapies for cardiovascular disease. However, clinical translation will require preclinical optimization and validation of large-animal iPSC models.To successfully derive endothelial cells from porcine iPSCs and demonstrate their potential utility for the treatment of myocardial ischemia.Porcine adipose stromal cells were reprogrammed to generate porcine iPSCs (piPSCs). Immunohistochemistry, quantitative PCR, microarray hybridization, and angiogenic assays confirmed that piPSC-derived endothelial cells (piPSC-ECs) shared similar morphological and functional properties as endothelial cells isolated from the autologous pig aorta. To demonstrate their therapeutic potential, piPSC-ECs were transplanted into mice with myocardial infarction. Compared with control, animals transplanted with piPSC-ECs showed significant functional improvement measured by echocardiography (fractional shortening at week 4: 27.2±1.3% versus 22.3±1.1%; P<0.001) and MRI (ejection fraction at week 4: 45.8±1.3% versus 42.3±0.9%; P<0.05). Quantitative protein assays and microfluidic single-cell PCR profiling showed that piPSC-ECs released proangiogenic and antiapoptotic factors in the ischemic microenvironment, which promoted neovascularization and cardiomyocyte survival, respectively. Release of paracrine factors varied significantly among subpopulations of transplanted cells, suggesting that transplantation of specific cell populations may result in greater functional recovery.In summary, this is the first study to successfully differentiate piPSCs-ECs from piPSCs and demonstrate that transplantation of piPSC-ECs improved cardiac function after myocardial infarction via paracrine activation. Further development of these large animal iPSC models will yield significant insights into their therapeutic potential and accelerate the clinical translation of autologous iPSC-based therapy.
View details for DOI 10.1161/CIRCRESAHA.112.269001
View details for Web of Science ID 000308868800015
View details for PubMedID 22821929
Safe Genetic Modification of Cardiac Stem Cells Using a Site-Specific Integration Technique
2012; 126 (11): S20-?
Human cardiac progenitor cells (hCPCs) are a promising cell source for regenerative repair after myocardial infarction. Exploitation of their full therapeutic potential may require stable genetic modification of the cells ex vivo. Safe genetic engineering of stem cells, using facile methods for site-specific integration of transgenes into known genomic contexts, would significantly enhance the overall safety and efficacy of cellular therapy in a variety of clinical contexts.We used the phiC31 site-specific recombinase to achieve targeted integration of a triple fusion reporter gene into a known chromosomal context in hCPCs and human endothelial cells. Stable expression of the reporter gene from its unique chromosomal integration site resulted in no discernible genomic instability or adverse changes in cell phenotype. Namely, phiC31-modified hCPCs were unchanged in their differentiation propensity, cellular proliferative rate, and global gene expression profile when compared with unaltered control hCPCs. Expression of the triple fusion reporter gene enabled multimodal assessment of cell fate in vitro and in vivo using fluorescence microscopy, bioluminescence imaging, and positron emission tomography. Intramyocardial transplantation of genetically modified hCPCs resulted in significant improvement in myocardial function 2 weeks after cell delivery, as assessed by echocardiography (P=0.002) and MRI (P=0.001). We also demonstrated the feasibility and therapeutic efficacy of genetically modifying differentiated human endothelial cells, which enhanced hind limb perfusion (P<0.05 at day 7 and 14 after transplantation) on laser Doppler imaging.The phiC31 integrase genomic modification system is a safe, efficient tool to enable site-specific integration of reporter transgenes in progenitor and differentiated cell types.
View details for DOI 10.1161/CIRCULATIONAHA.111.084913
View details for Web of Science ID 000314150200003
View details for PubMedID 22965984
Biomaterial applications in cardiovascular tissue repair and regeneration.
Expert review of cardiovascular therapy
2012; 10 (8): 1039-1049
Cardiovascular disease physically damages the heart, resulting in loss of cardiac function. Medications can help alleviate symptoms, but it is more beneficial to treat the root cause by repairing injured tissues, which gives patients better outcomes. Besides heart transplants, cardiac surgeons use a variety of methods for repairing different areas of the heart such as the ventricular septal wall and valves. A multitude of biomaterials are used in the repair and replacement of impaired heart tissues. These biomaterials fall into two main categories: synthetic and natural. Synthetic materials used in cardiovascular applications include polymers and metals. Natural materials are derived from biological sources such as human donor or harvested animal tissues. A new class of composite materials has emerged to take advantage of the benefits of the strengths and minimize the weaknesses of both synthetic and natural materials. This article reviews the current and prospective applications of biomaterials in cardiovascular therapies.
View details for DOI 10.1586/erc.12.99
View details for PubMedID 23030293
The manipulation of natural killer cells to target tumor sites using magnetic nanoparticles
2012; 33 (22): 5584-5592
The present work demonstrates that Cy5.5 conjugated Fe(3)O(4)/SiO(2) core/shell nanoparticles could allow us to control movement of human natural killer cells (NK-92MI) by an external magnetic field. Required concentration of the nanoparticles for the cell manipulation is as low as ~20 ?g Fe/mL. However, the relative ratio of the nanoparticles loaded NK-92MI cells infiltrated into the target tumor site is enhanced by 17-fold by applying magnetic field and their killing activity is still maintained as same as the NK-92MI cells without the nanoparticles. This approach allows us to open alternative clinical treatment with reduced toxicity of the nanoparticles and enhanced infiltration of immunology to the target site.
View details for DOI 10.1016/j.biomaterials.2012.04.041
View details for Web of Science ID 000305366500010
View details for PubMedID 22575830
Early Stem Cell Engraftment Predicts Late Cardiac Functional Recovery Preclinical Insights From Molecular Imaging
2012; 5 (4): 481-490
Human cardiac progenitor cells have demonstrated great potential for myocardial repair in small and large animals, but robust methods for longitudinal assessment of their engraftment in humans is not yet readily available. In this study, we sought to optimize and evaluate the use of positron emission tomography (PET) reporter gene imaging for monitoring human cardiac progenitor cell (hCPC) transplantation in a mouse model of myocardial infarction.hCPCs were isolated and expanded from human myocardial samples and stably transduced with herpes simplex virus thymidine kinase (TK) PET reporter gene. Thymidine kinase-expressing hCPCs were characterized in vitro and transplanted into murine myocardial infarction models (n=57). Cardiac echocardiographic, magnetic resonance imaging and pressure-volume loop analyses revealed improvement in left ventricular contractile function 2 weeks after transplant (hCPC versus phosphate-buffered saline, P<0.03). Noninvasive PET imaging was used to track hCPC fate over a 4-week time period, demonstrating a substantial decline in surviving cells. Importantly, early cell engraftment as assessed by PET was found to predict subsequent functional improvement, implying a "dose-effect" relationship. We isolated the transplanted cells from recipient myocardium by laser capture microdissection for in vivo transcriptome analysis. Our results provide direct evidence that hCPCs augment cardiac function after their transplantation into ischemic myocardium through paracrine secretion of growth factors.PET reporter gene imaging can provide important diagnostic and prognostic information regarding the ultimate success of hCPC treatment for myocardial infarction.
View details for DOI 10.1161/CIRCIMAGING.111.969329
View details for Web of Science ID 000313573500014
View details for PubMedID 22565608
Inefficient Reprogramming of Fibroblasts into Cardiomyocytes Using Gata4, Mef2c, and Tbx5
2012; 111 (1): 50-55
Direct reprogramming of fibroblasts into cardiomyocytes is a novel strategy for cardiac regeneration. However, the key determinants involved in this process are unknown.To assess the efficiency of direct fibroblast reprogramming via viral overexpression of GATA4, Mef2c, and Tbx5 (GMT).We induced GMT overexpression in murine tail tip fibroblasts (TTFs) and cardiac fibroblasts (CFs) from multiple lines of transgenic mice carrying different cardiomyocyte lineage reporters. We found that the induction of GMT overexpression in TTFs and CFs is inefficient at inducing molecular and electrophysiological phenotypes of mature cardiomyocytes. In addition, transplantation of GMT infected CFs into injured mouse hearts resulted in decreased cell survival with minimal induction of cardiomyocyte genes.Significant challenges remain in our ability to convert fibroblasts into cardiomyocyte-like cells and a greater understanding of cardiovascular epigenetics is needed to increase the translational potential of this strategy.
View details for DOI 10.1161/CIRCRESAHA.112.270264
View details for Web of Science ID 000306061700012
View details for PubMedID 22581928
Large animal induced pluripotent stem cells as pre-clinical models for studying human disease
JOURNAL OF CELLULAR AND MOLECULAR MEDICINE
2012; 16 (6): 1196-1202
The path to induced pluripotency Discovery of a pan-species pluripotency network Animal iPSCs and disease modelling Issues with large animal iPSCs Conclusions The derivation of human embryonic stem cells and subsequently human induced pluripotent stem cells (iPSCs) has energized regenerative medicine research and enabled seemingly limitless applications. Although small animal models, such as mouse models, have played an important role in the progression of the field, typically, they are poor representations of the human disease phenotype. As an alternative, large animal models should be explored as a potentially better approach for clinical translation of cellular therapies. However, only fragmented information regarding the derivation, characterization and clinical usefulness of pluripotent large animal cells is currently available. Here, we briefly review the latest advances regarding the derivation and use of large animal iPSCs.
View details for DOI 10.1111/j.1582-4934.2012.01521.x
View details for Web of Science ID 000304468600005
View details for PubMedID 22212700
Atomic Force Mechanobiology of Pluripotent Stem Cell-Derived Cardiomyocytes
2012; 7 (5)
We describe a method using atomic force microscopy (AFM) to quantify the mechanobiological properties of pluripotent, stem cell-derived cardiomyocytes, including contraction force, rate, duration, and cellular elasticity. We measured beats from cardiomyocytes derived from induced pluripotent stem cells of healthy subjects and those with dilated cardiomyopathy, and from embryonic stem cell lines. We found that our AFM method could quantitate beat forces of single cells and clusters of cardiomyocytes. We demonstrate the dose-responsive, inotropic effect of norepinephrine and beta-adrenergic blockade of metoprolol. Cardiomyocytes derived from subjects with dilated cardiomyopathy showed decreased force and decreased cellular elasticity compared to controls. This AFM-based method can serve as a screening tool for the development of cardiac-active pharmacological agents, or as a platform for studying cardiomyocyte biology.
View details for DOI 10.1371/journal.pone.0037559
View details for Web of Science ID 000305343500135
View details for PubMedID 22624048
Dynamic microRNA expression during the transition from right ventricular hypertrophy to failure
2012; 44 (10): 562-575
MicroRNAs (miRs) are small, noncoding RNAs that are emerging as crucial regulators of cardiac remodeling in left ventricular hypertrophy (LVH) and failure (LVF). However, there are no data on their role in right ventricular hypertrophy (RVH) and failure (RVF). This is a critical question given that the RV is uniquely at risk in patients with congenital right-sided obstructive lesions and in those with systemic RVs. We have developed a murine model of RVH and RVF using pulmonary artery constriction (PAC). miR microarray analysis of RV from PAC vs. control demonstrates altered miR expression with gene targets associated with cardiomyocyte survival and growth during hypertrophy (miR 199a-3p) and reactivation of the fetal gene program during heart failure (miR-208b). The transition from hypertrophy to heart failure is characterized by apoptosis and fibrosis (miRs-34, 21, 1). Most are similar to LVH/LVF. However, there are several key differences between RV and LV: four miRs (34a, 28, 148a, and 93) were upregulated in RVH/RVF that are downregulated or unchanged in LVH/LVF. Furthermore, there is a corresponding downregulation of their putative target genes involving cell survival, proliferation, metabolism, extracellular matrix turnover, and impaired proteosomal function. The current study demonstrates, for the first time, alterations in miRs during the process of RV remodeling and the gene regulatory pathways leading to RVH and RVF. Many of these alterations are similar to those in the afterload-stressed LV. miRs differentially regulated between the RV and LV may contribute to the RVs increased susceptibility to heart failure.
View details for DOI 10.1152/physiolgenomics.00163.2011
View details for Web of Science ID 000304367600003
View details for PubMedID 22454450
Imaging Stem Cell Therapy for the Treatment of Peripheral Arterial Disease
CURRENT VASCULAR PHARMACOLOGY
2012; 10 (3): 361-373
Arteriosclerotic cardiovascular diseases are among the leading causes of morbidity and mortality worldwide. Therapeutic angiogenesis aims to treat ischemic myocardial and peripheral tissues by delivery of recombinant proteins, genes, or cells to promote neoangiogenesis. Concerns regarding the safety, side effects, and efficacy of protein and gene transfer studies have led to the development of cell-based therapies as alternative approaches to induce vascular regeneration and to improve function of damaged tissue. Cell-based therapies may be improved by the application of imaging technologies that allow investigators to track the location, engraftment, and survival of the administered cell population. The past decade of investigations has produced promising clinical data regarding cell therapy, but design of trials and evaluation of treatments stand to be improved by emerging insight from imaging studies. Here, we provide an overview of pre-clinical and clinical experience using cell-based therapies to promote vascular regeneration in the treatment of peripheral arterial disease. We also review four major imaging modalities and underscore the importance of in vivo analysis of cell fate for a full understanding of functional outcomes.
View details for Web of Science ID 000303258600011
View details for PubMedID 22239638
Microfluidic single-cell real-time PCR for comparative analysis of gene expression patterns
2012; 7 (5): 829-838
Single-cell quantitative real-time PCR (qRT-PCR) combined with high-throughput arrays allows the analysis of gene expression profiles at a molecular level in approximately 11 h after cell sample collection. We present here a high-content microfluidic real-time platform as a powerful tool for comparatively investigating the regulation of developmental processes in single cells. This approach overcomes the limitations involving heterogeneous cell populations and sample amounts, and may shed light on differential regulation of gene expression in normal versus disease-related contexts. Furthermore, high-throughput single-cell qRT-PCR provides a standardized, comparative assay for in-depth analysis of the mechanisms underlying human pluripotent stem cell self-renewal and differentiation.
View details for DOI 10.1038/nprot.2012.021
View details for Web of Science ID 000303359300002
View details for PubMedID 22481529
Production of De Novo Cardiomyocytes: Human Pluripotent Stem Cell Differentiation and Direct Reprogramming
CELL STEM CELL
2012; 10 (1): 16-28
Cardiovascular disease is a leading cause of death worldwide. The limited capability of heart tissue to regenerate has prompted methodological developments for creating de novo cardiomyocytes, both in vitro and in vivo. Beyond uses in cell replacement therapy, patient-specific cardiomyocytes may find applications in drug testing, drug discovery, and disease modeling. Recently, approaches for generating cardiomyocytes have expanded to encompass three major sources of starting cells: human pluripotent stem cells (hPSCs), adult heart-derived cardiac progenitor cells (CPCs), and reprogrammed fibroblasts. We discuss state-of-the-art methods for generating de novo cardiomyocytes from hPSCs and reprogrammed fibroblasts, highlighting potential applications and future challenges.
View details for DOI 10.1016/j.stem.2011.12.013
View details for Web of Science ID 000299449500008
View details for PubMedID 22226352
In Vivo Functional and Transcriptional Profiling of Bone Marrow Stem Cells After Transplantation Into Ischemic Myocardium
ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY
2012; 32 (1): 92-102
Clinical trials of bone marrow-derived stem cell therapy for the heart have yielded variable results. The basic mechanism(s) that underlies their potential efficacy remains unknown. In the present study, we evaluated the survival kinetics, transcriptional response, and functional outcome of intramyocardial bone marrow mononuclear cell (BMMC) transplantation for cardiac repair in a murine myocardial infarction model.We used bioluminescence imaging and high-throughput transcriptional profiling to evaluate the in vivo survival kinetics and gene expression changes of transplanted BMMCs after their engraftment into ischemic myocardium. Our results demonstrate short-lived survival of cells following transplant, with less than 1% of cells surviving by 6 weeks posttransplantation. Moreover, transcriptomic analysis of BMMCs revealed nonspecific upregulation of various cell regulatory genes, with a marked downregulation of cell differentiation and maturation pathways. BMMC therapy caused limited improvement of heart function as assessed by echocardiography, invasive hemodynamics, and positron emission tomography. Histological evaluation of cell fate further confirmed findings of the in vivo cell tracking and transcriptomic analysis.Collectively, these data suggest that BMMC therapy, in its present iteration, may be less efficacious than once thought. Additional refinement of existing cell delivery protocols should be considered to induce better therapeutic efficacy.
View details for DOI 10.1161/ATVBAHA.111.238618
View details for Web of Science ID 000298288700014
View details for PubMedID 22034515
Enhancement of Human Adipose-Derived Stromal Cell Angiogenesis through Knockdown of a BMP-2 Inhibitor
PLASTIC AND RECONSTRUCTIVE SURGERY
2012; 129 (1): 53-66
Previous studies have demonstrated the role of noggin, a bone morphogenetic protein-2 inhibitor, in vascular development and angiogenesis. The authors hypothesized that noggin suppression in human adipose-derived stromal cells would enhance vascular endothelial growth factor secretion and angiogenesis in vitro and in vivo to a greater extent than bone morphogenetic protein-2 alone.Human adipose-derived stromal cells were isolated from human lipoaspirate (n = 6) noggin was knocked down using lentiviral techniques. Knockdown was confirmed and angiogenesis was assessed by tubule formation and quantitative real-time polymerase chain reaction. Cells were seeded onto scaffolds and implanted into a 4-mm critical size calvarial defect. In vivo angiogenic signaling was assessed by immunofluorescence and immunohistochemistry.Human adipose-derived stromal cells with noggin suppression secreted significantly higher amounts of angiogenic proteins, expressed higher levels of angiogenic genes, and formed more tubules in vitro. In vivo, calvarial defects seeded with noggin shRNA human adipose-derived stromal cells exhibited a significantly higher number of vessels in the defect site than controls by immunohistochemistry (p < 0.05). In addition, bone morphogenetic protein-2-releasing scaffolds significantly enhanced vascular signaling in the defect site.Human adipose-derived stromal cells demonstrate significant increases in angiogenesis in vitro and in vivo with both noggin suppression and BMP-2 supplementation. By creating a cell with noggin suppressed and by using a scaffold with increased bone morphogenetic protein-2 signaling, a more angiogenic niche can be created.
View details for DOI 10.1097/PRS.0b013e3182361ff5
View details for Web of Science ID 000298857100075
View details for PubMedID 21915082
Molecular Imaging of Bone Marrow Mononuclear Cell Survival and Homing in Murine Peripheral Artery Disease
2012; 5 (1): 46-55
This study aims to provide insight into cellular kinetics using molecular imaging after different transplantation methods of bone marrow-derived mononuclear cells (MNCs) in a mouse model of peripheral artery disease (PAD).MNC therapy is a promising treatment for PAD. Although clinical translation has already been established, there is a lack of knowledge about cell behavior after transplantation and about the mechanism whereby MNC therapy might ameliorate complaints of PAD.MNCs were isolated from F6 transgenic mice (FVB background) that express firefly luciferase (Fluc) and green fluorescence protein (GFP). Male FVB and C57Bl6 mice (n = 50) underwent femoral artery ligation and were randomized into 4 groups receiving the following: 1) single intramuscular (IM) injection of 2 × 10(6) MNCs; 2) 4 weekly IM injections of 5 × 10(5) MNCs; 3) 2 × 10(6) MNCs intravenously; and 4) phosphate-buffered saline as control. Cells were characterized by flow cytometry and in vitro bioluminescence imaging (BLI). Cell survival, proliferation, and migration were monitored by in vivo BLI, which was validated by ex vivo BLI, post-mortem immunohistochemistry, and flow cytometry. Paw perfusion and neovascularization was measured with laser Doppler perfusion imaging (LDPI) and histology, respectively.In vivo BLI revealed near-complete donor cell death 4 weeks after IM transplantation. After intravenous transplantation, BLI revealed that cells migrated to the injured area in the limb, as well as to the liver, spleen, and bone marrow. Ex vivo BLI showed presence of MNCs in the scar tissue and adductor muscle. However, no significant effects on neovascularization were observed, as monitored by LDPI and histology.This is one of the first studies to assess kinetics of transplanted MNCs in PAD using in vivo molecular imaging. MNC survival is short-lived, MNCs do not preferentially home to injured areas, and MNCs do not significantly stimulate perfusion in this particular model.
View details for DOI 10.1016/j.jcmg.2011.07.011
View details for Web of Science ID 000299392300007
View details for PubMedID 22239892
Nonintegrating Knockdown and Customized Scaffold Design Enhances Human Adipose-Derived Stem Cells in Skeletal Repair
2011; 29 (12): 2018-2029
An urgent need exists in clinical medicine for suitable alternatives to available techniques for bone tissue repair. Human adipose-derived stem cells (hASCs) represent a readily available, autogenous cell source with well-documented in vivo osteogenic potential. In this article, we manipulated Noggin expression levels in hASCs using lentiviral and nonintegrating minicircle short hairpin ribonucleic acid (shRNA) methodologies in vitro and in vivo to enhance hASC osteogenesis. Human ASCs with Noggin knockdown showed significantly increased bone morphogenetic protein (BMP) signaling and osteogenic differentiation both in vitro and in vivo, and when placed onto a BMP-releasing scaffold embedded with lentiviral Noggin shRNA particles, hASCs more rapidly healed mouse calvarial defects. This study therefore suggests that genetic targeting of hASCs combined with custom scaffold design can optimize hASCs for skeletal regenerative medicine.
View details for DOI 10.1002/stem.757
View details for Web of Science ID 000297220000012
View details for PubMedID 21997852
Endothelial progenitor cells in cardiovascular disease and chronic inflammation: from biomarker to therapeutic agent
BIOMARKERS IN MEDICINE
2011; 5 (6): 731-744
The discovery of endothelial progenitor cells in the 1990s challenged the paradigm of angiogenesis by showing that cells derived from hematopoietic stem cells are capable of forming new blood vessels even in the absence of a pre-existing vessel network, a process termed vasculogenesis. Since then, the majority of studies in the field have found a strong association between circulating endothelial progenitor cells and cardiovascular risk. Several studies have also reported that inflammation influences the mobilization and differentiation of endothelial progenitor cells. In this review, we discuss the emerging role of endothelial progenitor cells as biomarkers of cardiovascular disease as well as the interplay between inflammation and endothelial progenitor cell biology. We will also review the challenges in the field of endothelial progenitor cell-based therapy.
View details for DOI 10.2217/BMM.11.92
View details for Web of Science ID 000298488200005
View details for PubMedID 22103609
Non-invasive Bioluminescence Imaging of Myoblast-Mediated Hypoxia-Inducible Factor-1 Alpha Gene Transfer
MOLECULAR IMAGING AND BIOLOGY
2011; 13 (6): 1124-1132
We tested a novel imaging strategy, in which both the survival of transplanted myoblasts and their therapeutic transgene expression, a recombinant hypoxia-inducible factor-1? (HIF-1?-VP2), can be monitored using firefly luciferase (fluc) and Renilla luciferase (hrl) bioluminescence reporter genes, respectively.The plasmid pUbi-hrl-pUbi-HIF-1?-VP2, which expresses both hrl and HIF-1?-VP2 using two ubiquitin promoters, was characterized in vitro. C2c12 myoblasts stably expressing fluc and transiently transfected with pUbi-hrl-pUbi-HIF-1?-VP2 were injected into the mouse hindlimb. Both hrl and fluc expression were monitored using bioluminescence imaging (BLI).Strong correlations existed between the expression of hRL and each of HIF-1?-VP2, VEGF, and PlGF (r(2) > 0.83, r(2) > 0.82, and r(2) > 0.97, respectively). In vivo, both transplanted cells and HIF-1?-VP2 transgene expression were successfully imaged using BLI.An objective evaluation of myoblast-mediated gene transfer in living mice can be performed by monitoring both the survival and the transgene expression of transplanted myoblasts using the techniques developed herein.
View details for DOI 10.1007/s11307-011-0471-9
View details for Web of Science ID 000296794400009
View details for PubMedID 21267661
Site-Specific Recombinase Strategy to Create Induced Pluripotent Stem Cells Efficiently with Plasmid DNA
2011; 29 (11): 1696-1704
Induced pluripotent stem cells (iPSCs) have revolutionized the stem cell field. iPSCs are most often produced by using retroviruses. However, the resulting cells may be ill-suited for clinical applications. Many alternative strategies to make iPSCs have been developed, but the nonintegrating strategies tend to be inefficient, while the integrating strategies involve random integration. Here, we report a facile strategy to create murine iPSCs that uses plasmid DNA and single transfection with sequence-specific recombinases. PhiC31 integrase was used to insert the reprogramming cassette into the genome, producing iPSCs. Cre recombinase was then used for excision of the reprogramming genes. The iPSCs were demonstrated to be pluripotent by in vitro and in vivo criteria, both before and after excision of the reprogramming cassette. This strategy is comparable with retroviral approaches in efficiency, but is nonhazardous for the user, simple to perform, and results in nonrandom integration of a reprogramming cassette that can be readily deleted. We demonstrated the efficiency of this reprogramming and excision strategy in two accessible cell types, fibroblasts and adipose stem cells. This simple strategy produces pluripotent stem cells that have the potential to be used in a clinical setting.
View details for DOI 10.1002/stem.730
View details for Web of Science ID 000296565500007
View details for PubMedID 21898697
Functional Characterization and Expression Profiling of Human Induced Pluripotent Stem Cell- and Embryonic Stem Cell-Derived Endothelial Cells
STEM CELLS AND DEVELOPMENT
2011; 20 (10): 1701-1710
With regard to human induced pluripotent stem cells (hiPSCs), in which adult cells are reprogrammed into embryonic-like cells using defined factors, their functional and transcriptional expression pattern during endothelial differentiation has yet to be characterized. In this study, hiPSCs and human embryonic stem cells (hESCs) were differentiated using the embryoid body method, and CD31(+) cells were sorted. Fluorescence activated cell sorting analysis of hiPSC-derived endothelial cells (hiPSC-ECs) and hESC-derived endothelial cells (hESC-ECs) demonstrated similar endothelial gene expression patterns. We showed functional vascular formation by hiPSC-ECs in a mouse Matrigel plug model. We compared the gene profiles of hiPSCs, hESCs, hiPSC-ECs, hESC-ECs, and human umbilical vein endothelial cells (HUVECs) using whole genome microarray. Our analysis demonstrates that gene expression variation of hiPSC-ECs and hESC-ECs contributes significantly to biological differences between hiPSC-ECs and hESC-ECs as well as to the "distances" among hiPSCs, hESCs, hiPSC-ECs, hESC-ECs, and HUVECs. We further conclude that hiPSCs can differentiate into functional endothelial cells, but with limited expansion potential compared with hESC-ECs; thus, extensive studies should be performed to explore the cause and extent of such differences before clinical application of hiPSC-ECs can begin.
