Sean M Wu, MD, PhD is a board certified cardiologist who specializes in treating men and women with cardiac diseases such as coronary artery disease, cardiac valve disorder, rhythm disorders, cardio-oncology/cancer drug toxicity, and cardiac preventive management.
Dr. Wu also conduct research in cardiac developmental biology/congenital heart disease, stem cell biology and translation of stem cells into new treatments for congenital heart disease, adult heart failure and rhythm disorders.
In addition to completion of residency program and board certification in internal medicine, Dr. Wu has also completed a 3-year ACGME-accredited fellowship in cardiovascular disease with board certification and additional clinical training in echocardiography at Massachusetts General Hospital and cardiac developmental biology research training at Boston Children's Hospital/Harvard Medical School in Boston, MA.
- Cardiovascular Disease
- Coronary Artery Disease
- Arrhythmias, Cardiac
- Cardiac prevention
- Valve disorders
Member, Editorial Board, Journal of Cardiovascular Development and Disease (2021 - Present)
Member, Editorial Board, Cardiology Discovery (2020 - Present)
Chair, Faculty Search Committee, Surgical & Basic Science Faculty, Dept. of Cardiothoracic Surgery, Stanford SoM (2020 - Present)
Chair, Faculty Search Committee, Basic Sci & Enginr (BASE) Program, Moore Heart Center, LPCH (2018 - 2019)
Associate Member, Stanford Diabetes Research Center (2017 - Present)
Section Editor, Current Cardiology Reports (2016 - Present)
Associate Professor of Medicine (with tenure) and (by courtesy) Pediatrics, Stanford University (2016 - Present)
Editorial Consultant, Journal of American College of Cardiology: Basic to Translational Science (2015 - Present)
Guest Editor, Journal of Cardiovascular Development and Differentiation (2015 - 2016)
Consulting Editor, Circulation Research (2015 - 2019)
Editorial Board - General, Circulation Research (2014 - Present)
Section Editor, Current Treatment Options in Cardiovascular Medicine (2013 - 2017)
Assistant Professor of Medicine, Stanford University, School of Medicine (2012 - 2015)
Associate Editor, BMC Cardiovascular Disease (2011 - 2014)
Organizing Committee, NIH/NHLBI Cardiovascular Regenerative Medicine Symposium (2011 - 2013)
Editorial Board, Frontiers in Pharmacology and Smooth Muscle Biology (2010 - 2013)
Editorial Board, World Journal of Stem Cell (2009 - 2012)
Assistant Physician, Massachusetts General Hospital (2009 - 2012)
Assistant Professor of Medicine, Harvard Medical School (2009 - 2012)
Editorial Board, Clinical Medicine Insights: Cardiology (2007 - 2012)
Director, Mouse Microinjection Core, Massachusetts General Hospital (2007 - 2012)
Instructor in Medicine, Harvard Medical School (2006 - 2009)
Honors & Awards
Joan and Sanford I. Weill Scholar, Stanford Cardiovascular Institute (2020-)
2018 Kenneth D. Bloch Memorial Lecturer in Vascular Biology, American Heart Association (2018)
Consulting Editors of the Year, Circulation Research (2018)
Established Investigator Award, American Heart Association (2017-2021)
Superior Editorial Consultant, Circulation Research (2017)
Elected Member, American Society for Clinical Investigation (ASCI) (2016)
Cardiovascular Medicine Division Teaching Award, Department of Medicine, Stanford University School of Medicine (2015)
NIH Director's Pioneer Award, National Institutes of Health, Office of the Director (2014-2019)
David Lawrence Stein Award, American Heart Association-Western Affiliate (2014)
Endowed Faculty Scholar, Child Health Research Institute/ Lucile Packard Foundation for Children's Health (2013-2018)
Seed Grant Award (Co-Recipient with Dr. Beth Pruitt), Stanford Cardiovascular Institute (2013-2014)
SPARK Research Award, Division of Cardiology, Massachusetts General Hospital (2010-2011)
Fellow, American College of Cardiology (2010)
Progenitor Cell Biology Consortium, Co-Principal Investigator, NIH/NHLBI (2009-2016)
NIH Director's New Innovator Award, National Institutes of Health, Office of the Director (2008-2013)
Seed Grant Recipient, Harvard Stem Cell Institute (2008-2010)
Young Investigator Competitive Award in Cardiovascular Medicine, GlaxoSmithKline Education and Research Foundation (2007-2009)
de Gunzburg Family Scholar, Massachusetts General Hospital (2006)
K08 Mentored Clinical Scientist Award, NIH/NHLBI (2005-2011)
Abstract of Distinction, Research Symposium - Massachusetts General Hospital (2005)
NIH/NHLBI Scholarship, Keystone Symposium on Molecular Mechanism of Cardiac Disease and Regeneration (2005)
Career Development Award in Cardiovascular Medicine, American College of Cardiology Foundation/Pfizer (2004-2007)
ACCF/Bristol Meyers Travel Award, American College of Cardiology (2002)
Merck/ACC Young Investigator Award - 2nd Place, American College of Cardiology (2001)
Henry Christian Award for Research Excellence, American Federation for Medical Research (1999)
Experimental Pathologist-in-Training, American Society for Investigative Pathology (1998)
Award for Academic Excellence and Achievement, American Society of Clinical Pathologists (1996, 1997)
Tau Beta Pi, Stanford University School of Engineering (1992)
Terman Award, Stanford University School of Engineering (1992)
President's Award for Academic Excellence, Stanford University (1989)
Boards, Advisory Committees, Professional Organizations
Chair, American Heart Association National Research Committee, Bioethics Subcommittee (2020 - Present)
Chair, AHA-BCVS Committee on Early Career Development (2020 - Present)
Member, Scientific Committee, Sarnoff Cardiovascular Research Foundation (2020 - Present)
Vice Chair, AHA-BCVS Committee on Early Career Development (2018 - 2020)
Vice-Chair, American Heart Association National Research Committee, Bioethics Subcommittee (2017 - 2020)
Member, AHA - Committee on Scientific Session Programming (CSSP) (2016 - Present)
Member, AHA - BCVS Committee on Scientific and Clinical Education Lifelong Learning Committee (2016 - Present)
Member, American Heart Association - BCVS Committee on Early Career Development (2015 - 2018)
Member, American Heart Association National Research Committee, Stem Cell Research Subgroup (2013 - 2017)
Member, American Heart Association National Stem Cell Therapy Writing Group (2012 - Present)
Member, Research Administration Advisory Committee, Massachusette General Hospital (2010 - 2012)
Research Fellowship, Boston Children's Hospital/Harvard Medical School, Stem Cell Biology (2006)
Board Certification: American Board of Internal Medicine, Cardiovascular Disease (2005)
Fellowship: Massachusetts General Hospital (2005) MA
Board Certification, Internal Medicine, ABIM (2003)
Residency: Duke University Medical Center (2001) NC
Medical Education: Duke University School of Medicine (1999) NC
PhD, Duke University School of Arts and Sciences, Pathology (1998)
BS, Stanford University, Mechanical Engineering (1992)
BS, Stanford University, Biological Science (1992)
Community and International Work
National Asian Pacific American Medical Student Association
Opportunities for Student Involvement
Sean Wu, William Goodyer, Benjamin Beyersdorf, Eben Rosenthal, Nynke van den Berg. "United States Patent Application No. 62/871,551 NOVEL MOLECULAR TOOLS TO VISUALIZE AND TARGET THE CARDIAC CONDUCTION SYSTEM (CCS)", Leland Stanford Junior University, Jul 8, 2019
Sean Wu, Jan Buikema, Arun Sharma. "United States Patent Applicaiton No. 62/644,091 REAGENTS AND METHODS WITH WNT AGONISTS AND BIOACTIVE LIPIDS FOR GENERATING AND EXPANDING CARDIOMYOCYTES", Leland Stanford Junior University, Mar 16, 2018
Sean M. Wu. "United States Patent Application No. 13/552,975; US Patent No. 9393221 Methods and compounds for reducing intracellular lipid storage", Massachusetts General Hospital, Jul 19, 2016
Current Research and Scholarly Interests
Cardiovascular Developmental Biology
A major focus of the Wu Laboratory is to define the earliest steps in heart formation. We use experimentally-modified mice as our live model to take advantage of a broad range of molecular tools available. The similarity between a mouse heart and a human heart allows us to connect our results directly into finding ways to treat human heart diseases. We seek to understand what genes are responsible for making the heart chamber form in the right way. We are also interested in finding out what disturbances in the normal process of heart formation is responsible for devastating congenital heart diseases that lead to fetal demise or death shortly after birth. We have utilized the most state-of-the-art tools to try to understand the process of normal heart formation and have made significant discoveries in this area of research.