View details for DOI 10.1089/scd.2010.0426
View details for Web of Science ID 000295324900006
View details for PubMedID 21235328
Imaging Guiding the Clinical Translation of Cardiac Stem Cell Therapy
2011; 109 (8): 962-979
Stem cells have been touted as the holy grail of medical therapy, with promises to regenerate cardiac tissue, but it appears the jury is still out on this novel therapy. Using advanced imaging technology, scientists have discovered that these cells do not survive nor engraft long-term. In addition, only marginal benefit has been observed in large-animal studies and human trials. However, all is not lost. Further application of advanced imaging technology will help scientists unravel the mysteries of stem cell therapy and address the clinical hurdles facing its routine implementation. In this review, we will discuss how advanced imaging technology will help investigators better define the optimal delivery method, improve survival and engraftment, and evaluate efficacy and safety. Insights gained from this review may direct the development of future preclinical investigations and clinical trials.
View details for DOI 10.1161/CIRCRESAHA.111.242909
View details for Web of Science ID 000295368300015
View details for PubMedID 21960727
Preclinical Derivation and Imaging of Autologously Transplanted Canine Induced Pluripotent Stem Cells
JOURNAL OF BIOLOGICAL CHEMISTRY
2011; 286 (37): 32697-32704
Derivation of patient-specific induced pluripotent stem cells (iPSCs) opens a new avenue for future applications of regenerative medicine. However, before iPSCs can be used in a clinical setting, it is critical to validate their in vivo fate following autologous transplantation. Thus far, preclinical studies have been limited to small animals and have yet to be conducted in large animals that are physiologically more similar to humans. In this study, we report the first autologous transplantation of iPSCs in a large animal model through the generation of canine iPSCs (ciPSCs) from the canine adipose stromal cells and canine fibroblasts of adult mongrel dogs. We confirmed pluripotency of ciPSCs using the following techniques: (i) immunostaining and quantitative PCR for the presence of pluripotent and germ layer-specific markers in differentiated ciPSCs; (ii) microarray analysis that demonstrates similar gene expression profiles between ciPSCs and canine embryonic stem cells; (iii) teratoma formation assays; and (iv) karyotyping for genomic stability. Fate of ciPSCs autologously transplanted to the canine heart was tracked in vivo using clinical positron emission tomography, computed tomography, and magnetic resonance imaging. To demonstrate clinical potential of ciPSCs to treat models of injury, we generated endothelial cells (ciPSC-ECs) and used these cells to treat immunodeficient murine models of myocardial infarction and hindlimb ischemia.
View details for DOI 10.1074/jbc.M111.235739
View details for Web of Science ID 000294726800078
View details for PubMedID 21719696
An antibody against SSEA-5 glycan on human pluripotent stem cells enables removal of teratoma-forming cells
2011; 29 (9): 829-U86
An important risk in the clinical application of human pluripotent stem cells (hPSCs), including human embryonic and induced pluripotent stem cells (hESCs and hiPSCs), is teratoma formation by residual undifferentiated cells. We raised a monoclonal antibody against hESCs, designated anti-stage-specific embryonic antigen (SSEA)-5, which binds a previously unidentified antigen highly and specifically expressed on hPSCs--the H type-1 glycan. Separation based on SSEA-5 expression through fluorescence-activated cell sorting (FACS) greatly reduced teratoma-formation potential of heterogeneously differentiated cultures. To ensure complete removal of teratoma-forming cells, we identified additional pluripotency surface markers (PSMs) exhibiting a large dynamic expression range during differentiation: CD9, CD30, CD50, CD90 and CD200. Immunohistochemistry studies of human fetal tissues and bioinformatics analysis of a microarray database revealed that concurrent expression of these markers is both common and specific to hPSCs. Immunodepletion with antibodies against SSEA-5 and two additional PSMs completely removed teratoma-formation potential from incompletely differentiated hESC cultures.
View details for DOI 10.1038/nbt.1947
View details for Web of Science ID 000294718400024
View details for PubMedID 21841799
In vivo bioluminescence for tracking cell fate and function
AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY
2011; 301 (3): H663-H671
Tracking the fate and function of cells in vivo is paramount for the development of rational therapies for cardiac injury. Bioluminescence imaging (BLI) provides a means for monitoring physiological processes in real time, ranging from cell survival to gene expression to complex molecular processes. In mice and rats, BLI provides unmatched sensitivity because of the absence of endogenous luciferase expression in mammalian cells and the low background luminescence emanating from animals. In the field of stem cell therapy, BLI provides an unprecedented means to monitor the biology of these cells in vivo, giving researchers a greater understanding of their survival, migration, immunogenicity, and potential tumorigenicity in a living animal. In addition to longitudinal monitoring of cell survival, BLI is a useful tool for semiquantitative measurements of gene expression in vivo, allowing a better optimization of drug and gene therapies. Overall, this technology not only enables rapid, reproducible, and quantitative monitoring of physiological processes in vivo but also can measure the influences of therapeutic interventions on the outcome of cardiac injuries.
View details for DOI 10.1152/ajpheart.00337.2011
View details for Web of Science ID 000294431400006
View details for PubMedID 21666118
- Imaging Atherosclerosis With F18-Fluorodeoxyglucose Positron Emission Tomography What Are We Actually Seeing? JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY 2011; 58 (6): 615-617
Differences in Osteogenic Differentiation of Adipose-Derived Stromal Cells from Murine, Canine, and Human Sources In Vitro and In Vivo
PLASTIC AND RECONSTRUCTIVE SURGERY
2011; 128 (2): 373-386
Given the diversity of species from which adipose-derived stromal cells are derived and studied, the authors set out to delineate the differences in the basic cell biology that may exist across species. Briefly, the authors found that significant differences exist with regard to proliferation and osteogenic potentials of adipose-derived stromal cells across species.Adipose-derived stromal cells were derived from human, mouse, and canine sources as previously described. Retinoic acid, insulin-like growth factor-1, and bone morphogenetic protein-2 were added to culture medium; proliferation and osteogenic differentiation were assessed by standardized assays. In vivo methods included seeding 150,000 adipose-derived stromal cells on a biomimetic scaffold and analyzing healing by micro-computed tomography and histology.Adipose-derived stromal cells from all species had the capability to undergo osteogenic differentiation. Canine adipose-derived stromal cells were the most proliferative, whereas human adipose-derived stromal cells were the most osteogenic (p < 0.05). Human cells, however, had the most significant osteogenic response to osteogenic media. Retinoic acid stimulated osteogenesis in mouse and canine cells but not in human adipose-derived stromal cells. Insulin-like growth factor-1 enhanced osteogenesis across all species, most notably in human- and canine-derived cells.Adipose-derived stromal cells derived from human, mouse, and canine all have the capacity to undergo osteogenic differentiation. Canine adipose-derived stromal cells appear to be the most proliferative, whereas human adipose-derived stromal cells appear to be the most osteogenic. Different cytokines and chemicals can be used to modulate this osteogenic response. These results are promising as attempts are made to optimize tissue-engineered bone using adipose-derived stromal cells.
View details for DOI 10.1097/PRS.0b013e31821e6e49
View details for Web of Science ID 000293464400032
View details for PubMedID 21788829
Imaging Cardiac Stem Cell Therapy: Translations to Human Clinical Studies
JOURNAL OF CARDIOVASCULAR TRANSLATIONAL RESEARCH
2011; 4 (4): 514-522
Stem cell therapy promises to open exciting new options in the treatment of cardiovascular diseases. Although feasible and clinically safe, the in vivo behavior and integration of stem cell transplants still remain largely unknown. Thus, the development of innovative non-invasive imaging techniques capable of effectively tracking such therapy in vivo is vital for a more in-depth investigation into future clinical applications. Such imaging modalities will not only generate further insight into the mechanisms behind stem cell-based therapy, but also address some major concerns associated with translational cardiovascular stem cell therapy. In the present review, we summarize the principles underlying three major stem cell tracking methods: (1) radioactive labeling for positron emission tomography (PET) and single photon emission computed tomography (SPECT) imaging, (2) iron particle labeling for magnetic resonance imaging (MRI), and (3) reporter gene labeling for bioluminescence, fluorescence, MRI, SPECT, and PET imaging. We then discuss recent clinical studies that have utilized these modalities to gain biological insights into stem cell fate.
View details for DOI 10.1007/s12265-011-9281-3
View details for Web of Science ID 000292680400013
View details for PubMedID 21538182
Pretreatment with angiotensin-converting enzyme inhibitor improves doxorubicin-induced cardiomyopathy via preservation of mitochondrial function
JOURNAL OF THORACIC AND CARDIOVASCULAR SURGERY
2011; 142 (2): 396-U529
Doxorubicin is a widely used chemotherapy drug, but its application is associated with cardiotoxicity. Free radical generation and mitochondrial dysfunction are thought to contribute to doxorubicin-induced cardiac failure. Angiotensin-converting enzyme inhibitors are commonly used as cardioprotective agents and have recently been shown in clinical studies to be efficacious in the prevention of anthracycline-induced heart failure. This study evaluated a mechanism for these protective effects by testing the ability of the angiotensin-converting enzyme inhibitor enalapril to preserve mitochondrial function in a model of chronic doxorubicin treatment in rats.Sprague Dawley rats were divided into 3 groups and followed for a total of 10 weeks: (1) control-untreated, (2) doxorubicin treated, and (3) doxorubicin + enalapril treated. Doxorubicin was administered via intraperitoneal injection at weekly intervals from weeks 2 to 7. Enalapril was administered in the drinking water of the doxorubicin + enalapril group for the study duration.Doxorubicin treatment produced a significant loss in left ventricular contractility (P < .05), decrease in mitochondrial function via impairment of state-3 respiration, decrease in the cytosolic fraction of adenosine triphosphate, and up-regulation of free radical production. Enalapril significantly attenuated the decrease in percent fractional shortening (P < .05) and prevented the doxorubicin-associated reduction in respiratory efficiency and cytosolic adenosine triphosphate content (P < .05). Enalapril also abolished the robust doxorubicin-induced increase in free radical formation.Administration of enalapril attenuates doxorubicin-induced cardiac dysfunction via preservation of mitochondrial respiratory efficiency and reduction in doxorubicin-associated free radical generation.
View details for DOI 10.1016/j.jtcvs.2010.07.097
View details for Web of Science ID 000292775200035
View details for PubMedID 21094500
Dissecting the Oncogenic and Tumorigenic Potential of Differentiated Human Induced Pluripotent Stem Cells and Human Embryonic Stem Cells
2011; 71 (14): 5030-5039
Pluripotent stem cells, both human embryonic stem cells (hESC) and human-induced pluripotent stem cells (hiPSC), can give rise to multiple cell types and hence have tremendous potential for regenerative therapies. However, the tumorigenic potential of these cells remains a great concern, as reflected in the formation of teratomas by transplanted pluripotent cells. In clinical practice, most pluripotent cells will be differentiated into useful therapeutic cell types such as neuronal, cardiac, or endothelial cells prior to human transplantation, drastically reducing their tumorigenic potential. Our work investigated the extent to which these differentiated stem cell derivatives are truly devoid of oncogenic potential. In this study, we analyzed the gene expression patterns from three sets of hiPSC- and hESC-derivatives and the corresponding primary cells, and compared their transcriptomes with those of five different types of cancer. Our analysis revealed a significant gene expression overlap of the hiPSC- and hESC-derivatives with cancer, whereas the corresponding primary cells showed minimum overlap. Real-time quantitative PCR analysis of a set of cancer-related genes (selected on the basis of rigorous functional and pathway analyses) confirmed our results. Overall, our findings suggested that pluripotent stem cell derivatives may still bear oncogenic properties even after differentiation, and additional stringent functional assays to purify these cells should be done before they can be used for regenerative therapy.
View details for DOI 10.1158/0008-5472.CAN-10-4402
View details for Web of Science ID 000292763700029
View details for PubMedID 21646469
Efficient gene delivery of primary human cells using peptide linked polyethylenimine polymer hybrid
2011; 32 (20): 4647-4658
Polyethylenimine (PEI) based polymers are efficient agents for cell transfection. However, their use has been hampered due to high cell death associated with transfection thereby resulting in low efficiency of gene delivery within the cells. To circumvent the problem of cellular toxicity, metal binding peptides were linked to PEI. Eight peptide-PEI derivatives were synthesized to improve cell survival and transfection efficiency. TAT linked PEI was used as a control polymer. Peptides linked with PEI amines formed nanogels as shown by electron microscopy and atomic force microscopic measurements. Polymers were characterized by spectroscopic methods and their ability to form complexes with plasmids was tested using electrophoretic studies. These modifications improved polymer biocompatibility as well as cell survival markedly, when compared to PEI alone. A subset of the modified peptide-polymers also showed significantly higher transfection efficiency in primary human cells with respect to the widely used transfection agent, lipofectamine. Study of the underlying mechanism of the observed phenomena revealed lower levels of 'reactive oxygen species' (ROS) in the presence of the peptide-polymers when compared to PEI alone. This was further corroborated with global gene expression analysis which showed upregulation of multiple genes and pathways involved in regulating intracellular oxidative stress.
View details for DOI 10.1016/j.biomaterials.2011.03.016
View details for Web of Science ID 000291193700019
View details for PubMedID 21477858
- Report of the National Heart, Lung, and Blood Institute Working Group on the Translation of Cardiovascular Molecular Imaging CIRCULATION 2011; 123 (19): 2157-2163
Derivation of Human Induced Pluripotent Stem Cells for Cardiovascular Disease Modeling
2011; 108 (9): 1146-1156
The successful derivation of human induced pluripotent stem cells (hiPSCs) by dedifferentiation of somatic cells offers significant potential to overcome obstacles in the field of cardiovascular disease. hiPSC derivatives offer incredible potential for new disease models and regenerative medicine therapies. However, many questions remain regarding the optimal starting materials and methods to enable safe, efficient derivation of hiPSCs suitable for clinical applications. Initial reprogramming experiments were carried out using lentiviral or retroviral gene delivery methods. More recently, various nonviral methods that avoid permanent and random transgene insertion have emerged as alternatives. These include transient DNA transfection using plasmids or minicircles, protein transduction, or RNA transfection. In addition, several small molecules have been found to significantly augment hiPSC derivation efficiency, allowing the use of a fewer number of genes during pluripotency induction. We review these various methods for the derivation of hiPSCs, focusing on their ultimate clinical applicability, with an emphasis on their potential for use as cardiovascular therapies and disease-modeling platforms.
View details for DOI 10.1161/CIRCRESAHA.111.240374
View details for Web of Science ID 000289983600014
View details for PubMedID 21527744
- Comparison of Human Induced Pluripotent and Embryonic Stem Cells: Fraternal or Identical Twins? MOLECULAR THERAPY 2011; 19 (4): 635-638
Studies in Adipose-Derived Stromal Cells: Migration and Participation in Repair of Cranial Injury after Systemic Injection
PLASTIC AND RECONSTRUCTIVE SURGERY
2011; 127 (3): 1130-1140
Adipose-derived stromal cells are a multipotent cell type with the ability to undergo osteogenic differentiation. The authors sought to examine whether systemically administered adipose-derived stromal cells would migrate to and heal surgically created defects of the mouse cranial skeleton.Mouse adipose-derived stromal cells were harvested from luciferase-positive transgenic mice; human adipose-derived stromal cells were harvested from human lipoaspirate and labeled with luciferase and green fluorescent protein. A 4-mm calvarial defect (critical sized) was made in the mouse parietal bone; skin incisions alone were used as a control (n = 5 per group). Adipose-derived stromal cells were injected intravenously (200,000 cells per animal) and compared with saline injection only. Methods of analyses included micro-computed tomographic scanning, in vivo imaging system detection of luciferase activity, and standard histology.Migration of adipose-derived stromal cells to calvarial defect sites was confirmed by accumulation of luciferase activity and green fluorescent protein stain as early as 4 days and persisting up to 4 weeks. Little activity was observed among control groups. Intravenous administration of either mouse or human adipose-derived stromal cells resulted in histologic evidence of bone formation within the defect site, in comparison with an absence of bone among control defects. By micro-computed tomographic analysis, human but not mouse adipose-derived stromal cells stimulated significant osseous healing.Intravenously administered adipose-derived stromal cells migrate to sites of calvarial injury. Thereafter, intravenous human adipose-derived stromal cells contribute to bony calvarial repair. Intravenous administration of adipose-derived stromal cells may be an effective delivery method for future efforts in skeletal regeneration.
View details for DOI 10.1097/PRS.0b013e3182043712
View details for Web of Science ID 000287680200013
View details for PubMedID 21364416
Single cell transcriptional profiling reveals heterogeneity of human induced pluripotent stem cells
JOURNAL OF CLINICAL INVESTIGATION
2011; 121 (3): 1217-1221
Human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) are promising candidate cell sources for regenerative medicine. However, despite the common ability of hiPSCs and hESCs to differentiate into all 3 germ layers, their functional equivalence at the single cell level remains to be demonstrated. Moreover, single cell heterogeneity amongst stem cell populations may underlie important cell fate decisions. Here, we used single cell analysis to resolve the gene expression profiles of 362 hiPSCs and hESCs for an array of 42 genes that characterize the pluripotent and differentiated states. Comparison between single hESCs and single hiPSCs revealed markedly more heterogeneity in gene expression levels in the hiPSCs, suggesting that hiPSCs occupy an alternate, less stable pluripotent state. hiPSCs also displayed slower growth kinetics and impaired directed differentiation as compared with hESCs. Our results suggest that caution should be exercised before assuming that hiPSCs occupy a pluripotent state equivalent to that of hESCs, particularly when producing differentiated cells for regenerative medicine aims.
View details for DOI 10.1172/JCI44635
View details for Web of Science ID 000287991000039
View details for PubMedID 21317531
Human germ cell differentiation from fetal- and adult-derived induced pluripotent stem cells
HUMAN MOLECULAR GENETICS
2011; 20 (4): 752-762
Historically, our understanding of molecular genetic aspects of human germ cell development has been limited, at least in part due to inaccessibility of early stages of human development to experimentation. However, the derivation of pluripotent stem cells may provide the necessary human genetic system to study germ cell development. In this study, we compared the potential of human induced pluripotent stem cells (iPSCs), derived from adult and fetal somatic cells to form primordial and meiotic germ cells, relative to human embryonic stem cells. We found that ?5% of human iPSCs differentiated to primordial germ cells (PGCs) following induction with bone morphogenetic proteins. Furthermore, we observed that PGCs expressed green fluorescent protein from a germ cell-specific reporter and were enriched for the expression of endogenous germ cell-specific proteins and mRNAs. In response to the overexpression of intrinsic regulators, we also observed that iPSCs formed meiotic cells with extensive synaptonemal complexes and post-meiotic haploid cells with a similar pattern of ACROSIN staining as observed in human spermatids. These results indicate that human iPSCs derived from reprogramming of adult somatic cells can form germline cells. This system may provide a useful model for molecular genetic studies of human germline formation and pathology and a novel platform for clinical studies and potential therapeutical applications.
View details for DOI 10.1093/hmg/ddq520
View details for Web of Science ID 000286469400011
View details for PubMedID 21131292
Elastic Properties of Induced Pluripotent Stem Cells
TISSUE ENGINEERING PART A
2011; 17 (3-4): 495-502
The recent technique of transducing key transcription factors into unipotent cells (fibroblasts) to generate pluripotent stem cells (induced pluripotent stem cells [iPSCs]) has significantly changed the stem cell field. These cells have great promise for many clinical applications, including that of regenerative medicine. Our findings show that iPSCs can be derived from human adipose-derived stromal cells (hASCs), a notable advancement in the clinical applicability of these cells. To investigate differences between two iPS cell lines (fibroblast-iPSC and hASC-iPSC), and also the gold standard human embryonic stem cell, we looked at cell stiffness as a possible indicator of cell differentiation-potential differences. We used atomic force microscopy as a tool to determine stem cell stiffness, and hence differences in material properties between cells. Human fibroblast and hASC stiffness was also ascertained for comparison. Interestingly, cells exhibited a noticeable difference in stiffness. From least to most stiff, the order of cell stiffness was as follows: hASC-iPSC, human embryonic stem cell, fibroblast-iPSC, fibroblasts, and, lastly, as the stiffest cell, hASC. In comparing hASC-iPSCs to their origin cell, the hASC, the reprogrammed cell is significantly less stiff, indicating that greater differentiation potentials may correlate with a lower cellular modulus. The stiffness differences are not dependent on cell culture density; hence, material differences between cells cannot be attributed solely to cell-cell constraints. The change in mechanical properties of the cells in response to reprogramming offers insight into how the cell interacts with its environment and might lend clues to how to efficiently reprogram cell populations as well as how to maintain their pluripotent state.
View details for DOI 10.1089/ten.tea.2010.0211
View details for Web of Science ID 000286661600023
View details for PubMedID 20807017
Radiation exposure from imaging tests: is there an increased cancer risk?
Expert review of cardiovascular therapy
2011; 9 (2): 177-183
The increasing exposure to low-dose radiation from diagnostic testing has prompted renewed interest in evaluating its carcinogenic risk, but quantifying health risk from low-dose radiation exposure remains controversial. The current approach is to adopt the linear non-threshold model, which is commonly applied to high-dose exposure, and apply it to assess risk from low-dose exposure. However, existing data are conflicting and limited to epidemiological studies and/or in vitro analyses. In this article, we will discuss the potential cancer risk from low- and high-dose radiation, their effects on DNA repair response pathways, and the best course of action for patients and providers to minimize risk.
View details for DOI 10.1586/erc.10.184
View details for PubMedID 21453214
- Cardiovascular Molecular Imaging Focus on Clinical Translation CIRCULATION 2011; 123 (4): 425-443
- Sex differences in the diagnostic evaluation of coronary artery disease JOURNAL OF NUCLEAR CARDIOLOGY 2011; 18 (1): 144-152
Generation of adult human induced pluripotent stem cells using nonviral minicircle DNA vectors
2011; 6 (1): 78-88
Human induced pluripotent stem cells (hiPSCs) derived from patient samples have tremendous potential for innovative approaches to disease pathology investigation and regenerative medicine therapies. However, most hiPSC derivation techniques use integrating viruses, which may leave residual transgene sequences as part of the host genome, thereby unpredictably altering cell phenotype in downstream applications. In this study, we describe a protocol for hiPSC derivation by transfection of a simple, nonviral minicircle DNA construct into human adipose stromal cells (hASCs). Minicircle DNA vectors are free of bacterial DNA and thus capable of high expression in mammalian cells. Their repeated transfection into hASCs, abundant somatic cell sources that are amenable to efficient reprogramming, results in transgene-free hiPSCs. This protocol requires only readily available molecular biology reagents and expertise, and produces hiPSC colonies from an adipose tissue sample in ?4 weeks.
View details for DOI 10.1038/nprot.2010.173
View details for Web of Science ID 000285965000008
View details for PubMedID 21212777
Molecular imaging of RNA interference therapy targeting PHD2 for treatment of myocardial ischemia.
Methods in molecular biology (Clifton, N.J.)
2011; 709: 211-221
Coronary artery disease is the number one cause of morbidity and mortality in the Western world. It typically occurs when heart muscle receives inadequate blood supply due to rupture of atherosclerotic plaques. During ischemia, up-regulation of hypoxia inducible factor-1 alpha (HIF-1?) transcriptional factor can activate several downstream angiogenic genes. However, HIF-1? is naturally degraded by prolyl hydroxylase-2 (PHD2) protein. Recently, we cloned the mouse PHD2 gene by comparing the homolog gene in human and rat. The best candidate shRNA sequence for inhibiting PHD2 was inserted behind H1 promoter, followed by a separate hypoxia response element (HRE)-incorporated promoter driving a firefly luciferase (Fluc) reporter gene. This construct allowed us to monitor gene expression noninvasively and was used to test the hypothesis that inhibition of PHD2 by short hairpin RNA interference (shRNA) can lead to significant improvement in angiogenesis and contractility as revealed by in vitro and in vivo experiments.
View details for DOI 10.1007/978-1-61737-982-6_13
View details for PubMedID 21194030
Imaging of embryonic stem cell migration in vivo.
Methods in molecular biology (Clifton, N.J.)
2011; 750: 101-114
Conventional reporter gene technology and histological methods cannot routinely be used to track the in vivo behavior of embryonic stem (ES) cells longitudinally after cellular transplantation. Here we describe a protocol for monitoring the in vivo survival, proliferation, and migration of ES cells without necessitating animal sacrifice. Stable ES cell lines containing double fusion (DF; enhanced green fluorescent protein and firefly luciferase) or triple fusion (TF; monomeric red fluorescent protein, firefly luciferase, and herpes simplex virus thymidine kinase) reporter genes can be established within 4-6 weeks by lentiviral transduction followed by fluorescence-activated cell sorting. The cell fate and behavior of these DF or TF ES cells can subsequently be tracked noninvasively by bioluminescence and microPET imaging for a prolonged period of time.
View details for DOI 10.1007/978-1-61779-145-1_7
View details for PubMedID 21618086
Effects of Long-Term Culture on Human Embryonic Stem Cell Aging
STEM CELLS AND DEVELOPMENT
2011; 20 (1): 127-138
In recent years, human embryonic stem (hES) cells have become a promising cell source for regenerative medicine. Although hES cells have the ability for unlimited self-renewal, potential adverse effects of long-term cell culture upon hES cells must be investigated before therapeutic applications of hES cells can be realized. Here we investigated changes in molecular profiles associated with young (<60 passages) and old (>120 passages) cells of the H9 hES cell line as well as young (<85 passages) and old (>120 passages) cells of the PKU1 hES cell line. Our results show that morphology, stem cell markers, and telomerase activity do not differ significantly between young and old passage cells. Cells from both age groups were also shown to differentiate into derivatives of all 3 germ layers upon spontaneous differentiation in vitro. Interestingly, mitochondrial dysfunction was found to occur with prolonged culture. Old passage cells of both the H9 and PKU1 lines were characterized by higher mitochondrial membrane potential, larger mitochondrial morphology, and higher reactive oxygen species content than their younger counterparts. Teratomas derived from higher passage cells were also found to have an uneven preference for differentiation compared with tumors derived from younger cells. These findings suggest that prolonged culture of hES cells may negatively impact mitochondrial function and possibly affect long-term pluripotency.
View details for DOI 10.1089/scd.2009.0475
View details for Web of Science ID 000285870800012
View details for PubMedID 20629482
Tumorigenicity of pluripotent stem cells: biological insights from molecular imaging
JOURNAL OF THE ROYAL SOCIETY INTERFACE
2010; 7: S753-S763
Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have the ability (i) to duplicate indefinitely while maintaining pluripotency and (ii) to differentiate into cell types of all three embryonic germ layers. These two properties of ESCs and iPSCs make them potentially suitable for tissue engineering and cell replacement therapy for many different diseases, including Parkinson's disease, diabetes and heart disease. However, one critical obstacle in the clinical application of ESCs or iPSCs is the risk of teratoma formation. The emerging field of molecular imaging is allowing researchers to track transplanted ESCs or iPSCs in vivo, enabling early detection of teratomas.
View details for DOI 10.1098/rsif.2010.0353.focus
View details for Web of Science ID 000284505100007
View details for PubMedID 20880852
Dynamic MicroRNA Expression Programs During Cardiac Differentiation of Human Embryonic Stem Cells Role for miR-499
2010; 3 (5): 426-U97
MicroRNAs (miRNAs) are a newly discovered endogenous class of small, noncoding RNAs that play important posttranscriptional regulatory roles by targeting messenger RNAs for cleavage or translational repression. Human embryonic stem cells are known to express miRNAs that are often undetectable in adult organs, and a growing body of evidence has implicated miRNAs as important arbiters of heart development and disease.To better understand the transition between the human embryonic and cardiac "miRNA-omes," we report here the first miRNA profiling study of cardiomyocytes derived from human embryonic stem cells. Analyzing 711 unique miRNAs, we have identified several interesting miRNAs, including miR-1, -133, and -208, that have been previously reported to be involved in cardiac development and disease and that show surprising patterns of expression across our samples. We also identified novel miRNAs, such as miR-499, that are strongly associated with cardiac differentiation and that share many predicted targets with miR-208. Overexpression of miR-499 and -1 resulted in upregulation of important cardiac myosin heavy-chain genes in embryoid bodies; miR-499 overexpression also caused upregulation of the cardiac transcription factor MEF2C.Taken together, our data give significant insight into the regulatory networks that govern human embryonic stem cell differentiation and highlight the ability of miRNAs to perturb, and even control, the genes that are involved in cardiac specification of human embryonic stem cells.
View details for DOI 10.1161/CIRCGENETICS.109.934281
View details for Web of Science ID 000283163100006
View details for PubMedID 20733065
Methods to assess stem cell lineage, fate and function
ADVANCED DRUG DELIVERY REVIEWS
2010; 62 (12): 1175-1186
Stem cell therapy has the potential to regenerate injured tissue. For stem cells to achieve their full therapeutic potential, stem cells must differentiate into the target cell, reach the site of injury, survive, and engraft. To fully characterize these cells, evaluation of cell morphology, lineage specific markers, cell specific function, and gene expression must be performed. To monitor survival and engraftment, cell fate imaging is vital. Only then can organ specific function be evaluated to determine the effectiveness of therapy. In this review, we will discuss methods for evaluating the function of transplanted cells for restoring the heart, nervous system, and pancreas. We will also highlight the specific challenges facing these potential therapeutic areas.
View details for DOI 10.1016/j.addr.2010.08.008
View details for Web of Science ID 000285325900006
View details for PubMedID 20816906
The use of human mesenchymal stem cells encapsulated in RGD modified alginate microspheres in the repair of myocardial infarction in the rat
2010; 31 (27): 7012-7020
The combination of scaffold material and cell transplantation therapy has been extensively investigated in cardiac tissue engineering. However, many polymers are difficult to administer or lack the structural integrity to restore LV function. Additionally, polymers need to be biological friendly, favorably influence the microenvironment and increase stem cell retention and survival. This study determined whether human mesenchymal stem cells (hMSCs) encapsulated in RGD modified alginate microspheres are capable of facilitating myocardial repair. The in vitro study of hMSCs demonstrated that the RGD modified alginate can improve cell attachment, growth and increase angiogenic growth factor expression. Alginate microbeads and hMSCs encapsulated in microbeads successfully maintained LV shape and prevented negative LV remodeling after an MI. Cell survival was significantly increased in the encapsulated hMSC group compared with PBS control or cells alone. Microspheres, hMSCs, and hMSCs in microspheres groups reduced infarct area and enhanced arteriole formation. In summary, surface modification and microencapsulation techniques can be combined with cell transplantation leading to the maintenance of LV geometry, preservation of LV function, increase of angiogenesis and improvement of cell survival.
View details for DOI 10.1016/j.biomaterials.2010.05.078
View details for Web of Science ID 000280616300013
View details for PubMedID 20566215
Role of Molecular Imaging in Stem Cell Therapy for Myocardial Restoration
TRENDS IN CARDIOVASCULAR MEDICINE
2010; 20 (6): 183-188
During the past two decades, stem cells have created enthusiasm as a regenerative therapy for ischemic heart disease. Transplantation of bone marrow stem cells, skeletal myoblasts, and endothelial progenitor cells has shown to improve myocardial function after infarction. Recently, attention has focused on the potential use of embryonic stem cells and induced pluripotent stem cells because they possess the capacity to differentiate into various cell types, including cardiac and endothelial cells. Clinical trials have shown positive effects on the functional recovery of heart after myocardial infarction and have answered questions on timing, dosage, and cell delivery route of stem cells such as those derived from bone marrow. Despite the current advances in stem cell research, one main hurdle remains the lack of reliable information about the fate of cell engraftment, survival, and proliferation after transplantation. This review discusses the different cell types used in cardiac cell therapy as well as molecular imaging modalities relevant to survival issues.
View details for Web of Science ID 000298222000001
View details for PubMedID 22137639
Indirect imaging of cardiac-specific transgene expression using a bidirectional two-step transcriptional amplification strategy
2010; 17 (7): 827-838
Transcriptional targeting for cardiac gene therapy is limited by the relatively weak activity of most cardiac-specific promoters. We have developed a bidirectional plasmid vector, which uses a two-step transcriptional amplification (TSTA) strategy to enhance the expression of two optical reporter genes, firefly luciferase (fluc) and Renilla luciferase (hrluc), driven by the cardiac troponin T (cTnT) promoter. The vector was characterized in vitro and in living mice using luminometry and bioluminescence imaging to assess its ability to mediate strong, correlated reporter gene expression in a cardiac cell line and the myocardium, while minimizing expression in non-cardiac cell lines and the liver. In vitro, the TSTA system significantly enhanced cTnT-mediated reporter gene expression with moderate preservation of cardiac specificity. After intramyocardial and hydrodynamic tail vein delivery of an hrluc-enhanced variant of the vector, long-term fluc expression was observed in the heart, but not in the liver. In both the cardiac cell line and the myocardium, fluc expression correlated well with hrluc expression. These results show the vector's ability to effectively amplify and couple transgene expression in a cardiac-specific manner. Further replacement of either reporter gene with a therapeutic gene should allow non-invasive imaging of targeted gene therapy in living subjects.
View details for DOI 10.1038/gt.2010.30
View details for Web of Science ID 000279614600002
View details for PubMedID 20237511
Advances in cardiovascular molecular imaging for tracking stem cell therapy
THROMBOSIS AND HAEMOSTASIS
2010; 104 (1): 13-22
The high mortality rate associated with cardiovascular disease is partially due to the lack of proliferative cells in the heart. Without adequate repair following myocardial infarction, progressive dilation can lead to heart failure. Stem cell therapies present one promising option for treating cardiovascular disease, though the specific mechanisms by which they benefit the heart remain unclear. Before stem cell therapies can be used safely in human populations, their biology must be investigated using innovative technologies such as multi-modality molecular imaging. The present review will discuss the basic principles, labelling techniques, clinical applications, and drawbacks associated with four major modalities: radionuclide imaging, magnetic resonance imaging, bioluminescence imaging, and fluorescence imaging.
View details for DOI 10.1160/TH09-08-0530
View details for Web of Science ID 000280298300004
View details for PubMedID 20458434
Effects of Ionizing Radiation on Self-Renewal and Pluripotency of Human Embryonic Stem Cells
2010; 70 (13): 5539-5548
Human embryonic stem cells (hESC) present a novel platform for in vitro investigation of the early embryonic cellular response to ionizing radiation. Thus far, no study has analyzed the genome-wide transcriptional response to ionizing radiation in hESCs, nor has any study assessed their ability to form teratomas, the definitive test of pluripotency. In this study, we use microarrays to analyze the global gene expression changes in hESCs after low-dose (0.4 Gy), medium-dose (2 Gy), and high-dose (4 Gy) irradiation. We identify genes and pathways at each radiation dose that are involved in cell death, p53 signaling, cell cycling, cancer, embryonic and organ development, and others. Using Gene Set Enrichment Analysis, we also show that the expression of a comprehensive set of core embryonic transcription factors is not altered by radiation at any dose. Transplantation of irradiated hESCs to immune-deficient mice results in teratoma formation from hESCs irradiated at all doses, definitive proof of pluripotency. Further, using a bioluminescence imaging technique, we have found that irradiation causes hESCs to initially die after transplantation, but the surviving cells quickly recover by 2 weeks to levels similar to control. To conclude, we show that similar to somatic cells, irradiated hESCs suffer significant death and apoptosis after irradiation. However, they continue to remain pluripotent and are able to form all three embryonic germ layers. Studies such as this will help define the limits for radiation exposure for pregnant women and also radiotracer reporter probes for tracking cellular regenerative therapies.
View details for DOI 10.1158/0008-5472.CAN-09-4238
View details for Web of Science ID 000279396800036
View details for PubMedID 20530673
- Micro-CT for Characterization of Murine CV Disease Models JACC-CARDIOVASCULAR IMAGING 2010; 3 (7): 783-785
Antioxidants Improve Early Survival of Cardiomyoblasts After Transplantation to the Myocardium
MOLECULAR IMAGING AND BIOLOGY
2010; 12 (3): 325-334
We tested the hypothesis that modulation of the microenvironment (using antioxidants) will increase stem cell survival in hypoxia and after transplantation to the myocardium.Rat cardiomyoblasts were stably transfected with a reporter gene (firefly luciferase) for bioluminescence imaging (BLI). First, we examined the role of oxidative stress in cells under hypoxic conditions. Subsequently, stem cells were transplanted to the myocardium of rats using high-resolution ultrasound, and their survival was monitored daily using BLI.Under hypoxia, oxidative stress was increased together with decreased cell survival compared to control cells, both of which were preserved by antioxidants. In living subjects, oxidative stress blockade increased early cell survival after transplantation to the myocardium, compared to untreated cells/animals.Modulation of the local microenvironment (with antioxidants) improves stem cell survival. Increased understanding of the interaction between stem cells and their microenvironment will be critical to advance the field of regenerative medicine.
View details for DOI 10.1007/s11307-009-0274-4
View details for Web of Science ID 000277375300011
View details for PubMedID 20013064
- Gene Correction in Human Embryonic and Induced Pluripotent Stem Cells: Promises and Challenges Ahead MOLECULAR THERAPY 2010; 18 (6): 1061-1063
Embryonic Stem Cell-Derived Endothelial Cells Engraft Into the Ischemic Hindlimb and Restore Perfusion
ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY
2010; 30 (5): 984-U224
We examined the effect of delivery modality on the survival, localization, and functional effects of exogenously administered embryonic stem cells (ESCs) or endothelial cells derived from them (ESC-ECs) in the ischemic hindlimb.Murine ESCs or ESC-ECs were stably transduced with a construct for bioluminescence imaging (BLI) and fluorescent detection. In a syngeneic murine model of limb ischemia, ESCs or ESC-ECs were delivered by intramuscular (IM), intrafemoral artery (IA), or intrafemoral vein injections (n=5 in each group). For 2 weeks, cell survival and localization were tracked by BLI and confirmed by immunohistochemistry, and functional improvement was assessed by laser Doppler perfusion. BLI showed that ESCs localized to the ischemic limb after IM or IA, but not after intrafemoral vein administration. Regardless of the route of administration, ESCs were detected outside the hindlimb circulation in the spleen or lungs. ESCs did not improve limb perfusion and generated teratomas. In contrast, ESC-ECs delivered by all 3 modalities localized to the ischemic limb, as assessed by BLI. Most surprisingly, ESC-EC injected intrafemoral vein eventually localized to the ischemic limb after initially lodging in the pulmonary circulation. Immunohistochemical studies confirmed the engraftment of ESC-ECs into the limb vasculature after 2 weeks. Notably, ESC-ECs were not detected in the spleen or lungs after 2 weeks, regardless of route of administration. Furthermore, ESC-ECs significantly improved limb perfusion and neovascularization compared with the parental ESCs or the vehicle control group.In contrast to parental ESCs, ESC-ECs preferentially localized in the ischemic hindlimb by IA, IM, and intrafemoral vein delivery. ESC-ECs engrafted into the ischemic microvasculature, enhanced neovascularization, and improved limb perfusion.
View details for DOI 10.1161/ATVBAHA.110.202796
View details for Web of Science ID 000276677700015
View details for PubMedID 20167654
Current Perspectives on Imaging Cardiac Stem Cell Therapy
JOURNAL OF NUCLEAR MEDICINE
2010; 51: 128S-136S
Molecular imaging is a new discipline that makes possible the noninvasive visualization of cellular and molecular processes in living subjects. In the field of cardiovascular regenerative therapy, imaging cell fate after transplantation is a high priority in both basic research and clinical translation. For cell-based therapy to truly succeed, we must be able to track the locations of delivered cells, the duration of cell survival, and any potential adverse effects. The insights gathered from basic research imaging studies will yield valuable insights into better designs for clinical trials. This review highlights the different types of stem cells used for cardiovascular repair, the development of various imaging modalities to track their fate in vivo, and the challenges of clinical translation of cardiac stem cell imaging in the future.
View details for DOI 10.2967/jnumed.109.068239
View details for Web of Science ID 000277414600013
View details for PubMedID 20395348
- Rosiglitazone Increases Myocardial Glucose Metabolism in Insulin-Resistant Cardiomyopathy JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY 2010; 55 (9): 926-927
Human iPS cell-based therapy Considerations before clinical applications
2010; 9 (5): 880-885
Generation of induced pluripotent stem (iPS) cells has revolutionized the field of regenerative medicine. With the exponential increase in iPS cell research in the past three years, human iPS cells have been derived with different technologies and from various cell types. From a translational perspective, however, a number of issues must be addressed before safe and high quality patient-specific iPS cells can be derived for clinical applications. In addition, iPS cell-based therapies also need to be thoroughly evaluated in pre-clinical animal models before they can be applied to human subjects.
View details for Web of Science ID 000276307700017
View details for PubMedID 20160515
Human Neural Stem Cell Grafts Modify Microglial Response and Enhance Axonal Sprouting in Neonatal Hypoxic-Ischemic Brain Injury
2010; 41 (3): 516-523
Hypoxic-ischemic (HI) brain injury in newborn infants represents a major cause of cerebral palsy, development delay, and epilepsy. Stem cell-based therapy has the potential to rescue and replace the ischemic tissue caused by HI and to restore function. However, the mechanisms by which stem cell transplants induce functional recovery are yet to be elucidated. In the present study, we sought to investigate the efficacy of human neural stem cells derived from human embryonic stem cells in a rat model of neonatal HI and the mechanisms enhancing brain repair.The human neural stem cells were genetically engineered for in vivo molecular imaging and for postmortem histological tracking. Twenty-four hours after the induction of HI, animals were grafted with human neural stem cells into the forebrain. Motor behavioral tests were performed the fourth week after transplantation. We used immunocytochemistry and neuroanatomical tracing to analyze neural differentiation, axonal sprouting, and microglia response. Treatment-induced changes in gene expression were investigated by microarray and quantitative polymerase chain reaction.Bioluminescence imaging permitted real time longitudinal tracking of grafted human neural stem cells. HI transplanted animals significantly improved in their use of the contralateral impeded forelimb and in the Rotorod test. The grafts showed good survival, dispersion, and differentiation. We observed an increase of uniformly distributed microglia cells in the grafted side. Anterograde neuroanatomical tracing demonstrated significant contralesional sprouting. Microarray analysis revealed upregulation of genes involved in neurogenesis, gliogenesis, and neurotrophic support.These results suggest that human neural stem cell transplants enhance endogenous brain repair through multiple modalities in response to HI.
View details for DOI 10.1161/STROKEAHA.109.573691
View details for Web of Science ID 000274799600019
View details for PubMedID 20075340
A nonviral minicircle vector for deriving human iPS cells
2010; 7 (3): 197-U46
Owing to the risk of insertional mutagenesis, viral transduction has been increasingly replaced by nonviral methods to generate induced pluripotent stem cells (iPSCs). We report the use of 'minicircle' DNA, a vector type that is free of bacterial DNA and capable of high expression in cells, for this purpose. Here we use a single minicircle vector to generate transgene-free iPSCs from adult human adipose stem cells.
View details for DOI 10.1038/NMETH.1426
View details for Web of Science ID 000275058200018
View details for PubMedID 20139967
Comparison of Gene-Transfer Efficiency in Human Embryonic Stem Cells
MOLECULAR IMAGING AND BIOLOGY
2010; 12 (1): 15-24
Technologies designed to allow manipulation and modification of human embryonic stem (hES) cells are numerous and vary in the complexity of their methods, efficiency, reliability, and safety. The most commonly studied and practiced of these methods include electroporation, lipofection, nucleofection, and lentiviral transduction. However, at present, it is unclear which protocol offers the most efficient and reliable method of gene transfer to hES cells. In this study, a bi-fusion construct with ubiquitin promoter driving enhanced green fluorescent protein reporter and the firefly luciferase (pUb-eGFP-Fluc) along with neomycin selection marker was used for in vitro and in vivo studies. In vitro studies examined the transfection efficiency and viability of each technique using two hES cell lines (male H1 and female H9 cells). Lentiviral transduction demonstrated the highest efficiency (H1: 25.3 +/- 4.8%; H9: 22.4 +/- 6.5%) with >95% cell viability. Nucleofection demonstrated transfection efficiency of 16.1 +/- 3.6% (H1) and 5.8 +/- 3.2% (H9). However, minimal transfection efficiency was observed with electroporation (2.1 +/- 0.4% (H1) and 1.9 +/- 0.3% (H9)) and lipofection (1.5 +/- 0.5% (H1) and 1.3 +/- 0.2% (H9); P < 0.05 vs. lentiviral transduction). Electroporation also demonstrated the highest cell death (62 +/- 11% (H1) and 42 +/- 10% (H9)) followed by nucleofection (25 +/- 9% (H1) and 30 +/- 15 (H9)). Importantly, lentiviral transduction generated a greater number of hES cell lines stably expressing the double-fusion reporter gene (hES-DF) compared to other transfection techniques. Finally, following subcutaneous transplantation into immunodeficient nude mice, the hES-eGFP-Fluc cells showed robust proliferation as determined by longitudinal bioluminescence imaging. In summary, this study demonstrates that lentiviral transduction and nucleofection are efficient, simple, and safe techniques for reliable gene transfer in hES cells. The double-fusion construct provides an attractive approach for generating stable hES cell lines and monitoring engraftment and proliferation in vitro and in vivo.
View details for DOI 10.1007/s11307-009-0236-x
View details for Web of Science ID 000273479300004
View details for PubMedID 19551446
Persistent Donor Cell Gene Expression among Human Induced Pluripotent Stem Cells Contributes to Differences with Human Embryonic Stem Cells
2010; 5 (2)
Human induced pluripotent stem cells (hiPSCs) generated by de-differentiation of adult somatic cells offer potential solutions for the ethical issues surrounding human embryonic stem cells (hESCs), as well as their immunologic rejection after cellular transplantation. However, although hiPSCs have been described as "embryonic stem cell-like", these cells have a distinct gene expression pattern compared to hESCs, making incomplete reprogramming a potential pitfall. It is unclear to what degree the difference in tissue of origin may contribute to these gene expression differences. To answer these important questions, a careful transcriptional profiling analysis is necessary to investigate the exact reprogramming state of hiPSCs, as well as analysis of the impression, if any, of the tissue of origin on the resulting hiPSCs. In this study, we compare the gene profiles of hiPSCs derived from fetal fibroblasts, neonatal fibroblasts, adipose stem cells, and keratinocytes to their corresponding donor cells and hESCs. Our analysis elucidates the overall degree of reprogramming within each hiPSC line, as well as the "distance" between each hiPSC line and its donor cell. We further identify genes that have a similar mode of regulation in hiPSCs and their corresponding donor cells compared to hESCs, allowing us to specify core sets of donor genes that continue to be expressed in each hiPSC line. We report that residual gene expression of the donor cell type contributes significantly to the differences among hiPSCs and hESCs, and adds to the incompleteness in reprogramming. Specifically, our analysis reveals that fetal fibroblast-derived hiPSCs are closer to hESCs, followed by adipose, neonatal fibroblast, and keratinocyte-derived hiPSCs.
View details for DOI 10.1371/journal.pone.0008975
View details for Web of Science ID 000274209700007
View details for PubMedID 20126639
Timing of Bone Marrow Cell Delivery Has Minimal Effects on Cell Viability and Cardiac Recovery After Myocardial Infarction
2010; 3 (1): 77-U109
Despite ongoing clinical trials, the optimal time for delivery of bone marrow mononuclear cells (BMCs) after myocardial infarction is unclear. We compared the viability and effects of transplanted BMCs on cardiac function in the acute and subacute inflammatory phases of myocardial infarction.The time course of acute inflammatory cell infiltration was quantified by FACS analysis of enzymatically digested hearts of FVB mice (n=12) after left anterior descending artery ligation. Mac-1(+)Gr-1(high) neutrophil infiltration peaked at day 4. BMCs were harvested from transgenic FVB mice expressing firefly luciferase (Fluc) and green fluorescent protein (GFP). Afterward, 2.5x10(6) BMCs were injected into the left ventricle of wild-type FVB mice either immediately (acute BMC) or 7 days (subacute BMC) after myocardial infarction, or after a sham procedure (n=8 per group). In vivo bioluminescence imaging showed an early signal increase in both BMC groups at day 7, followed by a nonsignificant trend (P=0.203) toward improved BMC survival in the subacute BMC group that persisted until the bioluminescence imaging signal reached<0.01) and 6 weeks (both BMC groups versus saline; P<0.05) but no significant differences between the 2 BMC groups. FACS analysis of BMC-injected hearts at day 7 revealed that GFP(+) BMCs expressed hematopoietic (CD45, Mac-1, Gr-1), minimal progenitor (Sca-1, c-kit), and no endothelial (CD133, Flk-1) or cardiac (Trop-T) cell markers.Timing of BMC delivery has minimal effects on intramyocardial retention and preservation of cardiac function. In general, there is poor long-term engraftment and BMCs tend to adopt inflammatory cell phenotypes.
View details for DOI 10.1161/CIRCIMAGING.109.872085
View details for Web of Science ID 000273736000011
View details for PubMedID 19920031
Embryonic stem cell biology: insights from molecular imaging.
Methods in molecular biology (Clifton, N.J.)
2010; 660: 185-199
Embryonic stem (ES) cells have therapeutic potential in disorders of cellular loss such as myocardial infarction, type I diabetes and neurodegenerative disorders. ES cell biology in living subjects was largely poorly understood until incorporation of molecular imaging into the field. Reporter gene imaging works by integrating a reporter gene into ES cells and using a reporter probe to induce a signal detectable by normal imaging modalities. Reporter gene imaging allows for longitudinal tracking of ES cells within the same host for a prolonged period of time. This has advantages over postmortem immunohistochemistry and traditional imaging modalities. The advantages include expression of reporter gene is limited to viable cells, expression is conserved between generations of dividing cells, and expression can be linked to a specific population of cells. These advantages were especially useful in studying a dynamic cell population such as ES cells and proved useful in elucidating the biology of ES cells. Reporter gene imaging identified poor integration of differentiated ES cells transplanted into host tissue as well as delayed donor cell death as reasons for poor long-term survival in vivo. This imaging technology also confirmed that ES cells indeed have immunogenic properties that factor into cell survival and differentiation. Finally, reporter gene imaging improved our understanding of the neoplastic risk of undifferentiated ES cells in forming teratomas. Despite such advances, much remains to be understood about ES cell biology to translate this technology to the bedside, and reporter gene imaging will certainly play a key role in formulating this understanding.