Cardiovascular Tissue Engineering
We have recently embarked on cardiac tissue engineering work due to the significant promise of this research direction in creating functional cardiac tissue for modeling of heart diseases and for generation a new organ that may be transplantable. By using stem cells that can be turned into cardiac cells, we have brought stem cell biology and tissue engineering together to begin making true functional heart tissue for screening drugs to treat heart diseases and to build new replacement tissues that may one day be used to replace the damaged heart muscle after heart attack. We have actively collaborated with material science engineers, vascular engineers, and mechanical engineers to make new discoveries in this research area. We currently employ 3D bioprinting as a tool to generate full-thickness, vascularized, and functional cardiac tissue.
Cardiovascular Disease Modeling
While mouse models are useful for studying the process of heart formation, they are not exactly like the human hearts in various ways. Since we cannot easily obtain human heart tissue, we have chosen to use stem cells as the next best source of material to study human heart formation and disease onset. We focus on a special type of stem cells call induced pluripotent stem cells (iPSCs) that behave exactly like embryonic stem cells but are made from regular human skin or blood cell. These human iPSCs make excellent model of heart formation inside a petri dish in the lab and can be turned into beating heart muscle cells by treating them with special factors. Furthermore, the steps that these iPSCs take to become heart muscle cells replicate exactly the way a human fetus goes through during early development in utero.
Cardiovascular Regenerative Biology
Ultimately, our work in developmental biology and tissue engineering seek to identify the most effective way to treat damage hearts. The regenerative potentials of stem cells is unlimited but requires careful guidance when given to a patient with heart disease. Many efforts that have failed in the past is due to the lack of understanding of what stem cells are capable of doing to treat damaged hearts. We have studied the role of stem cells in a fetal heart injury and recovery model (Sturzu et al, Circulation 2015) and have addressed the challenges that must be overcome in order to move the field forward (Wu et al, Cell 2008). We are currently seeking to find new cell types that may be useful for repairing damages to the muscle and the conduction system (i.e. the electrical network) in the heart using human iPSC-derived cells. In the future, we seek to generate transplantable organs using innovative strategies that involve tissue engineering and interspecies chimerism with pluripotent stem cells.
Independent Studies (16)
- Directed Investigation
BIOE 392 (Aut, Win, Spr, Sum)
- Directed Reading in Medicine
MED 299 (Aut, Win, Spr)
- Directed Reading in Pediatrics
PEDS 299 (Win, Spr)
- Directed Reading in Stem Cell Biology and Regenerative Medicine
STEMREM 299 (Aut, Win, Spr)
- Early Clinical Experience
PEDS 280 (Win, Spr)
- Early Clinical Experience in Medicine
MED 280 (Win, Spr)
- Graduate Research
MED 399 (Aut, Win, Spr)
- Graduate Research
PEDS 399 (Win, Spr)
- Graduate Research
STEMREM 399 (Aut, Win, Spr, Sum)
- Medical Scholars Research
MED 370 (Aut, Win, Spr)
- Medical Scholars Research
PEDS 370 (Win, Spr)
- Medical Scholars Research
STEMREM 370 (Aut, Win, Spr)
- Out-of-Department Advanced Research Laboratory in Bioengineering
BIOE 191X (Aut, Win, Spr)
- Undergraduate Directed Reading/Research
PEDS 199 (Win, Spr)
- Undergraduate Research
MED 199 (Aut, Win, Spr)
- Undergraduate Research
STEMREM 199 (Aut, Win, Spr)
- Directed Investigation
- Patient-Specific Induced Pluripotent Stem Cells Implicate Intrinsic Impaired Contractility in Hypoplastic Left Heart Syndrome. Circulation 2020; 142 (16): 1605–8
Intrinsic Endocardial Defects Contribute to Hypoplastic Left Heart Syndrome.