View details for DOI 10.1007/978-1-60761-705-1_12
View details for PubMedID 20680820
Functional and Transcriptional Characterization of Human Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Myocardial Infarction
2009; 4 (12)
Differentiation of human embryonic stem cells into endothelial cells (hESC-ECs) has the potential to provide an unlimited source of cells for novel transplantation therapies of ischemic diseases by supporting angiogenesis and vasculogenesis. However, the endothelial differentiation efficiency of the conventional embryoid body (EB) method is low while the 2-dimensional method of co-culturing with mouse embryonic fibroblasts (MEFs) require animal product, both of which can limit the future clinical application of hESC-ECs. Moreover, to fully understand the beneficial effects of stem cell therapy, investigators must be able to track the functional biology and physiology of transplanted cells in living subjects over time.In this study, we developed an extracellular matrix (ECM) culture system for increasing endothelial differentiation and free from contaminating animal cells. We investigated the transcriptional changes that occur during endothelial differentiation of hESCs using whole genome microarray, and compared to human umbilical vein endothelial cells (HUVECs). We also showed functional vascular formation by hESC-ECs in a mouse dorsal window model. Moreover, our study is the first so far to transplant hESC-ECs in a myocardial infarction model and monitor cell fate using molecular imaging methods.Taken together, we report a more efficient method for derivation of hESC-ECs that express appropriate patterns of endothelial genes, form functional vessels in vivo, and improve cardiac function. These studies suggest that hESC-ECs may provide a novel therapy for ischemic heart disease in the future.
View details for DOI 10.1371/journal.pone.0008443
View details for Web of Science ID 000273180200002
View details for PubMedID 20046878
Current-Controlled Electrical Point-Source Stimulation of Embryonic Stem Cells
CELLULAR AND MOLECULAR BIOENGINEERING
2009; 2 (4): 625-635
Stem cell therapy is emerging as a promising clinical approach for myocardial repair. However, the interactions between the graft and host, resulting in inconsistent levels of integration, remain largely unknown. In particular, the influence of electrical activity of the surrounding host tissue on graft differentiation and integration is poorly understood. In order to study this influence under controlled conditions, an in vitro system was developed. Electrical pacing of differentiating murine embryonic stem (ES) cells was performed at physiologically relevant levels through direct contact with microelectrodes, simulating the local activation resulting from contact with surrounding electroactive tissue. Cells stimulated with a charged balanced voltage-controlled current source for up to 4 days were analyzed for cardiac and ES cell gene expression using real-time PCR, immunofluorescent imaging, and genome microarray analysis. Results varied between ES cells from three progressive differentiation stages and stimulation amplitudes (nine conditions), indicating a high sensitivity to electrical pacing. Conditions that maximally encouraged cardiomyocyte differentiation were found with Day 7 EBs stimulated at 30 microA. The resulting gene expression included a sixfold increase in troponin-T and a twofold increase in beta-MHCwithout increasing ES cell proliferation marker Nanog. Subsequent genome microarray analysis revealed broad transcriptome changes after pacing. Concurrent to upregulation of mature gene programs including cardiovascular, neurological, and musculoskeletal systems is the apparent downregulation of important self-renewal and pluripotency genes. Overall, a robust system capable of long-term stimulation of ES cells is demonstrated, and specific conditions are outlined that most encourage cardiomyocyte differentiation.
View details for DOI 10.1007/s12195-009-0096-0
View details for Web of Science ID 000272671600015
View details for PubMedID 20652088
Poor Functional Recovery After Transplantation of Diabetic Bone Marrow Stem Cells in Ischemic Myocardium
JOURNAL OF HEART AND LUNG TRANSPLANTATION
2009; 28 (11): 1158-1165
Autologous bone marrow mononuclear cell (BMMC) therapy has shown promise for improving cardiac function after myocardial infarction. The efficiency of such therapy for diabetic patients remains unknown.BMMCs were harvested from type 2 diabetic male BKS.Cg-m+/+Lepr(db)/J mice or C57BLKS/J (non-diabetic control) mice and were isolated using Ficoll-based separation. Cell characterization was performed by flow cytometry. Cell viability was determined by apoptosis and proliferation assays. Female BKS.Cg-m+/+Lepr(db)/J mice underwent left anterior descending artery ligation and were randomized into 3 groups receiving 2.5 x 10(6) diabetic BMMCs (n = 8), 2.5 x 10(6) control BMMCs (n = 8), or phosphate-buffered saline (n = 6). At Week 5, cardiac function was assessed with echocardiography and invasive hemodynamic measurements. Post-mortem cell survival was quantified by TaqMan real-time transcription polymerase chain reaction (RT-PCR) for the male Sry gene.BKS.Cg-m+/+Lepr(db)/J BMMCs showed a significantly lower mononuclear fraction and a significantly lower proliferation rate compared with C57BLKS/J BMMCs. Fractional shorting (40.1% +/- 1.2% vs 30.3% +/- 1.9%; p = 0.001) and cardiac output (4,166 +/- 393 vs 2,246 +/- 462 microl/min; p = 0.016) significantly improved for mice treated with control BMMCs injection compared with those treated with diabetic BMMCs, respectively. This difference could not be attributed to difference in cell engraftment because TaqMan RT-PCR showed no significant difference in cell survival in infarcted hearts between the 2 groups.Diabetic BMMCs are significantly impaired in their ability to improve cardiac function after myocardial infarction compared with control BMMCs. These findings could have significant clinical implication regarding autologous BMMC therapy in diabetic patients.
View details for DOI 10.1016/j.healun.2009.06.018
View details for Web of Science ID 000271795900007
View details for PubMedID 19782602
Hepatocyte Growth Factor or Vascular Endothelial Growth Factor Gene Transfer Maximizes Mesenchymal Stem Cell-Based Myocardial Salvage After Acute Myocardial Infarction
2009; 120 (11): S247-S254
Mesenchymal stem cell (MSC)-based regenerative strategies were investigated to treat acute myocardial infarction and improve left ventricular function.Murine AMI was induced by coronary ligation with subsequent injection of MSCs, hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), or MSCs +HGF/VEGF into the border zone. Left ventricular ejection fraction was calculated using micro-computed tomography imaging after 6 months. HGF and VEGF protein injection (with or without concomitant MSC injection) significantly and similarly improved the left ventricular ejection fraction and reduced scar size compared with the MSC group, suggesting that myocardial recovery was due to the cytokines rather than myocardial regeneration. To provide sustained paracrine effects, HGF or VEGF overexpressing MSCs were generated (MSC-HGF, MSC-VEGF). MSC-HGF and MSC-VEGF showed significantly increased in vitro proliferation and increased in vivo proliferation within the border zone. Cytokine production correlated with MSC survival. MSC-HGF- and MSC-VEGF-treated animals showed smaller scar sizes, increased peri-infarct vessel densities, and better preserved left ventricular function when compared with MSCs transfected with empty vector. Murine cardiomyocytes were exposed to hypoxic in vitro conditions. The LDH release was reduced, fewer cardiomyocytes were apoptotic, and Akt activity was increased if cardiomyocytes were maintained in conditioned medium obtained from MSC-HGF or MSC-VEGF cultures.This study showed that (1) elevating the tissue levels of HGF and VEGF after acute myocardial infarction seems to be a promising reparative therapeutic approach, (2) HGF and VEGF are cardioprotective by increasing the tolerance of cardiomyocytes to ischemia, reducing cardiomyocyte apoptosis and increasing prosurvival Akt activation, and (3) MSC-HGF and MSC-VEGF are a valuable source for increased cytokine production and maximize the beneficial effect of MSC-based repair strategies.
View details for DOI 10.1161/CIRCULATIONAHA.108.843680
View details for Web of Science ID 000269773000035
View details for PubMedID 19752375
Novel Minicircle Vector for Gene Therapy in Murine Myocardial Infarction
2009; 120 (11): S230-S237
Conventional plasmids for gene therapy produce low-level and short-term gene expression. In this study, we develop a novel nonviral vector that robustly and persistently expresses the hypoxia-inducible factor-1 alpha (HIF-1alpha) therapeutic gene in the heart, leading to functional benefits after myocardial infarction.We first created minicircles (MC) carrying double-fusion reporter gene consisting of firefly luciferase and enhanced green fluorescent protein (Fluc-eGFP) for noninvasive measurement of transfection efficiency. Mouse C2C12 myoblasts and normal FVB/N mice were used for in vitro and in vivo confirmation, respectively. Bioluminescence imaging showed stable MC gene expression in the heart for >12 weeks and the activity level was 5.6+/-1.2-fold stronger than regular plasmid at day 4 (P<0.01). Next, we created MC carrying HIF-1alpha (MC-HIF-1alpha) therapeutic gene for treatment of myocardial infarction. Adult FVB/N mice underwent left anterior descending ligation and were injected intramyocardially with: (1) MC-HIF-1alpha; (2) regular plasmid carrying HIF-1alpha (PL-HIF-1alpha) as positive control; and (3) PBS as negative control (n=10/group). Echocardiographic study showed a significantly greater improvement of left ventricular ejection fraction in the MC group (51.3%+/-3.6%) compared to regular plasmid group (42.3%+/-4.1%) and saline group (30.5%+/-2.8%) at week 4 (P<0.05 for both). Histology demonstrated increased neoangiogenesis in both treatment groups. Finally, Western blot showed MC express >50% higher HIF-1alpha level than regular plasmid.Taken together, this is the first study to our knowledge to demonstrate that MC can significantly improve transfection efficiency, duration of transgene expression, and cardiac contractility. Given the serious drawbacks associated with most viral vectors, we believe this novel nonviral vector can be of great value for cardiac gene therapy protocols.
View details for DOI 10.1161/CIRCULATIONAHA.108.841155
View details for Web of Science ID 000269773000033
View details for PubMedID 19752373
Feeder-free derivation of induced pluripotent stem cells from adult human adipose stem cells
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2009; 106 (37): 15720-15725
Ectopic expression of transcription factors can reprogram somatic cells to a pluripotent state. However, most of the studies used skin fibroblasts as the starting population for reprogramming, which usually take weeks for expansion from a single biopsy. We show here that induced pluripotent stem (iPS) cells can be generated from adult human adipose stem cells (hASCs) freshly isolated from patients. Furthermore, iPS cells can be readily derived from adult hASCs in a feeder-free condition, thereby eliminating potential variability caused by using feeder cells. hASCs can be safely and readily isolated from adult humans in large quantities without extended time for expansion, are easy to maintain in culture, and therefore represent an ideal autologous source of cells for generating individual-specific iPS cells.
View details for DOI 10.1073/pnas.0908450106
View details for Web of Science ID 000269806600040
View details for PubMedID 19805220
Effects of cell number on teratoma formation by human embryonic stem cells
2009; 8 (16): 2608-2612
Teratoma formation is a critical obstacle to safe clinical translation of human embryonic stem (ES) cell-based therapies in the future. As current methods of isolation are unable to yield 100% pure population of differentiated cells from a pluripotent donor source, potential development of these tumors is a significant concern. Here we used non-invasive reporter gene imaging to investigate the relationship between human ES cell number and teratoma formation in a xenogenic model of ES cell transplantation. Human ES cells (H9 line) were stably transduced with a double fusion (DF) reporter construct containing firefly luciferase and enhanced green fluorescent protein (Fluc-eGFP) driven by a human ubiquitin promoter. Immunodeficient mice received intramyocardial (n = 35) or skeletal muscle (n = 35) injection of 1 x 10(2), 1 x 10(3), 1 x 10(4), 1 x 10(5) or 1 x 10(6) DF positive ES cells suspended in saline for myocardium and Matrigel for skeletal muscle. Cell survival and proliferation were monitored via bioluminescence imaging (BLI) for an 8 week period following transplantation. Mice negative for Fluc signal after 8 weeks were followed out to day 365 to confirm tumor absence. Significantly, in this study, a minimum of 1 x 10(5) ES cells in the myocardium and 1 x 10(4) cells in the skeletal muscle was observed to be requisite for teratoma development, suggesting that human ES cell number may be a critical factor in teratoma formation. Engraftment and tumor occurrence were also observed to be highly dependent on ES cell number. We anticipate these results should yield useful insights to the safe and reliable application of human ES cell derivatives in the clinic.
View details for Web of Science ID 000268983900028
View details for PubMedID 19597339
Imaging Gene Expression in Human Mesenchymal Stem Cells: From Small to Large Animals
2009; 252 (1): 117-127
To evaluate the feasibility of reporter gene imaging in implanted human mesenchymal stem cells (MSCs) in porcine myocardium by using clinical positron emission tomography (PET)-computed tomography (CT) scanning.Animal protocols were approved by the Institutional Administrative Panel on Laboratory Animal Care. Transduction of human MSCs by using different doses of adenovirus that contained a cytomegalovirus (CMV) promoter driving the mutant herpes simplex virus type 1 thymidine kinase reporter gene (Ad-CMV-HSV1-sr39tk) was characterized in a cell culture. A total of 2.25 x 10(6) transduced (n = 5) and control nontransduced (n = 5) human MSCs were injected into the myocardium of 10 rats, and reporter gene expression in human MSCs was visualized with micro-PET by using the radiotracer 9-(4-[fluorine 18]-fluoro-3-hydroxymethylbutyl)-guanine (FHBG). Different numbers of transduced human MSCs suspended in either phosphate-buffered saline (PBS) (n = 4) or matrigel (n = 5) were injected into the myocardium of nine swine, and gene expression was visualized with a clinical PET-CT. For analysis of cell culture experiments, linear regression analyses combined with a t test were performed. To test differences in radiotracer uptake between injected and remote myocardium in both rats and swine, one-sided paired Wilcoxon tests were performed. In swine experiments, a linear regression of radiotracer uptake ratio on the number of injected transduced human MSCs was performed.In cell culture, there was a viral dose-dependent increase of gene expression and FHBG accumulation in human MSCs. Human MSC viability was 96.7% (multiplicity of infection, 250). Cardiac FHBG uptake in rats was significantly elevated (P < .0001) after human MSC injection (0.0054% injected dose [ID]/g +/- 0.0007 [standard deviation]) compared with that in the remote myocardium (0.0003% ID/g +/- 0.0001). In swine, myocardial radiotracer uptake was not elevated after injection of up to 100 x 10(6) human MSCs (PBS group). In the matrigel group, signal-to-background ratio increased to 1.87 after injection of 100 x 10(6) human MSCs and positively correlated (R(2) = 0.97, P < .001) with the number of administered human MSCs.Reporter gene imaging in human MSCs can be translated to large animals. The study highlights the importance of co-administering a "scaffold" for increasing intramyocardial retention of human MSCs.
View details for DOI 10.1148/radiol.2513081616
View details for Web of Science ID 000268362900015
View details for PubMedID 19366903
Molecular and Magnetic Resonance Imaging of Human Embryonic Stem Cell-Derived Neural Stem Cell Grafts in Ischemic Rat Brain
2009; 17 (7): 1282-1291
Real-time imaging of transplanted stem cells is essential for understanding their interactions in vivo with host environments, for tracking cell fate and function and for successful delivery and safety monitoring in the clinical setting. In this study, we used bioluminescence (BLI) and magnetic resonance imaging (MRI) to visualize the fate of grafted human embryonic stem cell (hESC)-derived human neural stem cells (hNSCs) in stroke-damaged rat brain. The hNSCs were genetically engineered with a lentiviral vector carrying a double fusion (DF) reporter gene that stably expressed enhanced green fluorescence protein (eGFP) and firefly luciferase (fLuc) reporter genes. The hNSCs were self-renewable, multipotent, and expressed markers for neural stem cells. Cell survival was tracked noninvasively by MRI and BLI for 2 months after transplantation and confirmed histologically. Electrophysiological recording from grafted GFP(+) cells and immuno-electronmicroscopy demonstrated connectivity. Grafted hNSCs differentiated into neurons, into oligodendrocytes in stroke regions undergoing remyelination and into astrocytes extending processes toward stroke-damaged vasculatures. Our data suggest that the combination of BLI and MRI modalities provides reliable real-time monitoring of cell fate.
View details for DOI 10.1038/mt.2009.104
View details for Web of Science ID 000267785800021
View details for PubMedID 19436269
MicroRNA Profiling of Human-Induced Pluripotent Stem Cells
STEM CELLS AND DEVELOPMENT
2009; 18 (5): 749-757
MicroRNAs (miRNAs) are a newly discovered endogenous class of small noncoding RNAs that play important posttranscriptional regulatory roles by targeting mRNAs for cleavage or translational repression. Accumulating evidence now supports the importance of miRNAs for human embryonic stem cell (hESC) self-renewal, pluripotency, and differentiation. However, with respect to induced pluripotent stem cells (iPSC), in which embryonic-like cells are reprogrammed from adult cells using defined factors, the role of miRNAs during reprogramming has not been well-characterized. Determining the miRNAs that are associated with reprogramming should yield significant insight into the specific miRNA expression patterns that are required for pluripotency. To address this lack of knowledge, we use miRNA microarrays to compare the "microRNA-omes" of human iPSCs, hESCs, and fetal fibroblasts. We confirm the presence of a signature group of miRNAs that is up-regulated in both iPSCs and hESCs, such as the miR-302 and 17-92 clusters. We also highlight differences between the two pluripotent cell types, as in expression of the miR-371/372/373 cluster. In addition to histone modifications, promoter methylation, transcription factors, and other regulatory control elements, we believe these miRNA signatures of pluripotent cells likely represent another layer of regulatory control over cell fate decisions, and should prove important for the cellular reprogramming field.
View details for DOI 10.1089/scd.2008.0247
View details for Web of Science ID 000266237000009
View details for PubMedID 19284351
Comparison of Optical Bioluminescence Reporter Gene and Superparamagnetic Iron Oxide MR Contrast Agent as Cell Markers for Noninvasive Imaging of Cardiac Cell Transplantation
MOLECULAR IMAGING AND BIOLOGY
2009; 11 (3): 178-187
In this study, we compared firefly luciferase (Fluc) reporter gene and superparamagnetic iron oxide (Feridex) as cell markers for longitudinal monitoring of cardiomyoblast graft survival using optical bioluminescence imaging (BLI) and magnetic resonance imaging (MRI), respectively.Rats (n = 31) underwent an intramyocardial injection of cardiomyoblasts (2 x 10(6)) labeled with Fluc, Feridex, or no marker (control) or an injection of Feridex alone (75 microg). Afterward, rats were serially imaged with BLI or MRI and killed at different time points for histological analysis.BLI revealed a drastically different cell survival kinetics (half-life = 2.65 days over 6 days) than that revealed by MRI (half-life = 16.8 days over 80 days). Injection of Feridex alone led to prolonged tissue retention of Feridex (> or =16 days) and persistent MR signal (> or =42 days).Fluc BLI reporter gene imaging is a more accurate gauge of transplanted cell survival as compared to MRI of Feridex-labeled cells.
View details for DOI 10.1007/s11307-008-0182-z
View details for Web of Science ID 000265686900005
View details for PubMedID 19034584
Tracking cardiac engraftment and distribution of implanted bone marrow cells: Comparing intra-aortic, intravenous, and intramyocardial delivery
JOURNAL OF THORACIC AND CARDIOVASCULAR SURGERY
2009; 137 (5): 1225-U216
Cell therapy improved cardiac function after a myocardial infarction in several preclinical studies; however, the functional benefits were limited in the initial clinical trials, perhaps because of inadequate cell engraftment. We used noninvasive molecular imaging to compare the distribution and myocardial retention of cells implanted by using clinical delivery routes.Bone marrow stromal cells isolated from male rats and transfected with a firefly luciferase reporter gene were injected by using 3 increasingly invasive techniques (ie, intravenous, intra-aortic, and intramyocardial) into female rats 3 or 28 days after coronary ligation. Whole-body bioluminescence imaging was performed 2, 24, and 48 hours later; implanted cells were quantified at 48 hours in explanted organs by means of bioluminescence and real-time polymerase chain reaction.Variations in cell distribution among groups were profound, with nearly complete trapping of the injected cells in the lungs after intravenous delivery. Cell delivery into the aortic root (with the distal aorta occluded) produced minimal cell retention in the heart. Direct intramyocardial injection facilitated the best early targeting of the cells (P < .05 vs intravenous and intra-aortic injection). Rapid signal loss over 48 hours indicated very poor cell survival in all 3 groups, although implanted cell retention was greater in mature compared with acute infarcts.This is the first study to correlate live cell imaging with quantitative genetic and histologic techniques. Noninvasive molecular imaging tracked delivered cells and will permit the evaluation of new and improved delivery platforms designed to increase cell homing, retention, and engraftment.
View details for DOI 10.1016/j.jtcvs.2008.11.001
View details for Web of Science ID 000265299000029
View details for PubMedID 19379996
Imaging Survival and Function of Transplanted Cardiac Resident Stem Cells
JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY
2009; 53 (14): 1229-1240
The goal of this study is to characterize resident cardiac stem cells (CSCs) and investigate their therapeutic efficacy in myocardial infarction by molecular imaging methods.CSCs have been isolated and characterized in vitro. These cells offer a provocative method to regenerate the damaged myocardium. However, the survival kinetics and function of transplanted CSCs have not been fully elucidated.CSCs were isolated from L2G85 transgenic mice (FVB strain background) that constitutively express both firefly luciferase and enhanced green fluorescence protein reporter gene. CSCs were characterized in vitro and transplanted in vivo into murine infarction models. Multimodality noninvasive imaging techniques were used to assess CSC survival and therapeutic efficacy for restoration of cardiac function.CSCs can be isolated from L2G85 mice, and fluorescence-activated cell sorting analysis showed expression of resident CSC markers (Sca-1, c-Kit) and mesenchymal stem cell markers (CD90, CD106). Afterwards, 5 x 10(5) CSCs (n = 30) or phosphate-buffered saline control (n = 15) was injected into the hearts of syngeneic FVB mice undergoing left anterior descending artery ligation. Bioluminescence imaging showed poor donor cell survival by week 8. Echocardiogram, invasive hemodynamic pressure-volume analysis, positron emission tomography imaging with fluorine-18-fluorodeoxyglucose, and cardiac magnetic resonance imaging demonstrated no significant difference in cardiac contractility and viability between the CSC and control group. Finally, postmortem analysis confirmed transplanted CSCs integrated with host cardiomyocytes by immunohistology.In a mouse myocardial infarction model, Sca-1-positive CSCs provide no long-term engraftment and benefit to cardiac function as determined by multimodality imaging.
View details for DOI 10.1016/j.jacc.2008.12.036
View details for Web of Science ID 000264724500011
View details for PubMedID 19341866
Noninvasive De novo Imaging of Human Embryonic Stem Cell-Derived Teratoma Formation
2009; 69 (7): 2709-2713
Teratoma formation can be a serious drawback after the therapeutic transplantation of human embryonic stem (hES) cells. Therefore, noninvasive imaging of teratomas could be a valuable tool for monitoring patients undergoing hES cell treatment. Here, we investigated the angiogenic process within teratomas derived from hES cells and now report the first example of using (64)Cu-labeled RGD tetramer ((64)Cu-DOTA-RGD4) for positron emission tomography imaging of teratoma formation by targeting alpha(v)beta(3) integrin. H9 hES cells (2 x 10(6)), stably expressing firefly luciferase, and enhanced green fluorescence protein (Fluc-eGFP) were injected into adult nude mice (n=12) s.c. Eight weeks after transplantation, these hES cell grafts evolved into teratomas as confirmed by longitudinal bioluminescence imaging. Under micropositron emission tomography imaging, 2-deoxy-2-[(18)F]fluoro-D-glucose and 3'-deoxy-3'-[(18)F]-fluorothymidine both failed to detect hES cell-derived teratomas (0.8+/-0.5 versus 1.1+/-0.4 %ID/g, respectively; P=not significant versus background signals). By contrast, (64)Cu-DOTA-RGD4 revealed specific and prominent uptake in vascularized teratoma and significantly lower uptake in control tumors (human ovarian carcinoma 2008 cell line), which had low integrin expression (10.1+/-3.4 versus 1.4+/-1.2 %ID/g; P<0.01). Immunofluorescence staining of CD31 and beta(3) integrin also supported our in vivo imaging results (P<0.05). Moreover, we found that the cells dissociated from teratomas showed higher alpha(v)beta(3) integrin expression than the 2008 cells. In conclusion, by targeting alpha(v)beta(3) integrin, we successfully showed the ability of (64)Cu-DOTA-RGD4 to noninvasively visualize teratoma formation in vivo for the first time.
View details for DOI 10.1158/0008-5472.CAN-08-4122
View details for Web of Science ID 000264908100004
View details for PubMedID 19318556
Comparison of Transplantation of Adipose Tissue- and Bone Marrow-Derived Mesenchymal Stem Cells in the Infarcted Heart
2009; 87 (5): 642-652
Mesenchymal stem cells hold promise for cardiovascular regenerative therapy. Derivation of these cells from the adipose tissue might be easier compared with bone marrow. However, the in vivo fate and function of adipose stromal cells (ASC) in the infarcted heart has never been compared directly to bone marrow-derived mesenchymal cells (MSC).ASC and MSC were isolated from transgenic FVB mice with a beta-actin promoter driving firefly luciferase and green fluorescent protein double fusion reporter gene, and they were characterized using flow cytometry, microscopy, bioluminescence imaging and luminometry. FVB mice (n=8 per group) underwent myocardial infarction followed by intramyocardial injection of 5x10(5) ASC, MSC, fibroblasts (Fibro, positive control), or saline (negative control). Cell survival was measured using bioluminescence imaging for 6 weeks and cardiac function was monitored by echocardiography and pressure-volume analysis. Ventricular morphology was assessed using histology.ASC and MSC were CD34(-), CD45(-), c-Kit(-), CD90(+), Sca-1(+), shared similar morphology and had a population doubling time of approximately 2 days. Cells expressed Fluc reporter genes in a number-dependent fashion as confirmed by luminometry. After cardiac transplantation, both cell types showed drastic donor cell death within 4 to 5 weeks. Furthermore, transplantation of either cell type was not capable of preserving ventricular function and dimensions, as confirmed by pressure-volume-loops and histology.This is the first study comparing the in vivo behavior of both cell types in the infarcted heart. ASC and MSC do not tolerate well in the cardiac environment, resulting in acute donor cell death and a subsequent loss of cardiac function similar to control groups.
View details for DOI 10.1097/TP.0b013e31819609d9
View details for Web of Science ID 000264146100005
View details for PubMedID 19295307
Imaging of STAT3 Signaling Pathway During Mouse Embryonic Stem Cell Differentiation
STEM CELLS AND DEVELOPMENT
2009; 18 (2): 205-214
Signal transducers and activators of transcription 3 (STAT3) is a pleiotropic transcription factor involved in a variety of physiological processes. STAT3 acts as a key transcriptional determinant of mouse embryonic stem (ES) cell self-renewal and plays a pivotal function in early mammalian embryogenesis because the development of many organs requires STAT3 activation. However, little is known about the role of STAT3 function during ES cell differentiation. To answer this question, we built a lentiviral construct with 7-repeat STAT3-binding sequence (enhancer) and minimal TA (promoter) driving renilla luciferase and monomeric red fluorescence protein (Rluc-mRFP), followed by a constitutive cytomegalovirus promoter driving green fluorescent protein as a selection marker. The specificity of our custom-designed 7-repeat STAT3 reporter construct was first confirmed by cotransfection with constitutively active version of STAT3 (STAT3C) into human embryonic kidney 293T cells. Next, a mouse ES cell line stably transduced with STAT3 reporter construct was isolated. This ES cell line showed a tight response in reporter gene expression with leukemia inhibitory factor (LIF) induction and was chosen as a developmental model for the STAT3 functional study. Using serial noninvasive bioluminescence imaging, we showed that the onset of embryoid body (EB) formation involved inhibition of STAT3 activity. However, during differentiation, STAT3 activity steadily increased from day 5 to 14 and was reduced by day 21. STAT3 activity was also confirmed separately by Western blots. Finally, phosphorylation of STAT3 was also found to correspond with cardiomyocyte differentiation. In summary, this is the first study to monitor real-time STAT3 activity during ES cell differentiation. This genetically modified line can be used to study the biological role of STAT3 during ES cell differentiation into different derivatives.
View details for DOI 10.1089/scd.2008.0152
View details for Web of Science ID 000264171300002
View details for PubMedID 18576943
Transplantation of Human Embryonic Stem Cell-Derived Endothelial Cells for Vascular Diseases
JOURNAL OF CELLULAR BIOCHEMISTRY
2009; 106 (2): 194-199
Using endothelial cells for therapeutic angiogenesis/vasculogenesis of ischemia diseases has led to exploring human embryonic stem cells (hESCs) as a potentially unlimited source for endothelial progenitor cells. With their capacity for self-renewal and pluripotency, hESCs and their derived endothelial cells (hESC-ECs) may be more advantageous than other endothelial cells obtained from diseased populations. However, hESC-ECs' poor differentiation efficiency and poorly characterized in vivo function after transplantation present significant challenges for their future clinical application. This review will focus on the differentiation pathways of hESCs and their therapeutic potential for vascular diseases, as well as the monitoring of transplanted cells' fate via molecular imaging. Finally, cell enhancement strategies to improve the engraftment efficiency of hESC-ECs will be discussed.
View details for DOI 10.1002/jcb.22003
View details for Web of Science ID 000262921600002
View details for PubMedID 19097085
Trafficking Mesenchymal Stem Cell Engraftment and Differentiation in Tumor-Bearing Mice by Bioluminescence Imaging
2009; 27 (7): 1548-1558
The objective of the study was to track the distribution and differentiation of mesenchymal stem cells (MSCs) in tumor-bearing mice. The 4T1 murine breast cancer cells were labeled with renilla luciferase-monomeric red fluorescence protein (rLuc-mRFP) reporter gene. The MSCs labeled with firefly luciferase-enhanced green fluorescence protein (fLuc-eGFP) reporter gene (MSCs-R) were isolated from L2G85 transgenic mice that constitutively express fLuc-eGFP reporter gene. To study the tumor tropism of MSCs, we established both subcutaneous and lung metastasis models. In lung metastasis tumor mice, we injected MSCs-R intravenously either on the same day or 4 days after 4T1 tumor cell injection. In subcutaneous tumor mice, we injected MSCs-R intravenously 7 days after subcutaneous 4T1 tumor inoculation. The tumor growth was monitored by rLuc bioluminescence imaging (BLI). The fate of MSCs-R was monitored by fLuc BLI. The localization of MSCs-R in tumors was examined histologically. The osteogenic and adipogenic differentiation of MSCs-R was investigated by alizarin red S and oil red O staining, respectively. The mechanism of the dissimilar differentiation potential of MSCs-R under different tumor microenvironments was investigated. We found that the 4T1 cells were successfully labeled with rLuc-mRFP. The MSCs-R isolated from L2G85 transgenic mice constitutively express fLuc-eGFP reporter gene. When injected intravenously, MSCs-R survived, proliferated, and differentiated in tumor sites but not elsewhere. The localization of GFP(+) MSCs-R in tumor lesions was confirmed ex vivo. In conclusion, the MSCs-R can selectively localize, survive, and proliferate in both subcutaneous tumor and lung metastasis as evidenced by noninvasive bioluminescence imaging and ex vivo validation. The MSCs-R migrated to lung tumor differentiated into osteoblasts, whereas the MSCs-R targeting subcutaneous tumor differentiated into adipocytes.
View details for DOI 10.1002/stem.81
View details for Web of Science ID 000268257100011
View details for PubMedID 19544460
- Multimodality Cardiovascular Molecular Imaging, Part II CIRCULATION-CARDIOVASCULAR IMAGING 2009; 2 (1): 56-70
Molecular imaging of human embryonic stem cells.
Methods in molecular biology (Clifton, N.J.)
2009; 515: 13-32
Human embryonic stem cells (hESCs) are a renewable source of differentiated cell types that may be employed in various tissue regeneration strategies. However, clinical implementation of cell transplantation therapy is hindered by legitimate concerns regarding the in vivo teratoma formation of undifferentiated hESCs and host immune reactions to allogenic cells. Investigating in vivo hESC behaviour and the ultimate feasibility of cell transplantation therapy necessitates the development of novel molecular imaging techniques to longitudinally monitor hESC localization, proliferation, and viability in living subjects. An innovative approach to harness the respective strengths of various imaging platforms is the creation and use of a fusion reporter construct composed of red fluorescent protein (RFP), firefly luciferase (fluc), and herpes simplex virus thymidine kinase (HSV-tk). The imaging modalities made available by use of this construct, including optical fluorescence, bioluminescence, and positron emission tomography (PET), mat be adapted to investigate a variety of physiological phenomena, including the spatio-temporal kinetics of hESC engraftment and proliferation in living subjects. This chapter describes the applications of reporter gene imaging to accelerate basic science research and clinical studies involving hESCs through (1) isolation of a homogenous hESC population, (2) noninvasive, longitudinal tracking of the location and proliferation of hESCs administered to a living subject, and (3) ablation of the hESC graft in the event of cellular misbehavior.
View details for PubMedID 19405224
Long term non-invasive imaging of embryonic stem cells using reporter genes
2009; 4 (8): 1192-1201
Development of non-invasive and accurate methods to track cell fate after delivery will greatly expedite transition of embryonic stem (ES) cell therapy to the clinic. In this protocol, we describe the in vivo monitoring of stem cell survival, proliferation and migration using reporter genes. We established stable ES cell lines constitutively expressing double fusion (DF; enhanced green fluorescent protein and firefly luciferase) or triple fusion (TF; monomeric red fluorescent protein, firefly luciferase and herpes simplex virus thymidine kinase (HSVtk)) reporter genes using lentiviral transduction. We used fluorescence-activated cell sorting to purify these populations in vitro, bioluminescence imaging and positron emission tomography (PET) imaging to track them in vivo and fluorescence immunostaining to confirm the results ex vivo. Unlike other methods of cell tracking, such as iron particle and radionuclide labeling, reporter genes are inherited genetically and can be used to monitor cell proliferation and survival for the lifetime of transplanted cells and their progeny.
View details for DOI 10.1038/nprot.2009.100
View details for Web of Science ID 000268858800009
View details for PubMedID 19617890
Bioluminescence reporter gene imaging of human embryonic stem cell survival, proliferation, and fate.
Methods in molecular biology (Clifton, N.J.)
2009; 574: 87-103
The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability. Molecular imaging, and specifically bioluminescent reporter gene imaging, has given investigators a high-throughput, inexpensive, and sensitive means for tracking in vivo cell proliferation over days, weeks, and even months. This advancement has significantly increased the understanding of the spatiotemporal kinetics of hESC engraftment and proliferation in living subjects. In this chapter, the specific materials and methods needed for tracking stem cell proliferation with bioluminescence imaging will be described.
View details for DOI 10.1007/978-1-60327-321-3_8
View details for PubMedID 19685302
In Vivo Imaging of Embryonic Stem Cells Reveals Patterns of Survival and Immune Rejection Following Transplantation
STEM CELLS AND DEVELOPMENT
2008; 17 (6): 1023-1029
Embryonic stem cell (ESC)-based transplantation is considered a promising novel therapy for a variety of diseases. This is bolstered by the suggested immune-privileged properties of ESCs. In this study, we used in vivo bioluminescent imaging (BLI) to non-invasively track the fate of transplanted murine ESCs (mESCs), which are stably transduced with a double fusion reporter gene consisting of firefly luciferase (FLuc) and enhanced green fluorescent protein (eGFP). Following syngeneic intramuscular transplantation of 1 x 10(6) mESCs, the cells survived and differentiated into teratomas. In contrast, allogeneic mESC transplants were infiltrated by a variety of inflammatory cells, leading to rejection within 28 days. Acceleration of rejection was observed when mESCs were allotransplanted following prior sensitization of the host. Finally, we demonstrate that the mESC derivatives were more rapidly rejected compared to undifferentiated mESCs. These data show that mESCs do not retain immune-privileged properties in vivo and are subject to immunological rejection as assessed by novel molecular imaging approaches.
View details for DOI 10.1089/scd.2008.0091
View details for Web of Science ID 000261490700002
View details for PubMedID 18491958
- Molecular Imaging Antidote to Cardiac Stem Cell Controversy JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY 2008; 52 (20): 1661-1664
Hemodynamic forces regulate embryonic stem cell commitment to vascular progenitors.
Current cardiology reviews
2008; 4 (4): 269-274
Pluripotent embryonic stem can (ES) cells can differentiate into all cell lineages. During the process of embryonic development, ES cells are exposed to fluid flow or blood flow generated by the contracting heart. Absence of fluid flow results in the formation of abnormal cardiac chambers and valve formation. Thus, hemodynamic forces and ES cell differentiation to vascular progenitor cells (VPCs) are of emerging interests for restoring endothelial dysfunction, inducing angiogenesis, and forming blood vessel networks. Hemodynamic forces such as fluid shear stress increase the percentage of cells in the S and G(2)-M phases, and induce decondensation of chromatin for gene transcription. Fluid shear stress further accelerates ES commitment to CD31(+) VPC vascular progenitor cells. These ES-derived CD31(+) cells express endothelial nitric oxide synthase (eNOS) and von Willebrand factor (vWF). They are also capable of LDL uptake and tubular network formation. In this context, understanding hemodynamic forces and ES cell kinetics of differentiation towards endothelial lineage has potential therapeutic applications for repairing vascular damage and engineering vascular graft. Multidisciplinary team approach will likely garner momentum and synergize expertise to address the current road blocks in basic stem cell research for engraftable, restorative, low immunogenic, and non-tumorigenic endothelial progenitors in high purity and stability.
View details for DOI 10.2174/157340308786349471
View details for PubMedID 20066134
Multimodality Cardiovascular Molecular Imaging, Part I
2008; 1 (3): 244-256
In Part I of this consensus article, the imaging methodology, evolving imaging technology, and development of novel targeted molecular probes relevant to the developing field of cardiovascular molecular imaging were reviewed. Novel reporter gene and reporter probe imaging approaches for tracking of cardiac transgene expression were also discussed and have important future implications for evaluation of gene- and cell-based therapies for the failing heart. The current role of metabolic and receptor imaging was also briefly reviewed, as these represent the beginning of our clinical application of molecular imaging within the cardiovascular system. Part II will summarize the available targeted imaging probes as well as specific future applications of molecular imaging for identification and evaluation of critical pathophysiological processes of the cardiovascular system.
View details for DOI 10.1161/CIRCIMAGING.108.824359
View details for Web of Science ID 000266128800011
View details for PubMedID 19808549
Transcriptional and Functional Profiling of Human Embryonic Stem Cell-Derived Cardiomyocytes
2008; 3 (10)
Human embryonic stem cells (hESCs) can serve as a potentially limitless source of cells that may enable regeneration of diseased tissue and organs. Here we investigate the use of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) in promoting recovery from cardiac ischemia reperfusion injury in a mouse model. Using microarrays, we have described the hESC-CM transcriptome within the spectrum of changes that occur between undifferentiated hESCs and fetal heart cells. The hESC-CMs expressed cardiomyocyte genes at levels similar to those found in 20-week fetal heart cells, making this population a good source of potential replacement cells in vivo. Echocardiographic studies showed significant improvement in heart function by 8 weeks after transplantation. Finally, we demonstrate long-term engraftment of hESC-CMs by using molecular imaging to track cellular localization, survival, and proliferation in vivo. Taken together, global gene expression profiling of hESC differentiation enables a systems-based analysis of the biological processes, networks, and genes that drive hESC fate decisions, and studies such as this will serve as the foundation for future clinical applications of stem cell therapies.
View details for DOI 10.1371/journal.pone.0003474
View details for Web of Science ID 000265126100005
View details for PubMedID 18941512
Multimodal evaluation of in vivo magnetic resonance imaging of myocardial restoration by mouse embryonic stem cells
JOURNAL OF THORACIC AND CARDIOVASCULAR SURGERY
2008; 136 (4): 1028-U14
Mouse embryonic stem cells have demonstrated potential to restore infarcted myocardium after acute myocardial infarction. Although the underlying mechanism remains controversial, magnetic resonance imaging has provided reliable in vivo assessment of functional recovery after cellular transplants. Multimodal comparison of the restorative effects of mouse embryonic stem cells and mouse embryonic fibroblasts was performed to validate magnetic resonance imaging data and provide mechanistic insight.SCID-beige mice (n = 55) underwent coronary artery ligation followed by injection of 2.5 x 10(5) mouse embryonic stem cells, 2.5 x 10(5) mouse embryonic fibroblasts, or normal saline solution. In vivo magnetic resonance imaging of myocardial restoration by mouse embryonic stem cells was evaluated by (1) in vivo pressure-volume loops, (2) in vivo bioluminescence imaging, and (3) ex vivo TaqMan (Roche Molecular Diagnostics, Pleasanton, Calif) polymerase chain reaction and immunohistologic examination.In vivo magnetic resonance imaging demonstrated significant improvement in left ventricular ejection fraction at 1 week in the mouse embryonic stem cell group. This finding was validated with (1) pressure-volume loop analysis demonstrating significantly improved systolic and diastolic functions, (2) bioluminescence imaging and polymerase chain reaction showing superior posttransplant survival of mouse embryonic stem cells, (3) immunohistologic identification of cardiac phenotype within engrafted mouse embryonic stem cells, and (4) polymerase chain reaction measuring increased expressions of angiogenic and antiapoptotic genes and decreased expressions of antifibrotic genes.This study validates in vivo magnetic resonance imaging as an effective means of evaluating the restorative potential of mouse embryonic stem cells.
View details for DOI 10.1016/j.jtcvs.2007.12.053
View details for Web of Science ID 000260314800033
View details for PubMedID 18954646
Short hairpin RNA interference therapy for ischemic heart disease
2008; 118 (14): S226-U289
During hypoxia, upregulation of hypoxia inducible factor-1 alpha transcriptional factor can activate several downstream angiogenic genes. However, hypoxia inducible factor-1 alpha is naturally degraded by prolyl hydroxylase-2 (PHD2) protein. Here we hypothesize that short hairpin RNA (shRNA) interference therapy targeting PHD2 can be used for treatment of myocardial ischemia and this process can be followed noninvasively by molecular imaging.PHD2 was cloned from mouse embryonic stem cells by comparing the homolog gene in human and rat. The best candidate shRNA sequence for inhibiting PHD2 was inserted into the pSuper vector driven by the H1 promoter followed by a separate hypoxia response element-incorporated promoter driving a firefly luciferase reporter gene. This construct was used to transfect mouse C2C12 myoblast cell line for in vitro confirmation. Compared with the control short hairpin scramble (shScramble) as control, inhibition of PHD2 increased levels of hypoxia inducible factor-1 alpha protein and several downstream angiogenic genes by >30% (P<0.01). Afterward, shRNA targeting PHD2 (shPHD2) plasmid was injected intramyocardially following ligation of left anterior descending artery in mice. Animals were randomized into shPHD2 experimental group (n=25) versus shScramble control group (n=20). Bioluminescence imaging detected plasmid-mediated transgene expression for 4 to 5 weeks. Echocardiography showed the shPHD2 group had improved fractional shortening compared with the shScramble group at Week 4 (33.7%+/-1.9% versus 28.4%+/-2.8%; P<0.05). Postmortem analysis showed increased presence of small capillaries and venules in the infarcted zones by CD31 staining. Finally, Western blot analysis of explanted hearts also confirmed that animals treated with shPHD2 had significantly higher levels of hypoxia inducible factor-1 alpha protein.This is the first study to image the biological role of shRNA therapy for improving cardiac function. Inhibition of PHD2 by shRNA led to significant improvement in angiogenesis and contractility by in vitro and in vivo experiments. With further validation, the combination of shRNA therapy and molecular imaging can be used to track novel cardiovascular gene therapy applications in the future.
View details for DOI 10.1161/CIRCULATIONAHA.107.760785
View details for Web of Science ID 000259648600033
View details for PubMedID 18824759
Comparison of different adult stem cell types for treatment of myocardial ischemia
2008; 118 (14): S121-U166
A comparative analysis of the efficacy of different cell candidates for the treatment of heart disease remains to be described. This study is designed to evaluate the therapeutic efficacy of 4 cell types in a murine model of myocardial infarction.Bone marrow mononuclear cells (MN), mesenchymal stem cells (MSC), skeletal myoblasts (SkMb), and fibroblasts (Fibro) expressing firefly luciferase (Fluc) and green fluorescence protein (GFP) were characterized by flow cytometry, bioluminescence imaging (BLI), and luminometry. Female FVB mice (n=70) underwent LAD ligation and intramyocardially received one cell type (5x10(5)) or PBS. Cell survival was measured by BLI and by TaqMan PCR. Cardiac function was assessed by echocardiography and invasive hemodynamic measurements. Fluc expression correlated with cell number in all groups (r(2)>0.93). In vivo BLI revealed acute donor cell death of MSC, SkMb, and Fibro within 3 weeks after transplantation. By contrast, cardiac signals were still present after 6 weeks in the MN group, as confirmed by TaqMan PCR (P<0.01). Echocardiography showed significant preservation of fractional shortening in the MN group compared to controls (P<0.05). Measurements of left ventricular end-systolic/diastolic volumes revealed that the least amount of ventricular dilatation occurred in the MN group (P<0.05). Histology confirmed the presence of MN, although there was no evidence of transdifferentiation by donor MN into cardiomyocytes.This is the first study to show that compared to MSC, SkMB, and Fibro, MN exhibit a more favorable survival pattern, which translates into a more robust preservation of cardiac function.
View details for DOI 10.1161/CIRCULATIONAHA.107.759480
View details for Web of Science ID 000259648600018
View details for PubMedID 18824743
Immunosuppressive therapy mitigates immunological rejection of human embryonic stem cell xenografts
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2008; 105 (35): 12991-12996
Given their self-renewing and pluripotent capabilities, human embryonic stem cells (hESCs) are well poised as a cellular source for tissue regeneration therapy. However, the host immune response against transplanted hESCs is not well characterized. In fact, controversy remains as to whether hESCs have immune-privileged properties. To address this issue, we used in vivo bioluminescent imaging to track the fate of transplanted hESCs stably transduced with a double-fusion reporter gene consisting of firefly luciferase and enhanced GFP. We show that survival after transplant is significantly limited in immunocompetent as opposed to immunodeficient mice. Repeated transplantation of hESCs into immunocompetent hosts results in accelerated hESC death, suggesting an adaptive donor-specific immune response. Our data demonstrate that transplanted hESCs trigger robust cellular and humoral immune responses, resulting in intragraft infiltration of inflammatory cells and subsequent hESC rejection. Moreover, we have found CD4(+) T cells to be an important modulator of hESC immune-mediated rejection. Finally, we show that immunosuppressive drug regimens can mitigate the anti-hESC immune response and that a regimen of combined tacrolimus and sirolimus therapies significantly prolongs survival of hESCs for up to 28 days. Taken together, these data suggest that hESCs are immunogenic, trigger both cellular and humoral-mediated pathways, and, as a result, are rapidly rejected in xenogeneic hosts. This process can be mitigated by a combined immunosuppressive regimen as assessed by molecular imaging approaches.
View details for DOI 10.1073/pnas.0805802105
View details for Web of Science ID 000259343000067
View details for PubMedID 18728188
Surgically palliated double-inlet left ventricle with transposition of the great arteries mistaken for aortic aneurysm with dissection
INTERNATIONAL JOURNAL OF CARDIOLOGY
2008; 128 (2): E82-E84
We report a 23-year-old male with history of double-inlet single ventricle with transposition of the great arteries who is s/p pulmonary artery banding, a Damus-Kaye-Stanzel anastomosis, and Fontan procedure during infancy and childhood who now presents with chest pain. A chest CTA at an outside hospital was thought concerning for the presence of a thoracic aortic aneurysm with dissection, prompting immediate transfer to our institution. However, repeat chest CTA at our institution revealed the predicted anastomoses based on his surgical procedures, which was misinterpreted as aortic aneurysm and dissection. An understanding of the physiology of his surgically repaired congenital heart disease is critical in interpreting his subsequent chest CTA and arriving at the appropriate clinical conclusion.
View details for DOI 10.1016/j.ijcard.2007.04.165
View details for Web of Science ID 000257950500043
View details for PubMedID 17689761
Noninvasive radionuclide imaging of cardiac gene therapy: progress and potential
NATURE CLINICAL PRACTICE CARDIOVASCULAR MEDICINE
2008; 5: S87-S95
Over the past decade, several clinical trials have evaluated the efficacy of cardiac-specific gene therapy. Despite encouraging results in basic research and preclinical studies, most of the recent large, randomized, placebo-controlled cardiac gene therapy trials have failed to provide convincing evidence of improvements in clinical outcomes. Because many of these problems are due to the lack of appropriate monitoring techniques, there is a critical need to develop noninvasive imaging techniques that can verify vector delivery and gene expression in target and nontarget tissues. The field of molecular imaging of cardiac gene expression is rapidly advancing because it offers distinct advantages over conventional methods, including the ability to noninvasively measure the location, time course, and magnitude of gene expression. We aim to give readers a clear understanding of how molecular imaging can enable noninvasive tracking of cardiac gene transfer and expression. We discuss limitations of current methods for analyzing gene transfer and describe how reporter gene imaging works.
View details for DOI 10.1038/ncpcardio1113
View details for Web of Science ID 000257868000013
View details for PubMedID 18641612
Intracellular and extracellular targets of molecular imaging in the myocardium
NATURE CLINICAL PRACTICE CARDIOVASCULAR MEDICINE
2008; 5: S33-S41
Utilization of molecular imaging has significantly advanced the field of cardiovascular medicine. In addition to the targets currently in use, novel targets are being developed, including those involved in the processes of myocardial metabolism, myocardial injury, cardiac neurotransmission, and interstitial dysregulation. Further development of these imaging targets may lead to improved characterization of disease processes and guide provision of individualized therapies.
View details for DOI 10.1038/ncpcardio1161
View details for Web of Science ID 000257868000006
View details for PubMedID 18641605
Transcriptome Alteration in the Diabetic Heart by Rosiglitazone: Implications for Cardiovascular Mortality
2008; 3 (7)
Recently, the type 2 diabetes medication, rosiglitazone, has come under scrutiny for possibly increasing the risk of cardiac disease and death. To investigate the effects of rosiglitazone on the diabetic heart, we performed cardiac transcriptional profiling and imaging studies of a murine model of type 2 diabetes, the C57BL/KLS-lepr(db)/lepr(db) (db/db) mouse.We compared cardiac gene expression profiles from three groups: untreated db/db mice, db/db mice after rosiglitazone treatment, and non-diabetic db/+ mice. Prior to sacrifice, we also performed cardiac magnetic resonance (CMR) and echocardiography. As expected, overall the db/db gene expression signature was markedly different from control, but to our surprise was not significantly reversed with rosiglitazone. In particular, we have uncovered a number of rosiglitazone modulated genes and pathways that may play a role in the pathophysiology of the increase in cardiac mortality as seen in several recent meta-analyses. Specifically, the cumulative upregulation of (1) a matrix metalloproteinase gene that has previously been implicated in plaque rupture, (2) potassium channel genes involved in membrane potential maintenance and action potential generation, and (3) sphingolipid and ceramide metabolism-related genes, together give cause for concern over rosiglitazone's safety. Lastly, in vivo imaging studies revealed minimal differences between rosiglitazone-treated and untreated db/db mouse hearts, indicating that rosiglitazone's effects on gene expression in the heart do not immediately turn into detectable gross functional changes.This study maps the genomic expression patterns in the hearts of the db/db murine model of diabetes and illustrates the impact of rosiglitazone on these patterns. The db/db gene expression signature was markedly different from control, and was not reversed with rosiglitazone. A smaller number of unique and interesting changes in gene expression were noted with rosiglitazone treatment. Further study of these genes and molecular pathways will provide important insights into the cardiac decompensation associated with both diabetes and rosiglitazone treatment.
View details for DOI 10.1371/journal.pone.0002609
View details for Web of Science ID 000264065800015
View details for PubMedID 18648539
Seeing is believing: tracking cells to determine the effects of cell transplantation.
Seminars in thoracic and cardiovascular surgery
2008; 20 (2): 102-109
Stem cell therapy holds promise as a therapeutic option for cardiovascular disease. As the field of cellular transplantation matures, novel methodologies are needed to longitudinally track and evaluate the functional effects of transplanted cells. At present, available techniques that hold the greatest promise for tracking cell fate include radionuclide labeling, ferromagnetic particle labeling, and genetic modification with reporter genes. This review describes the benefits and limitations of each technique and provides a summary of critical issues regarding stem cell transplantation that have been addressed by each imaging modality.