Cell stem cell
Hypoplastic left heart syndrome (HLHS) is a complex congenital heart disease characterized by abnormalities in the left ventricle, associated valves, and ascending aorta. Studies have shown intrinsic myocardial defects but do not sufficiently explain developmental defects in the endocardial-derived cardiac valve, septum, and vasculature. Here, we identify a developmentally impaired endocardial population in HLHS through single-cell RNA profiling of hiPSC-derived endocardium and human fetal heart tissue with an underdeveloped left ventricle. Intrinsic endocardial defects contribute to abnormal endothelial-to-mesenchymal transition, NOTCH signaling, and extracellular matrix organization, key factors in valve formation. Endocardial abnormalities cause reduced cardiomyocyte proliferation and maturation by disrupting fibronectin-integrin signaling, consistent with recently described de novo HLHS mutations associated with abnormal endocardial gene and fibronectin regulation. Together, these results reveal a critical role for endocardium in HLHS etiology and provide a rationale for considering endocardial function in regenerative strategies.
View details for DOI 10.1016/j.stem.2020.07.015
View details for PubMedID 32810435
A single-cell transcriptomic atlas characterizes ageing tissues in the mouse.
Ageing is characterized by a progressive loss of physiological integrity, leading to impaired function and increased vulnerability to death1. Despite rapid advances over recent years, many of the molecular and cellular processes that underlie the progressive loss of healthy physiology are poorly understood2. To gain a better insight into these processes, here we generate a single-cell transcriptomic atlas across the lifespan of Mus musculus that includes data from 23 tissues and organs. We found cell-specific changes occurring across multiple cell types and organs, as well as age-related changes in the cellular composition of different organs. Using single-cell transcriptomic data, we assessed cell-type-specific manifestations of different hallmarks of ageing-such as senescence3, genomic instability4 and changes in the immune system2. This transcriptomic atlas-which we denote Tabula Muris Senis, or 'Mouse Ageing Cell Atlas'-provides molecular information about how the most important hallmarks of ageing are reflected in a broad range of tissues and cell types.
View details for DOI 10.1038/s41586-020-2496-1
View details for PubMedID 32669714
Next-Generation Surrogate Wnts Support Organoid Growth and Deconvolute Frizzled Pleiotropy In Vivo.
Cell stem cell
Modulation of Wnt signaling has untapped potential in regenerative medicine due to its essential functions in stem cell homeostasis. However, Wnt lipidation and Wnt-Frizzled (Fzd) cross-reactivity have hindered translational Wnt applications. Here, we designed and engineered water-soluble, Fzd subtype-specific "next-generation surrogate" (NGS) Wnts that hetero-dimerize Fzd and Lrp6. NGS Wnt supports long-term expansion of multiple different types of organoids, including kidney, colon, hepatocyte, ovarian, and breast. NGS Wnts are superior to Wnt3a conditioned media in organoid expansion and single-cell organoid outgrowth. Administration of Fzd subtype-specific NGS Wnt in vivo reveals that adult intestinal crypt proliferation can be promoted by agonism of Fzd5 and/or Fzd8 receptors, while a broad spectrum of Fzd receptors can induce liver zonation. Thus, NGS Wnts offer a unified organoid expansion protocol and a laboratory "tool kit" for dissecting the functions of Fzd subtypes in stem cell biology.
View details for DOI 10.1016/j.stem.2020.07.020
View details for PubMedID 32818433
Wnt Activation and Reduced Cell-Cell Contact Synergistically Induce Massive Expansion of Functional Human iPSC-Derived Cardiomyocytes.
Cell stem cell
2020; 27 (1): 50–63.e5
Modulating signaling pathways including Wnt and Hippo can induce cardiomyocyte proliferation in vivo. Applying these signaling modulators to human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in vitro can expand CMs modestly (<5-fold). Here, we demonstrate massive expansion of hiPSC-CMs in vitro (i.e., 100- to 250-fold) by glycogen synthase kinase-3β (GSK-3β) inhibition using CHIR99021 and concurrent removal of cell-cell contact. We show that GSK-3β inhibition suppresses CM maturation, while contact removal prevents CMs from cell cycle exit. Remarkably, contact removal enabled 10 to 25 times greater expansion beyond GSK-3β inhibition alone. Mechanistically, persistent CM proliferation required both LEF/TCF activity and AKT phosphorylation but was independent from yes-associated protein (YAP) signaling. Engineered heart tissues from expanded hiPSC-CMs showed comparable contractility to those from unexpanded hiPSC-CMs, demonstrating uncompromised cellular functionality after expansion. In summary, we uncovered a molecular interplay that enables massive hiPSC-CM expansion for large-scale drug screening and tissue engineering applications.
View details for DOI 10.1016/j.stem.2020.06.001
View details for PubMedID 32619518
Ageing hallmarks exhibit organ-specific temporal signatures.
Ageing is the single greatest cause of disease and death worldwide, and understanding the associated processes could vastly improve quality of life. Although major categories of ageing damage have been identified-such as altered intercellular communication, loss of proteostasis and eroded mitochondrial function1-these deleterious processes interact with extraordinary complexity within and between organs, and a comprehensive, whole-organism analysis of ageing dynamics has been lacking. Here we performed bulk RNA sequencing of 17 organs and plasma proteomics at 10 ages across the lifespan of Mus musculus, and integrated these findings with data from the accompanying Tabula Muris Senis2-or 'Mouse Ageing Cell Atlas'-which follows on from the original Tabula Muris3. We reveal linear and nonlinear shifts in gene expression during ageing, with the associated genes clustered in consistent trajectory groups with coherent biological functions-including extracellular matrix regulation, unfolded protein binding, mitochondrial function, and inflammatory and immune response. Notably, these gene sets show similar expression across tissues, differing only in the amplitude and the age of onset of expression. Widespread activation of immune cells is especially pronounced, and is first detectable in white adipose depots during middle age. Single-cell RNA sequencing confirms the accumulation of T cells and B cells in adipose tissue-including plasma cells that express immunoglobulin J-which also accrue concurrently across diverse organs. Finally, we show how gene expression shifts in distinct tissues are highly correlated with corresponding protein levels in plasma, thus potentially contributing to the ageing of the systemic circulation. Together, these data demonstrate a similar yet asynchronous inter- and intra-organ progression of ageing, providing a foundation from which to track systemic sources of declining health at old age.
View details for DOI 10.1038/s41586-020-2499-y
View details for PubMedID 32669715
- Single-Cell Delineation of Who's on First and Second Heart Fields During Development CIRCULATION RESEARCH 2019; 125 (4): 411–13
Transcriptomic Profiling of the Developing Cardiac Conduction System at Single-Cell Resolution.
RATIONALE: The cardiac conduction system (CCS) consists of distinct components including the sinoatrial node (SAN), atrioventricular node (AVN), His bundle, bundle branches (BB) and Purkinje fibers (PF). Despite an essential role for the CCS in heart development and function, the CCS has remained challenging to interrogate due to inherent obstacles including small cell numbers, large cell type heterogeneity, complex anatomy and difficulty in isolation. Single-cell RNA-sequencing (scRNA-seq) allows for genome-wide analysis of gene expression at single-cell resolution.OBJECTIVE: Assess the transcriptional landscape of the entire CCS at single-cell resolution by scRNA-seq within the developing mouse heart.METHODS AND RESULTS: Wild-type, embryonic day 16.5 mouse hearts (n=6 per zone) were harvested and three zones of microdissection were isolated, including: Zone I - SAN region; Zone II - AVN/His region; and Zone III - BB/PF region. Tissue was digested into single cell suspensions, isolated, reverse transcribed and barcoded prior to high-throughput sequencing and bioinformatics analyses. scRNA-seq was performed on over 22,000 cells and all major cell types of the murine heart were successfully captured including bona fide clusters of cells consistent with each major component of the CCS. Unsupervised weighted gene co-expression network analysis led to the discovery of a host of novel CCS genes, a subset of which were validated using fluorescent in situ hybridization as well as whole mount immunolabelling with volume imaging (iDISCO+) in three-dimensions on intact mouse hearts. Further, subcluster analysis unveiled isolation of distinct CCS cell subtypes, including the clinically-relevant but poorly characterized "transitional cells" that bridge the CCS and surrounding myocardium.CONCLUSIONS: Our study represents the first comprehensive assessment of the transcriptional profiles from the entire CCS at single-cell resolution and provides a gene atlas for facilitating future efforts in conduction cell identification, isolation and characterization in the context of development and disease.