View details for DOI 10.1053/j.semtcvs.2008.04.002
View details for PubMedID 18707641
Shear stress influences spatial variations in vascular Mn-SOD expression: implication for LDL nitration
AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY
2008; 294 (6): C1576-C1585
Fluid shear stress modulates vascular production of endothelial superoxide anion (O2*-) and nitric oxide (*NO). Whether the characteristics of shear stress influence the spatial variations in mitochondrial manganese superoxide dismutase (Mn-SOD) expression in vasculatures is not well defined. We constructed a three-dimensional computational fluid dynamics model simulating spatial variations in shear stress at the arterial bifurcation. In parallel, explants of arterial bifurcations were sectioned from the human left main coronary bifurcation and right coronary arteries for immunohistolocalization of Mn-SOD expression. We demonstrated that Mn-SOD staining was prominent in the pulsatile shear stress (PSS)-exposed and atheroprotective regions, but it was nearly absent in the oscillatory shear stress (OSS)-exposed regions and lateral wall of arterial bifurcation. In cultured bovine aortic endothelial cells, PSS at mean shear stress (tau ave) of 23 dyn/cm2 upregulated Mn-SOD mRNA expression at a higher level than did OSS at tau ave = 0.02 dyn/cm2 +/- 3.0 dyn.cm(-2).s(-1) and at 1 Hz (PSS by 11.3 +/- 0.4-fold vs. OSS by 5.0 +/- 0.5-fold vs. static condition; P < 0.05, n = 4). By liquid chromatography and tandem mass spectrometry, it was found that PSS decreased the extent of low-density lipoprotein (LDL) nitration, whereas OSS increased nitration (P < 0.05, n = 4). In the presence of LDL, treatment with Mn-SOD small interfering RNA increased intracellular nitrotyrosine level (P < 0.5, n = 4), a fingerprint for nitrotyrosine formation. Our findings indicate that shear stress in the atheroprone versus atheroprotective regions regulates spatial variations in mitochondrial Mn-SOD expression with an implication for modulating LDL nitration.
View details for DOI 10.1152/ajpcell.00518.2007
View details for Web of Science ID 000256574900031
View details for PubMedID 18434620
- Can radionuclide imaging predict future response to stem cell therapy? JOURNAL OF NUCLEAR CARDIOLOGY 2008; 15 (3): 308-310
Imaging of VEGF receptor in a rat myocardial infarction model using PET
JOURNAL OF NUCLEAR MEDICINE
2008; 49 (4): 667-673
Myocardial infarction (MI) leads to left ventricular (LV) remodeling, which leads to the activation of growth factors such as vascular endothelial growth factor (VEGF). However, the kinetics of a growth factor's receptor expression, such as VEGF, in the living subject has not yet been described. We have developed a PET tracer (64Cu-DOTA-VEGF121 [DOTA is 1,4,7,10-tetraazadodecane-N,N',N'',N'''-tetraacetic acid]) to image VEGF receptor (VEGFR) expression after MI in the living subject.In Sprague-Dawley rats, MI was induced by ligation of the left coronary artery and confirmed by ultrasound (n = 8). To image and study the kinetics of VEGFRs, 64Cu-DOTA-VEGF121 PET scans were performed before MI induction (baseline) and on days 3, 10, 17, and 24 after MI. Sham-operated animals served as controls (n = 3).Myocardial origin of the 64Cu-DOTA-VEGF121 signal was confirmed by CT coregistration and autoradiography. VEGFR specificity of the 64Cu-DOTA-VEGF121 probe was confirmed by in vivo use of a 64Cu-DOTA-VEGFmutant. Baseline myocardial uptake of 64Cu-DOTA-VEGF121 was minimal (0.30 +/- 0.07 %ID/g [percentage injected dose per gram of tissue]); it increased significantly after MI (day 3, 0.97 +/- 0.05 %ID/g; P < 0.05 vs. baseline) and remained elevated for 2 wk (up to day 17 after MI), after which time it returned to baseline levels.We demonstrate the feasibility of imaging VEGFRs in the myocardium. In summary, we imaged and described the kinetics of 64Cu-DOTA-VEGF121 uptake in a rat model of MI. Studies such as the one presented here will likely play a major role when studying pathophysiology and assessing therapies in different animal models of disease and, potentially, in patients.
View details for DOI 10.2967/jnumed.107.040576
View details for Web of Science ID 000254813600028
View details for PubMedID 18375924
Comparison of reporter gene and iron particle labeling for tracking fate of human embryonic stem cells and differentiated endothelial cells in living subjects
2008; 26 (4): 864-873
Human embryonic stem (hES) cells are pluripotent stem cells capable of self-renewal and differentiation into virtually all cell types. Thus, they hold tremendous potential as cell sources for regenerative therapies. The concurrent development of accurate, sensitive, and noninvasive technologies capable of monitoring hES cells engraftment in vivo can greatly expedite basic research prior to future clinical translation. In this study, hES cells were stably transduced with a lentiviral vector carrying a novel double-fusion reporter gene that consists of firefly luciferase and enhanced green fluorescence protein. Reporter gene expression had no adverse effects on cell viability, proliferation, or differentiation to endothelial cells (human embryonic stem cell-derived endothelial cells [hESC-ECs]). To compare the two popular imaging modalities, hES cells and hESC-ECs were then colabeled with superparamagnetic iron oxide particles before transplantation into murine hind limbs. Longitudinal magnetic resonance (MR) imaging showed persistent MR signals in both cell populations that lasted up to 4 weeks. By contrast, bioluminescence imaging indicated divergent signal patterns for hES cells and hESC-ECs. In particular, hESC-ECs showed significant bioluminescence signals at day 2, which decreased progressively over the following 4 weeks, whereas bioluminescence signals from undifferentiated hES cells increased dramatically during the same period. Post-mortem histology and immunohistochemistry confirmed teratoma formation after injection of undifferentiated hES cells but not hESC-ECs. From these data taken together, we concluded that reporter gene is a better marker for monitoring cell viability, whereas iron particle labeling is a better marker for high-resolution detection of cell location by MR. Furthermore, transplantation of predifferentiated rather than undifferentiated hES cells would be more suited for avoiding teratoma formation.
View details for DOI 10.1634/stemcells.2007-0843
View details for Web of Science ID 000255000000003
View details for PubMedID 18218820
- Reporter gene imaging following percutaneous delivery in swine - Moving toward clinical applications JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY 2008; 51 (5): 595-597
In vitro and in vivo bioluminescence reporter gene imaging of human embryonic stem cells.
Journal of visualized experiments : JoVE
The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability. Molecular imaging has given investigators a high-throughput, inexpensive, and sensitive means for tracking in vivo cell proliferation over days, weeks, and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment, proliferation, and teratoma-formation in living subjects. A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes, under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery, are introduced into cells using a variety of vector and non-vector methods. Once in the cell, reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions, depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive, noninvasive instrumentation (e.g., CCD cameras) using signal-generating probes such as D-luciferin. To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging, bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase, derived from the firefly Photinus pyralis, encodes an enzyme that catalyzes D-luciferin to the optically active metabolite, oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells, allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore, because expression of the reporter gene product is required for signal generation, only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. In this video, the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described.
View details for DOI 10.3791/740
View details for PubMedID 19066577
Cardiac Differentiation of Embryonic Stem Cells with Point-Source Electrical Stimulation
2008 30TH ANNUAL INTERNATIONAL CONFERENCE OF THE IEEE ENGINEERING IN MEDICINE AND BIOLOGY SOCIETY, VOLS 1-8
The use of pluripotent stem cells as a means to repair damaged heart tissue has recently emerged as a promising, yet controversial therapy. Despite the different approaches and the variety of cell types used, many of these procedures have been met with mixed success. The lack of understanding of the differentiation and integration process, notably with respect to electrical signaling, significantly hampers the development of these therapies. A system was thus developed allowing the use of point source electrical stimulation on embryonic stem (ES) cells to study the effect of physiologically-relevant electrical stimulus. When modulating the amplitude of the stimulus over various differentiation stages of embryonic stem cells, differences in the proportions of cardiomyocytes to embryonic stem cells were observed through quantitative PCR. The use of this technique might have larger applications in understanding molecular pathways towards the regeneration process.
View details for Web of Science ID 000262404501031
View details for PubMedID 19163013
A novel platform device for rodent echocardiography.
2008; 49: E1-7
Acquisition of echocardiographic data from rodents is subject to wide variability due to variations in technique. We hypothesize that a dedicated imaging platform can aid in standardization of technique and improve the quality of images obtained. We constructed a device consisting of a boom-mounted steel platform frame (25 x 35 x 3 cm) on which a transparent polyethylene membrane is mounted. The animal is placed onto the membrane and receives continual inhaled anesthesia via an integrated port. The membrane allows for probe positioning from beneath the animal to obtain standard echo-views in left lateral decubitus or prone positions. The frame can be set at any desired angle ranging from 0 to 360 degrees along either the long or short axis. Adult male Sprague-Dawley rats (n = 5) underwent echocardiography (General Electric, Vivid 7, 14 MHz) using the platform. The device allowed for optimal positioning of animals for a variety of standard echocardiographic measurements. Evaluations among all animals showed minimal variability between two different operators and time points. We tested the feasibility of the device for supporting the assessment of cardiac function in a disease model by evaluating a separate cohort of adult male spontaneously hypertensive rats (n = 5) that underwent left anterior descending coronary artery ligation. Serial echocardiography demonstrated statistically significant decreases of fractional shortening and ejection fraction (p < 0.01) 240 days after surgery. Our novel imaging platform allowed for consistent collection of high-quality echocardiographic data from rats. Future studies will focus on improving this technology to allow for standardized high-throughput echocardiographic analysis in small animal models of disease.
View details for PubMedID 18506056
- Comparison of imaging techniques for tracking cardiac stem cell therapy JOURNAL OF NUCLEAR MEDICINE 2007; 48 (12): 1916-1919
Spatial and temporal kinetics, of teratoma formation from murine embryonic stem cell transplantation
STEM CELLS AND DEVELOPMENT
2007; 16 (6): 883-891
Pluripotent embryonic stem (ES) cells have the potential to form teratomas composed of derivatives from all three germ layers in animal models. This tumorigenic potential prevents clinical translation of ES cell research. In order to understand the biology and physiology of teratoma formation, we investigated the influence of undifferentiated ES cell number, migration, and long-term follow up after transplantation. Murine ES cells were stably transduced with a self-inactivating (SIN) lentiviral vector with a constitutive ubiquitin promoter driving a double-fusion (DF) reporter gene that consists of firefly luciferase and enhanced green fluorescent protein (Fluc-eGFP). To assess effects of cell numbers, varying numbers of ES-DF cells (1, 10, 100, 1,000, and 10,000) were injected subcutaneously into the dorsal regions of adult nude mice. To assess cell migration, 1 x 10(6) ES-DF cells were injected intramyocardially into adult Sv129 mice, and leakage to other extracardiac sites was monitored. To assess effects of long-term engraftment, 1 x 10(4) ES-DF cells were injected intramyocardially into adult nude rats, and cell survival response was monitored for 10 months. Our results show that ES-DF cells caused extracardiac teratoma in both immunocompetent and immunodeficient hosts; the lowest number of undifferentiated ES cells capable of causing teratoma was 500-1,000; and long-term engraftment could be shown for >300 days. Collectively, these results illustrate the potent tumorigenic potential of ES cells, which presents an enormous obstacle for future clinical studies.
View details for DOI 10.1089/scd.2007.0160
View details for Web of Science ID 000252031000002
View details for PubMedID 17896868
In vivo imaging and evaluation of different biomatrices for improvement of stem cell survival
JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE
2007; 1 (6): 465-468
Therapeutic effects from injection of stem cells are often hampered by acute donor cell death as well as migration away from damaged areas. This is likely due to the fact that injected cells do not have the physical and biochemical cues for ordered engrafment. Here we evaluate 3 common biomatrices (Matrigel, Collagen I, Purmatrix) that has the potential of providing suitable scaffolds needed to enhance stem cell survival. The longitudinal fate of transplanted stem cells was monitored by reporter imaging techniques.
View details for DOI 10.1002/term.55
View details for Web of Science ID 000256520300008
View details for PubMedID 18163533
Quantum dot imaging for embryonic stem cells
Semiconductor quantum dots (QDs) hold increasing potential for cellular imaging both in vitro and in vivo. In this report, we aimed to evaluate in vivo multiplex imaging of mouse embryonic stem (ES) cells labeled with Qtracker delivered quantum dots (QDs).Murine embryonic stem (ES) cells were labeled with six different QDs using Qtracker. ES cell viability, proliferation, and differentiation were not adversely affected by QDs compared with non-labeled control cells (P = NS). Afterward, labeled ES cells were injected subcutaneously onto the backs of athymic nude mice. These labeled ES cells could be imaged with good contrast with one single excitation wavelength. With the same excitation wavelength, the signal intensity, defined as (total signal-background)/exposure time in millisecond was 11 +/- 2 for cells labeled with QD 525, 12 +/- 9 for QD 565, 176 +/- 81 for QD 605, 176 +/- 136 for QD 655, 167 +/- 104 for QD 705, and 1,713 +/- 482 for QD 800. Finally, we have shown that QD 800 offers greater fluorescent intensity than the other QDs tested.In summary, this is the first demonstration of in vivo multiplex imaging of mouse ES cells labeled QDs. Upon further improvements, QDs will have a greater potential for tracking stem cells within deep tissues. These results provide a promising tool for imaging stem cell therapy non-invasively in vivo.
View details for DOI 10.1186/1472-6750-7-67
View details for Web of Science ID 000252448600001
View details for PubMedID 17925032
Differentiation, survival, and function of embryonic stem cell-derived endothelial cells for ischemic heart disease
2007; 116 (11): I46-I54
Embryonic stem (ES) cells are distinguished by their capacity for self-renewal and pluripotency. Here we characterize the differentiation of ES cell-derived endothelial cells (ESC-ECs), use molecular imaging techniques to examine their survival in vivo, and determine the therapeutic efficacy of ESC-ECs for restoration of cardiac function after ischemic injury.Murine ES cells were transfected with a construct composed of a vascular endothelial cadherin promoter driving enhanced green fluorescence protein (pVE-cadherin-eGFP). Differentiation of ES cells to ECs was detected by FACS analysis using Flk-1 (early EC marker at day 4) and VE-cadherin (late EC marker at day 8). After isolation, these ESC-ECs express endothelial cell markers similar to adult mouse lung endothelial cells, form vascular-like channels, and incorporate DiI-labeled acetylated low-density lipoprotein (DiI-Ac-LDL). For in vivo imaging, ES cells were transduced with an ubiquitin promoter driving firefly luciferase and monomeric red fluorescence protein (pUb-Fluc-mRFP). A robust correlation exists between Fluc signals and cell numbers by ex vivo imaging analysis (R2=0.98) and by in vitro enzyme assay (R2=0.94). Afterward, 5x10(5) ESC-ECs or PBS (as control) was injected into the hearts of mice undergoing LAD ligation (n=15 per group). Bioluminescence imaging showed longitudinal survival of transplanted ESC-ECs for approximately 8 weeks. Echocardiogram demonstrated significant functional improvement in the ESC-EC group compared with control (P=0.04). Finally, postmortem analysis confirmed increased presence of small capillaries and venules in the infarcted zones by CD31 staining.This is the first study to track the fate and function of transplanted ESC-ECs in the heart. With further validation, these ESC-ECs could become a valuable source of cell therapy for induction of angiogenesis in the treatment of myocardial ischemia.
View details for DOI 10.1161/CIRCULATIONAHA.106.680561
View details for Web of Science ID 000249364500007
View details for PubMedID 17846325
Cardiovascular molecular imaging
2007; 244 (2): 337-355
The goal of this review is to highlight how molecular imaging will impact the management and improved understanding of the major cardiovascular diseases that have substantial clinical impact and research interest. These topics include atherosclerosis, myocardial ischemia, myocardial viability, heart failure, gene therapy, and stem cell transplantation. Traditional methods of evaluation for these diseases will be presented first, followed by methods that incorporate conventional and molecular imaging approaches.
View details for Web of Science ID 000248821400005
View details for PubMedID 17592037
Positron emission tomography imaging of conditional gene activation in the heart
JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY
2007; 43 (1): 18-26
The Cre-loxP system has been routinely used for conditional activation and deletion of gene expression. However, the spatiotemporal manner of these events in the heart has not yet been defined by in vivo imaging. Adenovirus (1 x 10(9 )pfu) carrying the silent positron emission tomography (PET) reporter gene, herpes simplex virus type 1 thymidine kinase (HSV1-tk), was injected into the left ventricular wall of male transgenic mice (n=15) or FVB controls (n=8). Transgenic mice expressed Cre recombinase driven by a cardiac-specific alpha-myosin heavy chain (alpha-MHC) promoter. Following injection of the 9-[4-fluoro-3-(hydroxymethyl)butyl]guanine ([18F]-FHBG; 137+/-25 microCi) reporter probe, microPET imaging was used to assess the expression of HSV1-tk reporter gene in the myocardium. Two days following adenoviral injection, cardiac HSV1-tk gene activation resulted in tracer uptake of 3.20+/-0.51% ID/g for alpha-MHC-Cre and 0.05+/-0.02%ID/g for control mice (P<0.01). The in vivo results were confirmed by RT-PCR and Western blot analysis. Similar transfections were evaluated in both cardiac-specific and non-cardiac-specific cell lines. Enzyme activity showed a robust correlation (r2=0.82) between in vivo molecular imaging technique and traditional in vitro enzyme assays. With further development and validation, PET imaging will likely play an important role in the noninvasive, repetitive, and quantitative measurement of conditional gene activation in the future.
View details for DOI 10.1016/j.yjmcc.2007.03.809
View details for Web of Science ID 000248001400004
View details for PubMedID 17467733
Integration of genomics, proteomics, and imaging for cardiac stem cell therapy
EUROPEAN JOURNAL OF NUCLEAR MEDICINE AND MOLECULAR IMAGING
2007; 34: S20-S26
Cardiac stem cell therapy is beginning to mature as a valid treatment for heart disease. As more clinical trials utilizing stem cells emerge, it is imperative to establish the mechanisms by which stem cells confer benefit in cardiac diseases. In this paper, we review three methods - molecular cellular imaging, gene expression profiling, and proteomic analysis - that can be integrated to provide further insights into the role of this emerging therapy.
View details for DOI 10.1007/s00259-007-0437-y
View details for Web of Science ID 000253384100003
View details for PubMedID 17464506
Magnetic resonance imaging of progressive cardiomyopathic changes in the db/db mouse
AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY
2007; 292 (5): H2106-H2118
The db/db mouse is a well-established model of diabetes. Previous reports have documented contractile dysfunction (i.e., cardiomyopathy) in these animals, although the extant literature provides limited insights into cardiac structure and function as they change over time. To better elucidate the natural history of cardiomyopathy in db/db mice, we performed cardiac magnetic resonance (CMR) scans on these animals. CMR imaging was conducted with a 4.7-T magnet on female db/db mice and control db/+ littermates at 5, 9, 13, 17, and 22 wk of age. Gated gradient echo sequences were used to obtain cineographic short-axis slices from apex to base. From these images left ventricular (LV) mass (LVM), wall thickness, end-diastolic volume (LVEDV), and ejection fraction (LVEF) were determined. Additionally, cardiac [(18)F]fluorodeoxyglucose ([(18)F]FDG) PET scanning, pressure-volume loops, and real-time quantitative PCR on db/db myocardium were performed. Relative to control, db/db mice developed significant increases in LVM and wall thickness as early as 9 wk of age. LVEDV diverged slightly later, at 13 wk. Interestingly, compared with the baseline level, LVEF in the db/db group did not decrease significantly until 22 wk. Additionally, [(18)F]FDG metabolic imaging showed a 40% decrease in glucose uptake in db/db mice. Furthermore, contractile dysfunction was observed in 15-wk db/db mice undergoing pressure-volume loops. Finally, real-time quantitative PCR revealed an age-dependent recapitulation of the fetal gene program, consistent with a myopathic process. In summary, as assessed by CMR, db/db mice develop characteristic structural and functional changes consistent with cardiomyopathy.
View details for DOI 10.1152/ajpheart.00856.2006
View details for Web of Science ID 000247777200012
View details for PubMedID 17122193
An unusual cause of stroke from a left atrial mass.
Journal of the American Society of Echocardiography
2007; 20 (5): 537 e1-2
A 59-year-old woman was admitted to the hospital after having severe headache for 1 day. Her medical history was significant for coronary artery disease, diabetes, atrial fibrillation, and rheumatic heart disease. The patient was in remission from stage II left breast cancer (T2 N0 M0) in 1997, treated with lumpectomy, 6 cycles of chemotherapy (cyclophosphamide, methotrexate, and 5-fluorouracil), and local radiation therapy (total 6000 cGy). Head magnetic resonance imaging scan showed left occipital hemorrhage. Subsequent angiogram showed a possible mycotic aneurysm of distal parietal occipital branch of the posterior cerebral artery. Cardiology was consulted to evaluate for a cardioembolic source.
View details for PubMedID 17484995
Molecular imaging of embryonic stem cell misbehavior and suicide gene ablation
CLONING AND STEM CELLS
2007; 9 (1): 107-117
Numerous studies have demonstrated the potential use of stem cells for the repair and regeneration of injured tissues. However, tracking transplanted stem cell fate and function in vivo remains problematic. To address these issues, murine embryonic stem (ES) cells were stably transduced with self-inactivating lentiviral vectors carrying either a triple fusion (TF) or double fusion (DF) reporter gene construct. The TF consisted of monomeric red fluorescence protein (mrfp), firefly luciferase (Fluc), and herpes simplex virus truncated thymidine kinase (HSV-ttk) reporter genes. The DF consisted of enhanced green fluorescence protein (egfp) and Fluc reporter genes but lacked HSV-ttk. Stably transduced ES-TF or ES-DF cells were selected by fluorescence activated cell sorting based on either mrfp (TF) or egfp (DF) expression. Afterwards, cells were injected subcutaneously into the right (ES-TF cells) and left (ES-DF cells) shoulders of adult female nude mice. Cell survival was tracked noninvasively by bioluminescence and positron emission tomography imaging of Fluc and HSV-ttk reporter genes, respectively. Imaging signals progressively increased from day 2 to day 14, consistent with ES cell survival and proliferation in vivo. However, teratoma formation occurred in all nude mice after 5 weeks. Administration of ganciclovir (GCV), targeting the HSV-ttk gene, resulted in selective ablation of teratomas arising from the ES-TF cells but not ES-DF cells. These data demonstrate the novel use of multimodality imaging techniques to (1) monitor transplanted ES cell survival and proliferation in vivo and (2) assess the efficacy of suicide gene therapy as a backup safety measure against teratoma formation.
View details for DOI 10.1089/clo.2006.0016
View details for Web of Science ID 000245390300015
View details for PubMedID 17386018
Clinical hurdles for the transplantation of cardiomyocytes derived from human embryonic stem cells: role of molecular imaging
CURRENT OPINION IN BIOTECHNOLOGY
2007; 18 (1): 38-45
Over the past few years, human embryonic stem cells (hESCs) have gained popularity as a potentially ideal cell candidate for tissue regeneration. In particular, hESCs are capable of cardiac lineage-specific differentiation and confer improvement of cardiac function following transplantation into animal models. Although such data are encouraging, there remain significant hurdles before safe and successful translation of hESC-based treatment into clinical therapy, including the ability to assess cells following transplant. To this end, molecular imaging has proven a reliable methodology for tracking the long-term fate of transplanted cells. Imaging reporter genes that are introduced into the cells before transplantation enable non-invasive and longitudinal studies of cell viability, location and behaviour in vivo. Therefore, molecular imaging is expected to play an increasing role in characterizing the biology and physiology of hESC-derived cardiac cells in living subjects.
View details for DOI 10.1016/j.copbio.2006.12.003
View details for Web of Science ID 000244593000007
View details for PubMedID 17196814
Clinical dilemmas in treating left ventricular thrombus.
International journal of cardiology
2007; 114 (3): e118-9
View details for PubMedID 17049652
Molecular Imaging of bone marrow mononuclear cell homing and engraftment in ischemic myocardium
2007; 25 (10): 2677-2684
Bone marrow mononuclear cell (BMMC) therapy shows promise as a treatment for ischemic heart disease. However, the ability to monitor long-term cell fate remains limited. We hypothesized that molecular imaging could be used to track stem cell homing and survival after myocardial ischemia-reperfusion (I/R) injury. We first harvested donor BMMCs from adult male L2G85 transgenic mice constitutively expressing both firefly luciferase (Fluc) and enhanced green fluorescence protein reporter gene. Fluorescence-activated cell sorting analysis revealed approximately 0.07% of the population to consist of classic hematopoietic stem cells (lin-, thy-int, c-kit+, Sca-1+). Afterward, adult female FVB recipients (n = 38) were randomized to sham surgery or acute I/R injury. Animals in the sham (n = 16) and I/R (n = 22) groups received 5 x 10(6) of the L2G85-derived BMMCs via tail vein injection. Bioluminescence imaging (BLI) was used to track cell migration and survival in vivo for 4 weeks. BLI showed preferential homing of BMMCs to hearts with I/R injury compared with sham hearts within the first week following cell injection. Ex vivo analysis of explanted hearts by histology confirmed BLI imaging results, and quantitative real-time polymerase chain reaction (for the male Sry gene) further demonstrated a greater number of BMMCs in hearts with I/R injury compared with the sham group. Functional evaluation by echocardiography demonstrated a trend toward improved left ventricular fractional shortening in animals receiving BMMCs. Taken together, these data demonstrate that molecular imaging can be used to successfully track BMMC therapy in murine models of heart disease. Specifically, we have demonstrated that systemically delivered BMMCs preferentially home to and are retained by injured myocardium. Disclosure of potential conflicts of interest is found at the end of this article.