View details for DOI 10.1161/CIRCRESAHA.118.314578
View details for PubMedID 31284824
- Prometheus Unbound in Ya(p) Heart DEVELOPMENTAL CELL 2019; 48 (6): 741–42
- Single-cell analysis of early progenitor cells that build coronary arteries NATURE 2018; 559 (7714): 356-+
Single-cell transcriptomics of 20 mouse organs creates a Tabula Muris
2018; 562: 367–372
View details for DOI 10.1038/s41586-018-0590-4
Transcriptomic Profiling Maps Anatomically Patterned Subpopulations among Single Embryonic Cardiac Cells
2016; 39 (4): 491-507
Embryonic gene expression intricately reflects anatomical context, developmental stage, and cell type. To address whether the precise spatial origins of cardiac cells can be deduced solely from their transcriptional profiles, we established a genome-wide expression database from 118, 949, and 1,166 single murine heart cells at embryonic day 8.5 (e8.5), e9.5, and e10.5, respectively. We segregated these cells by type using unsupervised bioinformatics analysis and identified chamber-specific genes. Using a random forest algorithm, we reconstructed the spatial origin of single e9.5 and e10.5 cardiomyocytes with 92.0% ± 3.2% and 91.2% ± 2.8% accuracy, respectively (99.4% ± 1.0% and 99.1% ± 1.1% if a ±1 zone margin is permitted) and predicted the second heart field distribution of Isl-1-lineage descendants. When applied to Nkx2-5(-/-) cardiomyocytes from murine e9.5 hearts, we showed their transcriptional alteration and lack of ventricular phenotype. Our database and zone classification algorithm will enable the discovery of novel mechanisms in early cardiac development and disease.
View details for DOI 10.1016/j.devcel.2016.10.014
View details for Web of Science ID 000389162800013
View details for PubMedID 27840109
Lift NIH restrictions on chimera research.
Science (New York, N.Y.)
2015; 350 (6261): 640
View details for PubMedID 26542560
Harnessing the potential of induced pluripotent stem cells for regenerative medicine
NATURE CELL BIOLOGY
2011; 13 (5): 497-505
The discovery of methods to convert somatic cells into induced pluripotent stem cells (iPSCs) through expression of a small combination of transcription factors has raised the possibility of producing custom-tailored cells for the study and treatment of numerous diseases. Indeed, iPSCs have already been derived from patients suffering from a large variety of disorders. Here we review recent progress that has been made in establishing iPSC-based disease models, discuss associated technical and biological challenges, and highlight possible solutions to overcome these barriers. We believe that a better understanding of the molecular basis of pluripotency, cellular reprogramming and lineage-specific differentiation of iPSCs is necessary for progress in regenerative medicine.
View details for DOI 10.1038/ncb0511-497
View details for Web of Science ID 000290148700004
View details for PubMedID 21540845
View details for PubMedCentralID PMC3617981
Generation of Functional Ventricular Heart Muscle from Mouse Ventricular Progenitor Cells
2009; 326 (5951): 426-429
The mammalian heart is formed from distinct sets of first and second heart field (FHF and SHF, respectively) progenitors. Although multipotent progenitors have previously been shown to give rise to cardiomyocytes, smooth muscle, and endothelial cells, the mechanism governing the generation of large numbers of differentiated progeny remains poorly understood. We have employed a two-colored fluorescent reporter system to isolate FHF and SHF progenitors from developing mouse embryos and embryonic stem cells. Genome-wide profiling of coding and noncoding transcripts revealed distinct molecular signatures of these progenitor populations. We further identify a committed ventricular progenitor cell in the Islet 1 lineage that is capable of limited in vitro expansion, differentiation, and assembly into functional ventricular muscle tissue, representing a combination of tissue engineering and stem cell biology.
View details for DOI 10.1126/science.1177350
View details for Web of Science ID 000270818600053
View details for PubMedID 19833966
View details for PubMedCentralID PMC2895998
Epicardial progenitors contribute to the cardiomyocyte lineage in the developing heart
2008; 454 (7200): 109-U5
The heart is formed from cardiogenic progenitors expressing the transcription factors Nkx2-5 and Isl1 (refs 1 and 2). These multipotent progenitors give rise to cardiomyocyte, smooth muscle and endothelial cells, the major lineages of the mature heart. Here we identify a novel cardiogenic precursor marked by expression of the transcription factor Wt1 and located within the epicardium-an epithelial sheet overlying the heart. During normal murine heart development, a subset of these Wt1(+) precursors differentiated into fully functional cardiomyocytes. Wt1(+) proepicardial cells arose from progenitors that express Nkx2-5 and Isl1, suggesting that they share a developmental origin with multipotent Nkx2-5(+) and Isl1(+) progenitors. These results identify Wt1(+) epicardial cells as previously unrecognized cardiomyocyte progenitors, and lay the foundation for future efforts to harness the cardiogenic potential of these progenitors for cardiac regeneration and repair.