View details for DOI 10.1634/stemcells.2007-0041
View details for Web of Science ID 000249929900031
View details for PubMedID 17628019
Genetic modification of embryonic stem cells with VEGF enhances cell survival and improves cardiac function
CLONING AND STEM CELLS
2007; 9 (4): 549-563
Cardiac stem cell therapy remains hampered by acute donor cell death posttransplantation and the lack of reliable methods for tracking cell survival in vivo. We hypothesize that cells transfected with inducible vascular endothelial growth factor 165 (VEGF(165)) can improve their survival as monitored by novel molecular imaging techniques. Mouse embryonic stem (ES) cells were transfected with an inducible, bidirectional tetracycline (Bi-Tet) promoter driving VEGF(165) and renilla luciferase (Rluc). Addition of doxycycline induced Bi-Tet expression of VEGF(165) and Rluc significantly compared to baseline (p<0.05). Expression of VEGF(165) enhanced ES cell proliferation and inhibited apoptosis as determined by Annexin-V staining. For noninvasive imaging, ES cells were transduced with a double fusion (DF) reporter gene consisting of firefly luciferase and enhanced green fluorescence protein (Fluc-eGFP). There was a robust correlation between cell number and Fluc activity (R(2)=0.99). Analysis by immunostaining, histology, and RT-PCR confirmed that expression of Bi-Tet and DF systems did not affect ES cell self-renewal or pluripotency. ES cells were differentiated into beating embryoid bodies expressing cardiac markers such as troponin, Nkx2.5, and beta-MHC. Afterward, 5 x 10(5) cells obtained from these beating embryoid bodies or saline were injected into the myocardium of SV129 mice (n=36) following ligation of the left anterior descending (LAD) artery. Bioluminescence imaging (BLI) and echocardiography showed that VEGF(165) induction led to significant improvements in both transplanted cell survival and cardiac function (p<0.05). This is the first study to demonstrate imaging of embryonic stem cell-mediated gene therapy targeting cardiovascular disease. With further validation, this platform may have broad applications for current basic research and further clinical studies.
View details for DOI 10.1089/clo.2007.0032
View details for Web of Science ID 000252027800010
View details for PubMedID 18154515
Proteomic analysis of reporter genes for molecular imaging of transplanted embryonic stem cells
2006; 6 (23): 6234-6249
Study of stem cells may reveal promising treatment for diseases. The fate and function of transplanted stem cells remain poorly defined. Recent studies demonstrate that reporter genes can monitor real-time survival of transplanted stem cells in living subjects. We examined the effects of a novel and versatile triple fusion (TF) reporter gene construction on embryonic stem (ES) cell function by proteomic analysis. Murine ES cells were stably transduced with a self-inactivating lentiviral vector containing fluorescence (firefly luciferase; Fluc), bioluminescence (monomeric red fluorescence protein; mRFP), and positron emission tomography (herpes simplex virus type 1 truncated thymidine kinase; tTK) reporter genes. Fluorescence-activated cell sorting (FACS) analysis isolated stably transduced populations. TF reporter gene effects on cellular function were evaluated by quantitative proteomic profiling of control ES cells versus ES cells stably expressing the TF construct (ES-TF). Overall, no significant changes in protein quantity were observed. TF reporter gene expression had no effect on ES cell viability, proliferation, and differentiation capability. Molecular imaging studies tracked ES-TF cell survival and proliferation in living animals. In summary, this is the first proteomic study, demonstrating the unique potential of reporter gene imaging for tracking ES cell transplantation non-invasively, repetitively, and quantitatively.
View details for DOI 10.1002/pmic.200600150
View details for Web of Science ID 000242879000011
View details for PubMedID 17080479
Molecular imaging of human embryonic stem cells - Keeping an eye on differentiation, tumorigenicity and immunogenicity
2006; 5 (23): 2748-2752
Human embryonic stem cells (hESCs) are capable of differentiation into every cell type of the human being. They are under extensive investigation for their regenerative potential in a variety of debilitating diseases. However, the field of hESC research is still in its infancy, as there are several critical issues that need to be resolved before clinical translation. Two major concerns are the ability of undifferentiated hESCs to form teratomas and the possibility of a provoked immune reaction after transplantation of hESCs into a new host. Therefore, it is imperative to develop noninvasive imaging modalities that allow for longitudinal, repetitive, and quantitative assessment of transplanted cell survival, proliferation, and migration in vivo. Reporter gene-based molecular imaging offers these characteristics and has great potential in the field of stem cell therapy. Moreover, it has recently been shown that reporter gene imaging can be combined with therapeutic strategies. Here, we provide an outline of the current status of hESC research and discuss the concerns of tumorigenicity and immunogenicity. Furthermore, we describe how molecular imaging can be utilized to follow and resolve these issues.
View details for Web of Science ID 000242897800011
View details for PubMedID 17172859
Right coronary cameral fistula resulting from surgery of double chamber right ventricle.
Journal of the American Society of Echocardiography
2006; 19 (9): 1191 e9-11
View details for PubMedID 16950481
Overview of stem cells and imaging modalities for cardiovascular diseases
JOURNAL OF NUCLEAR CARDIOLOGY
2006; 13 (4): 554-569
Stem cell therapy is emerging as a promising approach to treat heart diseases. Considerable evidence from experimental studies and initial clinical trials suggests that stem cell transplantation promotes systolic function and prevent ventricular remodeling. However, the specific mechanisms by which stem cells improve heart function remain largely unknown. In addition, interpreting the long-term effects of stem cell therapy is difficult because of the limitations of conventional techniques. The recent development of molecular imaging techniques offers great potential to address these critical issues by noninvasively tracking the fate of the transplanted cells. This review offers a focused discussion on the use of stem cell therapy and imaging in the context of cardiology.
View details for DOI 10.1016/j.nuclcard.2006.05.012
View details for Web of Science ID 000239736700014
View details for PubMedID 16919579
Noninvasive evaluation of immunosuppressive drug efficacy on acute donor cell survival
MOLECULAR IMAGING AND BIOLOGY
2006; 8 (3): 163-170
The therapeutic benefits of cell transplantation may depend on the survival of sufficient numbers of grafted cells. We evaluate four potent immunosuppressive medications aimed at preventing acute donor cell death. PROCEDURES AND RESULTS: Embryonic rat H9c2 myoblasts were stably transduced to express firefly luciferase reporter gene (H9c2-Fluc). H9c2-Fluc cells (3x10(6)) were injected into thigh muscles of Sprague-Dawley rats (N=30) treated with cyclosporine, dexamethasone, mycophenolate mofetil, tacrolimus, or saline from day -3 to day +14. Longitudinal optical bioluminescence imaging was performed over two weeks. Fluc activity was 40.0+/-12.1% (dexamethasone), 30.5+/-12.5% (tacrolimus), and 21.5+/-3.5% (mycophenolate) vs. 12.0+/-5.0% (control) and 8.3+/-5.0% (cyclosporine) at day 4 (P<0.05). However, by day 14, cell signals had decreased drastically to <10% for all groups despite drug therapy.This study demonstrates the ability of optical molecular imaging for tracking cell survival noninvasively and raises important questions with regard to the overall efficacy of immunosuppressives for prolonging transplanted cell survival.
View details for DOI 10.1007/s11307-006-0038-3
View details for Web of Science ID 000237754300003
View details for PubMedID 16555032
In vivo bioluminescence imaging of cord blood derived mesenchymal stem cell transplantation into rat myocardium
ANNALS OF NUCLEAR MEDICINE
2006; 20 (3): 165-170
The conventional method for the analysis of myocardial cell transplantation depends on postmortem histology. Here, we have sought to demonstrate the feasibility of a longitudinal monitoring of transplanted cell survival in living animals, accomplished with optical imaging techniques and pharmacological interventions.Human cord blood (50 ml) was donated with parental consent. After getting cord blood derived mesenchymal stem cells (CBMSCs), cells were transfected (MOI = 100) overnight with adenovirus encoding firefly luciferase gene (Ad-CMV-Fluc). Our experimental Sprague-Dawley rats (n = 12) were given intramyocardial injections containing 1 x 10(6) CBMSCs, which had been made to express the firefly luciferase (Fluc) reporter gene. Optical bioluminescence imaging was then conducted using a cooled charged-coupled device (CCD) camera (Xenogen), beginning on the day after the transplantation (day 1). Groups of mice were intraperitoneally injected with cyclosporine (5 mg/kg) or tacrolimus (1 mg/kg), in an attempt to determine the degree to which cell survival had been prolonged, and these values were then compared with the cell survival values of the negative control group. The presence of transplanted CBMSCs on in vivo images confirmed by in situ hybridization for human specific Alu in the myocardium.Cardiac bioluminescence signals were determined to be present for 6 days after transplantation: day 1 (97000 +/- 9100 x 10(5) p/s/cm2/sr), day 3 (9600 +/- 1110 p/s/cm2/sr), and day 5 (3200 +/- 550 p/s/cm2/sr). The six mice that received either cyclosporine or tacrolimus displayed cardiac bioluminescence signals for a period of 8 days after transplantation. We observed significant differences between the treated group and the non-treated group, beginning on day 3 (tacrolimus; 26500 +/- 4340 p/s/cm2/sr, cyclosporine; 27200 +/- 3340 p/s/cm2/sr, non-treated; 9630 +/- 1180 p/s/cm2/sr, p < 0.01), and persisting until day 7 (tacrolimus; 12500 +/- 2946 p/s/cm2/sr, cyclosporine; 7310 +/- 1258 p/s/cm2/sr, non-treated; 2460 +/- 160 p/s/cm2/sr, p < 0.01). The human-derived CBMSCs were detected in the myocardium 3 days after transplantation by in situ hybridization.The locations, magnitude, and survival duration of the CBMSCs were noninvasively monitored with a bioluminescence optical imaging system. We determined that optical molecular imaging expedites the fast throughput screening of pharmaceutical agents, allowing for the noninvasive tracking of cell survival within animals. In rat cardiac CBMSC transplant models, transient immunosuppressive treatment with tacrolimus or cyclosporine was shown to improve donor cell survival.
View details for Web of Science ID 000237375100001
View details for PubMedID 16715945
Transcriptional profiling of reporter genes used for molecular imaging of embryonic stem cell transplantation
2006; 25 (1): 29-38
Stem cell therapy offers exciting promise for treatment of ischemic heart disease. Recent advances in molecular imaging techniques now allow investigators to monitor cell fate noninvasively and repetitively. Here we examine the effects of a triple-fusion reporter gene on embryonic stem (ES) cell transcriptional profiles. Murine ES cells were stably transfected with a self-inactivating lentiviral vector carrying a triple-fusion (TF) construct consisting of fluorescence, bioluminescence, and positron emission tomography (PET) reporter genes. Fluorescence-activated cell sorting (FACS) analysis allowed isolation of stably transfected populations. Microarray studies comparing gene expression in nontransduced control ES cells vs. stably transduced ES cells expressing triple fusion (ES-TF) revealed some increases in transcriptional variability. Annotation analysis showed that ES-TF cells downregulated cell cycling, cell death, and protein and nucleic acid metabolism genes while upregulating homeostatic and anti-apoptosis genes. Despite these transcriptional changes, expression of the TF reporter gene had no significant effects on ES cell viability, proliferation, and differentiation capability. Importantly, transplantation studies in murine myocardium demonstrated the feasibility of tracking ES-TF cells in living subjects using bioluminescence and PET imaging. Taken together, this is the first study to analyze in detail the effects of reporter genes on molecular imaging of ES cells.
View details for DOI 10.1152/physiolgenomics.00254.2005
View details for Web of Science ID 000236722700004
View details for PubMedID 16390873
Molecular imaging of cardiac stem cell transplantation.
Current cardiology reports
2006; 8 (2): 147-154
In recent years, stem cell therapy for the treatment of heart disease has translated from the imagination of investigators to the bedside of patients. The initial results from trials evaluating cell therapy for the heart are encouraging. As this new field of cellular transplantation matures, it is imperative that novel methodologies for evaluating cell therapy are developed and applied to guide therapy. Molecular imaging is a discipline that is evolving to address these needs and is expected to play an increasing role in the characterization and assessment of cell therapy. This article provides a focused overview of clinical stem cell therapy for the heart, followed by a discussion of how novel molecular imaging techniques are presently being applied to monitor cell therapy.
View details for PubMedID 16524542
F-18-labeled bombesin analogs for targeting GRP receptor-expressing prostate cancer
JOURNAL OF NUCLEAR MEDICINE
2006; 47 (3): 492-501
The gastrin-releasing peptide receptor (GRPR) is found to be overexpressed in a variety of human tumors. The aim of this study was to develop 18F-labeled bombesin analogs for PET of GRPR expression in prostate cancer xenograft models.[Lys3]Bombesin ([Lys3]BBN) and aminocaproic acid-bombesin(7-14) (Aca-BBN(7-14)) were labeled with 18F by coupling the Lys3 amino group and Aca amino group, respectively, with N-succinimidyl-4-18F-fluorobenzoate (18F-SFB) under slightly basic condition (pH 8.5). Receptor-binding affinity of FB-[Lys3]BBN and FB-Aca-BBN(7-14) was tested in PC-3 human prostate carcinoma cells. Internalization and efflux of both radiotracers were also evaluated. Tumor-targeting efficacy and in vivo kinetics of both radiotracers were examined in male athymic nude mice bearing subcutaneous PC-3 tumors by means of biodistribution and dynamic microPET imaging studies. 18F-FB-[Lys3]BBN was also tested for orthotopic PC-3 tumor delineation. Metabolic stability of 18F-FB-[Lys3]BBN was determined in mouse blood, urine, liver, kidney, and tumor homogenates at 1 h after injection.The typical decay-corrected radiochemical yield was about 30%-40% for both tracers, with a total reaction time of 150 +/- 20 min starting from 18F-. 18F-FB-[Lys3]BBN had moderate stability in the blood and PC-3 tumor, whereas it was degraded rapidly in the liver, kidneys, and urine. Both radiotracers exhibited rapid blood clearance. 18F-FB-[Lys3]BBN had predominant renal excretion. 18F-FB-Aca-BBN(7-14) exhibited both hepatobiliary and renal clearance. Dynamic microPET imaging studies revealed that the PC-3 tumor uptake of 18F-FB-[Lys3]BBN in PC-3 tumor was much higher than that of 18F-FB-Aca-BBN(7-14) at all time points examined (P < 0.01). The receptor specificity of 18F-FB-[Lys3]BBN in vivo was demonstrated by effective blocking of tumor uptake in the presence of [Tyr4]BBN. No obvious blockade was found in PC-3 tumor when 18F-FB-Aca-BBN(7-14) was used as radiotracer under the same condition. 18F-FB-[Lys3]BBN was also able to visualize orthotopic PC-3 tumor at early time points after tracer administration, at which time minimal urinary bladder activity was present to interfere with the receptor-mediated tumor uptake.This study demonstrates that 18F-FB-[Lys3]BBN and PET are suitable for detecting GRPR-positive prostate cancer in vivo.
View details for Web of Science ID 000249695800020
View details for PubMedID 16513619
In vivo visualization of embryonic stem cell survival, proliferation, and migration after cardiac delivery
2006; 113 (7): 1005-1014
Recent studies have shown that stem cell therapy can promote tissue regeneration; however, monitoring stem cells in vivo remains problematic owing to limitations of conventional histological assays and imaging modalities.Murine embryonic stem (ES) cells were stably transduced with a lentiviral vector carrying a novel triple-fusion (TF) reporter gene that consists of firefly luciferase, monomeric red fluorescence protein, and truncated thymidine kinase (fluc-mrfp-ttk). ES cell viability, proliferation, and differentiation ability were not adversely affected by either reporter genes or reporter probes compared with nontransduced control cells (P=NS). Afterward, 1x10(7) of ES cells carrying the TF reporter gene (ES-TF) were injected into the myocardium of adult nude rats (n=20). Control animals received nontransduced ES cells (n=6). At day 4, the bioluminescence and positron emission tomography signals in study animals were 3.7x10(7)+/-5.8x10(6) photons.s(-1).cm(-2) per steradian (sr) and 0.08+/-0.03% injected dose/g, respectively (P<0.05 versus control). Both signals increased progressively from week 1 to week 4, which indicated ES cell survival and proliferation in the host. Histological analysis demonstrated the formation of intracardiac and extracardiac teratomas. Finally, animals (n=4) that were treated with intraperitoneal injection of ganciclovir (50 mg/kg) did not develop teratomas when compared with control animals (n=4) treated with saline (1 mL/kg).This is the first study to characterize ES cells that stably express fluorescence, bioluminescence, and positron emission tomography reporter genes and monitor the kinetics of ES cell survival, proliferation, and migration. This versatile imaging platform should have broad applications for basic research and clinical studies on stem cell therapy.
View details for DOI 10.1161/CIRCULATIONHA.105.588954
View details for Web of Science ID 000235403900015
View details for PubMedID 16476845
Imaging chemically modified adenovirus for targeting tumors expressing integrin alpha(v)beta(3) in living mice with mutant herpes simplex virus type 1 thymidine kinase PET reporter gene
JOURNAL OF NUCLEAR MEDICINE
2006; 47 (1): 130-139
The aim of this study was to change adenovirus tropism by chemical modification of the fiber knobs with PEGylated RGD peptide for targeting integrin alpha(v)beta(3) that is uniquely or highly expressed in tumor cells and neovasculature of tumors of various origins.The first generation Ad (Ad) vector, which expresses the herpes simplex virus type 1 mutant thymidine kinase (HSV1-sr39tk) gene under the control of cytomegalovirus (CMV) promoter was conjugated with poly(ethylene glycol) (PEG) or RGD-PEG. The transduction efficiency of Ads (Adtk, PEG-Adtk, and RGD-PEG-Adtk) into different types of cells (293T, MCF7, MDA-MB-435, and U87MG) was analyzed and quantified by thymidine kinase (TK) assay using 8-(3)H-penciclovir (8-(3)H-PCV) as substrate. The in vivo infectivity of the Ad vectors after intravenous administration into integrin alpha(v)beta(3)-positive U87MG and MDA-MB-435 tumor-bearing athymic nude mice was measured by both noninvasive microPET using 9-[4-(18)F-fluoro-3-(hydroxymethyl)butyl]guanine ((18)F-FHBG) as a reporter probe and ex vivo TK assay of the tumor and tissue homogenates.PEGylation completely abrogated coxsackievirus and adenovirus receptor (CAR)-knob interaction and the infectivity of PEG-Adtk is significantly lower than that of unmodified Adtk in CAR-positive cells. RGD-PEG-modified virus (RGD-PEG-Adtk) had significantly higher infectivity than PEG-Adtk and the extent of increase is related to both CAR and integrin alpha(v)beta(3) expression levels. (18)F-FHBG had minimal nonspecific uptake in the liver and tumors that are void of sr39tk. Mice preinjected intravenously with unmodified Adtk resulted in high hepatic uptake and moderate tumor accumulation of the tracer. In contrast, RGD-PEG-Adtk administration resulted in significantly lower liver uptake without compromising the tumor accumulation of (18)F-FHBG. Expression of TK in the liver and tumor homogenates corroborated with the magnitude of (18)F-FHBG uptake quantified by noninvasive microPET. Analysis of liver and tumor tissue integrin level confirmed that RGD-integrin interaction is responsible for the enhanced tumor infectivity of RGD-PEG-Adtk.The results of this study suggest that RGD-PEG conjugation is an effective way to modify Ad vector tropism for improved systemic gene delivery. Noninvasive PET and (18)F-FHBG are able to monitor in vivo transfectivity of both Adtk and RGD-PEG-Adtk vectors in the liver and tumors after intravenous injection.
View details for Web of Science ID 000234679300024
View details for PubMedID 16391197
Effects of epigenetic modulation on reporter gene expression: implications for stem cell imaging.
2006; 20 (1): 106-108
Tracking stem cell localization, survival, differentiation, and proliferation after transplantation in living subjects is essential for understanding stem cell biology and physiology. In this study, we investigated the long-term stability of reporter gene expression in an embryonic rat cardiomyoblast cell line and the role of epigenetic modulation on reversing reporter gene silencing. Cells were stably transfected with plasmids carrying cytomegalovirus promoter driving firefly luciferase reporter gene (CMV-Fluc) and passaged repeatedly for 3-8 months. Within the highest expressor clone, the firefly luciferase activity decreased progressively from passage 1 (843+/-28) to passage 20 (250+/-10) to passage 40 (44+/-3) to passage 60 (3+/-1 RLU/microg; P<0.05 vs. passage 1). Firefly luciferase activity was maximally rescued by treatment with 5-azacytidine (DNA methyltransferase inhibitor) compared with trichostatin A (histone deacetylase inhibitor) and retinoic acid (transcriptional activator; P<0.05). Increasing dosages of 5-azacytidine treatment led to higher levels of firefly luciferase mRNA (RT-PCR) and protein (Western blots) and inversely lower levels of methylation in the CMV promoter (DNA nucleotide sequence). These in vitro results were extended to in vivo bioluminescence imaging (BLI) of cell transplant in living animals. Cells treated with 5-azacytidine were monitored for 2 wk compared with 1 wk for untreated cells (P<0.05). These findings should have important implications for reporter gene-based imaging of stem cell transplantation.
View details for PubMedID 16246867
Image-guided cardiac cell delivery using high-resolution small-animal ultrasound
2005; 12 (6): 1142-1147
Open-chest cardiac injection is the preferred delivery method for cardiac gene and stem cell therapy in small animals, but it is invasive and the operator is unable to see the actual delivery site. High-resolution ultrasound has recently been developed for small-animal imaging. We tested the hypothesis that image-guided cardiac cell delivery using high-resolution ultrasound guidance is feasible and reproducible. Sprague-Dawley rats (n = 11) were imaged using high-resolution ultrasound, and stably transfected cardiomyoblasts (plasmid-CMV-firefly luciferase) were injected into the anterior cardiac wall under ultrasound guidance (parasternal long-axis view), using a 28-gauge needle. After injection, bioluminescence imaging was performed using a cooled charged-coupled camera. Injection was successful in all animals and was associated with no mortality. The signal detected was positively correlated with the amount of cells transplanted (R(2) = 0.94, P = 0.03) and highly correlated with ex vivo assays (R(2) = 0.82). In addition, the optical signal could be followed longitudinally using bioluminescence imaging. Ultrasound image-guided cardiac cell delivery is an effective, safe, and reproducible way to perform cell delivery to a specific myocardial region and can be combined with assessment of cardiac function. We are confident that the use of these technologies will play a significant role in the future of gene and cell therapy.
View details for DOI 10.1016/j.ymthe.2005.07.532
View details for Web of Science ID 000233864700017
View details for PubMedID 16111921
Human gene therapy and imaging: cardiology
EUROPEAN JOURNAL OF NUCLEAR MEDICINE AND MOLECULAR IMAGING
2005; 32: S346-S357
This review discusses the basics of cardiovascular gene therapy, the results of recent human clinical trials, and the rapid progress in imaging techniques in cardiology. Improved understanding of the molecular and genetic basis of coronary heart disease has made gene therapy a potential new alternative for the treatment of cardiovascular diseases. Experimental studies have established the proof-of-principle that gene transfer to the cardiovascular system can achieve therapeutic effects. First human clinical trials provided initial evidence of feasibility and safety of cardiovascular gene therapy. However, phase II/III clinical trials have so far been rather disappointing and one of the major problems in cardiovascular gene therapy has been the inability to verify gene expression in the target tissue. New imaging techniques could significantly contribute to the development of better gene therapeutic approaches. Although the exact choice of imaging modality will depend on the biological question asked, further improvement in image resolution and detection sensitivity will be needed for all modalities as we move from imaging of organs and tissues to imaging of cells and genes.
View details for DOI 10.1007/s00259-005-1897-6
View details for Web of Science ID 000203011200003
View details for PubMedID 16096829
- Effects of epigenetic modulation on reporter gene expression: implications for stem cell imaging FASEB JOURNAL 2005; 19 (12): 106-?
- Common congenital heart disorders in adults CURRENT PROBLEMS IN CARDIOLOGY 2004; 29 (11): 641-700
Molecular imaging of the kinetics of vascular endothelial growth factor gene expression in ischemic myocardium
2004; 110 (6): 685-691
Angiogenic gene therapy is a promising treatment paradigm for patients with ischemic heart disease. In this study, we used micro-positron emission tomography (microPET) to monitor the transgene expression, function, and effects in a whole-body system.Adenovirus with cytomegalovirus promoter driving an angiogenic gene (vascular endothelial growth factor [VEGF]) linked to a PET reporter gene (herpes simplex virus type 1 mutant thymidine kinase; Ad-CMV-VEGF121-CMV-HSV1-sr39tk) was used to transfect rat embryonic cardiomyoblasts in vitro. Expression of both genes correlated strongly (r=0.98; P<0.001). Afterward, rats underwent ligation of the left anterior descending artery followed by injection of 1x10(10) pfu of Ad-CMV-VEGF121-CMV-HSV1-sr39tk (study; n=35) or Ad-null (control; n=15) at the peri-infarct region. Noninvasive microPET imaging was used to assess the uptake of 9-(4-[18F]-fluoro-hydroxymethylbutyl)guanine ([18F]-FHBG) PET reporter probe by cells expressing the HSV1-sr39tk PET reporter gene. Cardiac transgene expression peaked at day 1 and declined over the next 2 weeks. Repeat adenoviral injections at day 60 yielded no detectable signal. The in vivo reporter gene expression (% injected dose/g of [18F]-FHBG) correlated well with ex vivo gamma counting (r=0.92), myocardial tissue HSV1-sr39TK enzyme activity (r=0.95), and myocardial tissue VEGF level (r=0.94; P<0.001 for all). The VEGF121 isoform induced significant increases in capillaries and small blood vessels. However, the level of neovasculature did not translate into significant improvements in functional parameters such as myocardial contractility by echocardiography, perfusion by nitrogen-13 ammonia imaging, and metabolism by [18F]-fluorodeoxyglucose imaging.Taken together, these findings establish the feasibility of molecular imaging for monitoring angiogenic gene expression with a PET reporter gene and probe noninvasively, quantitatively, and repetitively. The principles demonstrated here can be used to evaluate other therapeutic genes of interest in animal models before future clinical trials are initiated.
View details for DOI 10.1161/01.CIR.0000138153.02213.22
View details for Web of Science ID 000223194700008
View details for PubMedID 15302807
- Molecular imaging of cardiovascular gene products JOURNAL OF NUCLEAR CARDIOLOGY 2004; 11 (4): 491-505
Cardiac-Specific Gene Expression Facilitated by an Enhanced Myosin Light Chain Promoter
2004; 3 (2): 69-75
Adenoviral gene transfer has been shown to be effective in cardiac myocytes in vitro and in vivo. A major limitation of myocardial gene therapy is the extracardiac transgene expression.To minimize extracardiac gene expression, we have constructed a tissue-specific promoter for cardiac gene transfer, namely, the 250-bp fragment of the myosin light chain-2v (MLC-2v) gene, which is known to be expressed in a tissue-specific manner in ventricular myocardium followed by a luciferase (luc) reporter gene (Ad.4 x MLC250.Luc). Rat cardiomyocytes, liver and kidney cells were infected with Ad.4 x MLC.Luc or control vectors. For in vivo testing, Ad.4 x MLC250.Luc was injected into the myocardium or in the liver of rats. Kinetics of promoter activity were monitored over 8 days using a cooled CCD camera.In vitro: By infecting hepatic versus cardiomyocyte cells, we found that the promoter specificity ratio (luc activity in cardiomyocytes per liver cells) was 20.4 versus 0.9 (Ad.4 x MLC250.Luc vs. Ad.CMV). In vivo: Ad.4 x MLC250.Luc significantly reduced luc activity in liver (38.4-fold), lung (16.1-fold), and kidney (21.8-fold) versus Ad.CMV (p =.01); whereas activity in the heart was only 3.8-fold decreased. The gene expression rate of cardiomyocytes versus hepatocytes was 7:1 (Ad.4 x MLC.Luc) versus 1:1.4 (Ad.CMV.Luc).This new vector may be useful to validate therapeutic approaches in animal disease models and offers the perspective for selective expression of therapeutic genes in the diseased heart.
View details for Web of Science ID 000208288100001
View details for PubMedID 15296671
Micro-positron emission tomography imaging of cardiac gene expression in rats using bicistronic adenoviral vector-mediated gene delivery
2004; 109 (11): 1415-1420
We have previously validated the use of micro-positron emission tomography (microPET) for monitoring the expression of a single PET reporter gene in rat myocardium. We now report the use of a bicistronic adenoviral vector (Ad-CMV-D2R80a-IRES-HSV1-sr39tk) for linking the expression of 2 PET reporter genes, a mutant rat dopamine type 2 receptor (D2R80a) and a mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk), with the aid of an internal ribosomal entry site (IRES).Rat H9c2 cardiomyoblasts transduced with increasing titers of Ad-CMV-D2R80a-IRES-HSV1-sr39tk (0 to 2.5x10(8) pfu) were assayed 48 hours later for reporter protein activities, which were found to correlate well with viral titer (r2=0.96, P<0.001 for D2R80A; r2=0.98, P<0.001 for HSV1-sr39TK) and each other (r2=0.97; P<0.001). Experimental (n=8) and control (n=6) athymic rats underwent intramyocardial injection of up to 2x10(9) pfu of Ad-CMV-D2R80a-IRES-HSV1-sr39tk and saline, respectively. Forty-eight hours later and weekly thereafter, rats were assessed for D2R80a-dependent myocardial accumulation of 3-(2-[18F]fluoroethyl)spiperone ([18F]-FESP) and HSV1-sr39tk-dependent sequestration of 9-(4-[18F]fluoro-3-hydroxymethylbutyl)guanine ([18F]-FHBG) using microPET. Longitudinal [18F]-FESP and [18F]-FHBG imaging of experimental rats revealed a good correlation between the cardiac expressions of the 2 PET reporter genes (r2=0.73; P<0.001). The location of adenovirus-mediated transgene expression, as inferred from microPET images, was confirmed by ex vivo gamma counting of explanted heart.The IRES-based bicistronic adenoviral vector can potentially be used in conjunction with PET for indirect imaging of therapeutic gene expression by replacing 1 of the 2 PET reporter genes with a therapeutic gene of choice.
View details for DOI 10.1161/01.CIR.0000121727.59564.5B
View details for Web of Science ID 000220364700015
View details for PubMedID 15007006
Molecular imaging of cardiac cell transplantation in living animals using optical bioluminescence and positron emission tomography
2003; 108 (11): 1302-1305
The current method of analyzing myocardial cell transplantation relies on postmortem histology. We sought to demonstrate the feasibility of monitoring transplanted cell survival in living animals using molecular imaging techniques.For optical bioluminescence charged-coupled device imaging, rats (n=20) underwent intramyocardial injection of embryonic rat H9c2 cardiomyoblasts (3x10(6) to 5x10(5)) expressing firefly luciferase (Fluc) reporter gene. Cardiac bioluminescence signals were present for more than 2 weeks with 3x10(6) cells: day 1 (627 000+/-15%), day 2 (346 100+/-21%), day 4 (112 800+/-20%), day 8 (78 860+/-24%), day 12 (67 780+/-12%), and day 16 (62 200+/-5% p x s(-1) x cm(2-1) x sr(-1)). For micro-positron emission tomography imaging, rats (n=20) received cardiomyoblasts (3x10(6)) expressing mutant herpes simplex type 1 thymidine kinase (HSV1-sr39tk) reporter gene. Detailed tomography of transplanted cells is shown by 9-(4-[18F]-fluoro-3hydroxymethylbutyl)guanine ([18F]-FHBG) reporter probe and nitrogen-13 ammonia ([13N]-NH3) perfusion images. Within the transplanted region, there was a 4.48+/-0.71-fold increase of in vivo [18F]-FHBG activity and a 4.01+/-0.51-fold increase of ex vivo gamma counting compared with control animals. Finally, the in vivo images of cell survival were confirmed by ex vivo autoradiography, histology, immunohistochemistry, and reporter protein assays.The location(s), magnitude, and survival duration of embryonic cardiomyoblasts were monitored noninvasively. With further development, molecular imaging studies should add critical insights into cardiac cell transplantation biology.
View details for DOI 10.1161/01.CIR.0000091252.20010.6E
View details for Web of Science ID 000185328800006
View details for PubMedID 12963637
Positron-emission tomography reporter gene expression imaging in rat myocardium
2003; 107 (2): 326-332
This study examines the quantitative accuracy, detection sensitivity, and time course of imaging the expression of a mutant herpes simplex type-1 virus thymidine kinase (HSV1-sr39tk) PET reporter gene in rat myocardium by using the PET reporter probe 9-(4-[18F]-Fluoro-3-Hydroxymethylbutyl)-Guanine ([18F]-FHBG) and a small-animal PET (microPET).In 40 rats, adenovirus expressing HSV1-sr39tk driven by a cytomegalovirus promoter (Ad-CMV-HSV1-sr39tk, 1x10(6) to 1x10(9) pfu) was injected through a thoracotomy directly into the left ventricular myocardium. After 3 days, myocardial perfusion was imaged with [13N]-ammonia for delineating the left ventricular myocardium, followed by imaging the expression of the reporter gene with intravenous [18F]-FHBG. The total myocardial [18F]-FHBG accumulation was quantified in percent of injected dose (%ID). Immunohistochemistry and autoradiography demonstrated HSV1-sr39tk enzyme (HSV1-sr39TK) and accumulation of [18F]-FHBG in the inoculated myocardium in 3 rats each. In 24 rats with various viral titers, the %ID was correlated with ex vivo well counting (r2=0.981, P<0.0001) and myocardial HSV1-sr39TK activity by tissue enzyme activity assay (r2=0.790, P<0.0001). Myocardial [18F]-FHBG accumulation was identified at viral titers down to 1x10(7) pfu. In 6 rats serially imaged up to day 17, myocardial [18F]-FHBG accumulation on microPET peaked on days 3 to 5 and was no longer identified on days 10 to 17.HSV1-sr39tk reporter gene expression can be monitored with [18F]-FHBG and microPET in rat myocardium quantitatively and serially with high detection sensitivity. Cardiac PET reporter gene imaging offers the potential of monitoring the expression of therapeutic genes in cardiac gene therapy.
View details for DOI 10.1161/01.CIR.0000044385.60972.AE
View details for Web of Science ID 000180785700022
View details for PubMedID 12538436
Positron emission tomography imaging of cardiac reporter gene expression in living rats
2002; 106 (2): 180-183
Imaging reporter gene expression is useful for noninvasive monitoring of gene therapy. In this study, we imaged cardiac reporter gene expression in living rats using micro positron emission tomography (microPET).Rats (n=10) underwent intramyocardial injection with 1x10(9) pfu of adenovirus carrying cytomegalovirus promoter-driving herpes simplex virus type 1 mutant thymidine kinase (Ad-CMV-HSV1-sr39tk) as PET reporter gene. Control rats (n=4) received 1x10(9) pfu of adenovirus carrying cytomegalovirus promoter-driving firefly luciferase (Ad-CMV-Fluc). On days 2 to 4, microPET images were obtained after a tail vein injection of nitrogen-13 ammonia ([13N]-NH3) as myocardial perfusion tracer, followed by 9-(4-[18F]-fluoro-3 hydroxymethylbutyl) guanine ([18F]-FHBG) to assess HSV1-sr39tk expression. After imaging, hearts were removed for ex vivo [18F] gamma counting and thymidine kinase enzyme assay. Results show homogenous myocardial distribution of [13N]-NH3 on all microPET images. Rats injected with Ad-CMV-HSV1-sr39tk have significant [18F]-FHBG uptake in the anterolateral wall compared with background signal in controls. Gamma counting shows 20.0+/-4.4-fold increase of radioactivity, whereas enzyme assay shows 22.1+/-6.1-fold increase of thymidine kinase activity in Ad-CMV-HSV1-sr39tk injected rats (P<0.05).Successful imaging of cardiac HSV1-sr39tk expression was performed in living rats with microPET. The presence of [18F]-FHBG uptake is confirmed by gamma counting and the presence of HSV1-sr39TK protein by thymidine kinase enzyme assay. Cardiac reporter gene imaging by PET may eventually be applied toward human gene therapy studies.
View details for DOI 10.1161/01.CIR.0000023620.59633.53
View details for Web of Science ID 000176820500016
View details for PubMedID 12105155
Optical imaging of cardiac reporter gene expression in living rats
2002; 105 (14): 1631-1634
Studies of cardiac gene transfer rely on postmortem analysis using histologic staining or enzyme assays. Noninvasive imaging of the temporal and spatial characteristics of cardiac gene expression in the same subject offers significant advantages.Rats underwent direct myocardial injection via left thoracotomy with adenovirus-expressing firefly luciferase (Ad-CMV-Fluc; n=30). The reporter substrate D-luciferin was injected intraperitoneally. Serial images were acquired by use of a cooled charged couple detector (CCD) camera. Results are expressed as relative light unit per minute (RLU/min). Rats transduced with 1x10(9) plaque-forming units show decremental cardiac luciferase activity over time: 152 070+/-21 170 (day 2), 195 806+/-62 630 (day 5), 7250+/-2941 (day 8), and 2040+/-971 RLU/min (day 14). To assess the detection sensitivity, serially diluted titers of Ad-CMV-Fluc were injected: 1x10(9) (195 393+/-14 896), 1x10(8) (33 777+/-18 179), 1x10(7) (417+/-91), 1x10(6) (185+/-64), 1x10(5) (53+/-1), and control (54+/-1) (P<0.05 for 1x10(9), 1x10(8), and 1x10(7) plaque-forming units versus control adenovirus-expressing mutant thymidine kinase [Ad-CMV-HSV1-sr39tk]; n=3). Finally, rats were euthanized, and in vitro luciferase activity correlated with in vivo CCD signals (r2=0.92).This study demonstrates for the first time the feasibility of imaging the location, magnitude, and time course of cardiac reporter gene expression in living rats. Cardiac gene therapy studies could be aided with wider application of this approach.
View details for DOI 10.1161/01.CIR.0000014984.95520.AD
View details for Web of Science ID 000174987300185
View details for PubMedID 11940538
Noninvasive optical imaging of firefly luciferase reporter gene expression in skeletal muscles of living mice
2001; 4 (4): 297-306
The ability to monitor reporter gene expression noninvasively offers significant advantages over current techniques such as postmortem tissue staining or enzyme activity assays. Here we demonstrate a novel method of repetitively tracking in vivo gene expression of firefly luciferase (FL) in skeletal muscles of mice using a cooled charged coupled device (CCD) camera. We first show that the cooled CCD camera provides consistent and reproducible results within +/-8% standard deviation from mean values, and a detection sensitivity (range tested: 1 x 10(4) - 1 x 10(9) plaque form-ing units (pfu)) of 1 x 10(6) pfu of E1-deleted adenovirus expressing FL driven by a cytomegalovirus promoter (Ad-CMV-FL). The duration and magnitude of adenoviral mediated (1 x 10(9) pfu) FL gene expression were then followed over time. FL gene expression in immunocompetent Swiss Webster mice peaks within the first 48 hours, falls by 98% after 20 days, and persists for >150 days. In contrast, FL activity in nude mice remains elevated for >110 days. Finally, transduced Swiss Webster and nude mice were sacrificed to show that the in vivo CCD signals correlate well with in vitro luciferase enzyme assays (r(2)=0.91 and 0.96, respectively). Our findings demonstrate the ability of the cooled CCD camera to sensitively and noninvasively track the location, magnitude, and persistence of FL gene expression. Monitoring of gene therapy studies in small animals may be aided considerably with further extensions of this technique.
View details for Web of Science ID 000171403200006
View details for PubMedID 11592831
Severe regional ischemia alters coronary flow reserve in the remote perfusion area
JOURNAL OF NUCLEAR CARDIOLOGY
2000; 7 (1): 43-52
Clinical and experimental studies suggest that coronary flow reserve (CFR) may be abnormal in regions remote from myocardial infarction. We sought to determine the possible relation among stenosis severity, ischemic dysfunction, and impairment of CFR in remote regions.In 7 open-chest dogs, acute graded left circumflex (LCX) ischemia was created and maintained based on measurement of the transstenotic (aortic-distal LCX) pressure gradient (measured in millimeters of mercury). Regional thickening was assessed with sonomicrometers. Regional myocardial flow was assessed at rest with radiolabeled microspheres. Doppler flow probes were placed on proximal LCX and left anterior descending (LAD) arteries to measure resting flow and CFR in response to intracoronary injection of adenosine (36 microg). These parameters were assessed under baseline conditions and during transstenotic gradients of 10, 20, 30, and 40 mm Hg. Increasing LCX stenosis severity caused progressive impairment of LCX CFR: baseline (2.22+/-0.10), stenosis 10 (1.80+/-0.06), stenosis 20 (1.56+/-0.08), stenosis 30 (1.30+/-0.04), and stenosis 40 (1.17+/-0.06) (P<.01 vs. baseline). Remote LAD CFR was not altered by mild to moderate LCX stenosis (baseline [2.33+/-0.19]; stenosis 10 [2.30+/-0.25]; stenosis 20 [2.15+/-0.26]). However, critical LCX stenosis producing mild to moderate reduction in thickening in the ischemic region was associated with a significant impairment of LAD CFR: stenosis 30 (1.90+/-0.26) and stenosis 40 (1.80+/-0.22) (P<.01 vs. baseline). These changes in remote CFR persisted after correction for changes in the rate-pressure product.In an acute canine model of progressive LCX coronary stenosis, CFR was impaired in both ischemic and remote nonischemic regions in association with mild to moderate ischemic-induced regional myocardial dysfunction. Thus pharmacologic vasodilation provoked only mild heterogeneity in CFR in the presence of a critical LCX stenosis as a result of concurrent reduction of LAD CFR. This phenomenon warrants further clinical and experimental investigation because it may affect detection of flow heterogeneity during acute ischemia (which induced myocardial dysfunction).
View details for Web of Science ID 000085428600007
View details for PubMedID 10698234
Limitations of dobutamine for enhancing flow heterogeneity in the presence of single coronary stenosis: Implications for technetium-99m-sestamibi imaging
JOURNAL OF NUCLEAR MEDICINE
1998; 39 (3): 417-425
Dobutamine is used as an alternative to exercise in conjunction with 99mTc-sestamibi SPECT perfusion imaging for detection of coronary artery disease. However, the use of quantitative dobutamine 99mTc-sestamibi SPECT imaging for enhanced detection of coronary stenosis has not been established. The goal of this study is to examine the effects of dobutamine stress on regional myocardial blood flow and relative myocardial 99mTc-sestamibi activity in the presence of a single-vessel stenosis.In six open-chest dogs with left circumflex artery stenosis, radiolabeled microspheres were injected during baseline, severe stenosis and peak dobutamine stress (10 microg/kg/min). Technetium-99m-sestamibi was injected intravenously at peak dobutamine. Hearts were excised 20 min after 99mTc-sestamibi injection for SPECT imaging and post-mortem gamma-well counting.Dobutamine significantly increased heart rate, rate-pressure product and the first derivative of left ventricular pressure. Ischemic zone (left circumflex) myocardial blood flows (in ml/min/g) were: baseline, 0.92 +/- 0.15; stenosis, 0.65 +/- 0.16; and dobutamine, 1.19 +/- 0.38. Nonischemic zone myocardial blood flows were: baseline, 0.99 +/- 0.18; stenosis, 1.01 +/- 0.12; and dobutamine, 1.94 +/- 0.32 (p < 0.01 versus stenosis). Ischemic flows, expressed as percentages of nonischemic flows, were: baseline, 94% +/- 2%; stenosis, 63% +/- 11% (p < 0.05 versus baseline) and dobutamine, 60% +/- 12% (p was not significant versus stenosis). Technetium-99m-sestamibi activity in the ischemic zone (75% +/- 6% nonischemic) underestimated the relative flow deficit produced during dobutamine stress (p = 0.056). Myocardial 99mTc-sestamibi activity correlated with flow when flow was less than 1.0 ml/min/g. At higher flow ranges (1.0 ml/min/g-3.5 ml/min/g), 99mTc-sestamibi did not track flow.In a canine model of flow-limiting, single-vessel stenosis, dobutamine (10 microg/kg/min) did not augment flow heterogeneity. In addition, relative myocardial 99mTc-sestamibi activity underestimated microsphere flow at higher flows induced by dobutamine, leading to underestimation of ischemia. These findings suggest that dobutamine stress 99mTc-sestamibi scintigraphy may underestimate the relative flow deficit.
View details for Web of Science ID 000072421600012
View details for PubMedID 9529285
Double Knockdown of Prolyl Hydroxylase and Factor-Inhibiting Hypoxia-Inducible Factor With Nonviral Minicircle Gene Therapy Enhances Stem Cell Mobilization and Angiogenesis After Myocardial Infarction
LIPPINCOTT WILLIAMS & WILKINS. 2011: S46-S54
Under normoxic conditions, hypoxia-inducible factor (HIF)-1? is rapidly degraded by 2 hydroxylases: prolyl hydroxylase (PHD) and factor-inhibiting HIF-1 (FIH). Because HIF-1? mediates the cardioprotective response to ischemic injury, its upregulation may be an effective therapeutic option for ischemic heart failure.PHD and FIH were cloned from mouse embryonic stem cells. The best candidate short hairpin (sh) sequences for inhibiting PHD isoenzyme 2 and FIH were inserted into novel, nonviral, minicircle vectors. In vitro studies after cell transfection of mouse C2C12 myoblasts, HL-1 atrial myocytes, and c-kit(+) cardiac progenitor cells demonstrated higher expression of angiogenesis factors in the double-knockdown group compared with the single-knockdown and short hairpin scramble control groups. To confirm in vitro data, shRNA minicircle vectors were injected intramyocardially after left anterior descending coronary artery ligation in adult FVB mice (n=60). Functional studies using MRI, echocardiography, and pressure-volume loops showed greater improvement in cardiac function in the double-knockdown group. To assess mechanisms of this functional recovery, we performed a cell trafficking experiment, which demonstrated significantly greater recruitment of bone marrow cells to the ischemic myocardium in the double-knockdown group. Fluorescence-activated cell sorting showed significantly higher activation of endogenous c-kit(+) cardiac progenitor cells. Immunostaining showed increased neovascularization and decreased apoptosis in areas of injured myocardium. Finally, western blots and laser-capture microdissection analysis confirmed upregulation of HIF-1? protein and angiogenesis genes, respectively.We demonstrated that HIF-1? upregulation by double knockdown of PHD and FIH synergistically increases stem cell mobilization and myocardial angiogenesis, leading to improved cardiac function.
View details for DOI 10.1161/CIRCULATIONAHA.110.014019
View details for Web of Science ID 000294782800006
View details for PubMedID 21911818
Novel MicroRNA Prosurvival Cocktail for Improving Engraftment and Function of Cardiac Progenitor Cell Transplantation
LIPPINCOTT WILLIAMS & WILKINS. 2011: S27-S34
Although stem cell therapy has provided a promising treatment for myocardial infarction, the low survival of the transplanted cells in the infarcted myocardium is possibly a primary reason for failure of long-term improvement. Therefore, the development of novel prosurvival strategies to boost stem cell survival will be of significant benefit to this field.Cardiac progenitor cells (CPCs) were isolated from transgenic mice, which constitutively express firefly luciferase and green fluorescent protein. The CPCs were transduced with individual lentivirus carrying the precursor of miR-21, miR-24, and miR-221, a cocktail of these 3 microRNA precursors, or green fluorescent protein as a control. After challenge in serum free medium, CPCs treated with the 3 microRNA cocktail showed significantly higher viability compared with untreated CPCs. After intramuscular and intramyocardial injections, in vivo bioluminescence imaging showed that microRNA cocktail-treated CPCs survived significantly longer after transplantation. After left anterior descending artery ligation, microRNA cocktail-treated CPCs boost the therapeutic efficacy in terms of functional recovery. Histological analysis confirmed increased myocardial wall thickness and CPC engraftment in the myocardium with the microRNA cocktail. Finally, we used bioinformatics analysis and experimental validation assays to show that Bim, a critical apoptotic activator, is an important target gene of the microRNA cocktail, which collectively can bind to the 3'UTR region of Bim and suppress its expression.We have demonstrated that a microRNA prosurvival cocktail (miR-21, miR-24, and miR-221) can improve the engraftment of transplanted cardiac progenitor cells and therapeutic efficacy for treatment of ischemic heart disease.
View details for DOI 10.1161/CIRCULATIONAHA.111.017954
View details for Web of Science ID 000294782800004
View details for PubMedID 21911815
MicroRNA-210 as a Novel Therapy for Treatment of Ischemic Heart Disease
LIPPINCOTT WILLIAMS & WILKINS. 2010: S124-S131
MicroRNAs are involved in various critical functions, including the regulation of cellular differentiation, proliferation, angiogenesis, and apoptosis. We hypothesize that microRNA-210 can rescue cardiac function after myocardial infarction by upregulation of angiogenesis and inhibition of cellular apoptosis in the heart.Using microRNA microarrays, we first showed that microRNA-210 was highly expressed in live mouse HL-1 cardiomyocytes compared with apoptotic cells after 48 hours of hypoxia exposure. We confirmed by polymerase chain reaction that microRNA-210 was robustly induced in these cells. Gain-of-function and loss-of-function approaches were used to investigate microRNA-210 therapeutic potential in vitro. After transduction, microRNA-210 can upregulate several angiogenic factors, inhibit caspase activity, and prevent cell apoptosis compared with control. Afterward, adult FVB mice underwent intramyocardial injections with minicircle vector carrying microRNA-210 precursor, minicircle carrying microRNA-scramble, or sham surgery. At 8 weeks, echocardiography showed a significant improvement of left ventricular fractional shortening in the minicircle vector carrying microRNA-210 precursor group compared with the minicircle carrying microRNA-scramble control. Histological analysis confirmed decreased cellular apoptosis and increased neovascularization. Finally, 2 potential targets of microRNA-210, Efna3 and Ptp1b, involved in angiogenesis and apoptosis were confirmed through additional experimental validation.MicroRNA-210 can improve angiogenesis, inhibit apoptosis, and improve cardiac function in a murine model of myocardial infarction. It represents a potential novel therapeutic approach for treatment of ischemic heart disease.
View details for DOI 10.1161/CIRCULATIONAHA.109.928424
View details for Web of Science ID 000282294800019
View details for PubMedID 20837903
In vivo visualization of transplanted bone marrow mononuclear cell behavior in murine peripheral artery occlusive disease
WILEY-BLACKWELL. 2010: S9-S9
View details for Web of Science ID 000281136400032
Modeling Conduction in Host-Graft Interactions Between Stem Cell Grafts and Cardiomyocytes
IEEE. 2009: 6014-6017
Cell therapy has recently made great strides towards aiding heart failure. However, while transplanted cells may electromechanically integrate into host tissue, there may not be a uniform propagation of a depolarization wave between the heterogeneous tissue boundaries. A model using microelectrode array technology that maps the electrical interactions between host and graft tissues in co-culture is presented and sheds light on the effects of having a mismatch of conduction properties at the boundary. Skeletal myoblasts co-cultured with cardiomyocytes demonstrated that conduction velocity significantly decreases at the boundary despite electromechanical coupling. In an attempt to improve the uniformity of conduction with host cells, differentiating human embryonic stem cells (hESC) were used in co-culture. Over the course of four to seven days, synchronous electrical activity was observed at the hESC boundary, implying differentiation and integration. Activity did not extend far past the boundary, and conduction velocity was significantly greater than that of the host tissue, implying the need for other external measures to properly match the conduction properties between host and graft tissue.
View details for Web of Science ID 000280543604229
View details for PubMedID 19964687