View details for DOI 10.1038/nature07060
View details for Web of Science ID 000257308300047
View details for PubMedID 18568026
View details for PubMedCentralID PMC2574791
Origins and fates of cardiovascular progenitor cells
2008; 132 (4): 537-543
Multipotent cardiac progenitor cells are found in the fetal and adult heart of many mammalian species including humans and form as intermediates during the differentiation of embryonic stem cells. Despite similar biological properties, the molecular identities of these different cardiac progenitor cell populations appear to be distinct. Elucidating the origins and lineage relationships of these cell populations will accelerate clinical applications such as drug screening and cell therapy as well as shedding light on the pathogenic mechanisms underlying cardiac diseases.
View details for DOI 10.1016/j.cell.2008.02.002
View details for Web of Science ID 000253817900012
View details for PubMedID 18295570
View details for PubMedCentralID PMC2507768
Developmental origin of a bipotential myocardial and smooth muscle cell precursor in the mammalian heart
2006; 127 (6): 1137-1150
Despite recent advances in delineating the mechanisms involved in cardiogenesis, cellular lineage specification remains incompletely understood. To explore the relationship between developmental fate and potential, we isolated a cardiac-specific Nkx2.5(+) cell population from the developing mouse embryo. The majority of these cells differentiated into cardiomyocytes and conduction system cells. Some, surprisingly, adopted a smooth muscle fate. To address the clonal origin of these lineages, we isolated Nkx2.5(+) cells from in vitro differentiated murine embryonic stem cells and found approximately 28% of these cells expressed c-kit. These c-kit(+) cells possessed the capacity for long-term in vitro expansion and differentiation into both cardiomyocytes and smooth muscle cells from a single cell. We confirmed these findings by isolating c-kit(+)Nkx2.5(+) cells from mouse embryos and demonstrated their capacity for bipotential differentiation in vivo. Taken together, these results support the existence of a common precursor for cardiovascular lineages in the mammalian heart.
View details for DOI 10.1016/j.cell.2006.10.028
View details for Web of Science ID 000242991000013
View details for PubMedID 17123591
Massive expansion and cryopreservation of functional human induced pluripotent stem cell-derived cardiomyocytes.
2021; 2 (1): 100334
Since the discovery of human induced pluripotent stem cells (hiPSCs), numerous strategies have been established to efficiently derive cardiomyocytes from hiPSCs (hiPSC-CMs). Here, we describe a cost-effective strategy for the subsequent massive expansion (>250-fold) of high-purity hiPSC-CMs relying on two aspects: removal of cell-cell contacts and small-molecule inhibition with CHIR99021. The protocol maintains CM functionality, allows cryopreservation, and the cells can be used in downstream assays such as disease modeling, drug and toxicity screening, and cell therapy. For complete details on the use and execution of this protocol, please refer to Buikema (2020).
View details for DOI 10.1016/j.xpro.2021.100334
View details for PubMedID 33615277
- Myocarditis Surveillance with High-Sensitivity Troponin I During Cancer Treatment with Immune Checkpoint Inhibitors. JACC. CardioOncology 2021; 3 (1): 137–39
CRISPR/Cas9-based targeting of fluorescent reporters to human iPSCs to isolate atrial and ventricular-specific cardiomyocytes.
2021; 11 (1): 3026
Generating cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) has represented a significant advance in our ability to model cardiac disease. Current differentiation protocols, however, have limited use due to their production of heterogenous cell populations, primarily consisting of ventricular-like CMs. Here we describe the creation of two chamber-specific reporter hiPSC lines by site-directed genomic integration using CRISPR-Cas9 technology. In the MYL2-tdTomato reporter, the red fluorescent tdTomato was inserted upstream of the 3' untranslated region of the Myosin Light Chain 2 (MYL2) gene in order faithfully label hiPSC-derived ventricular-like CMs while avoiding disruption of endogenous gene expression. Similarly, in the SLN-CFP reporter, Cyan Fluorescent Protein (CFP) was integrated downstream of the coding region of the atrial-specific gene, Sarcolipin (SLN). Purification of tdTomato+ and CFP+ CMs using flow cytometry coupled with transcriptional and functional characterization validated these genetic tools for their use in the isolation of bona fide ventricular-like and atrial-like CMs, respectively. Finally, we successfully generated a double reporter system allowing for the isolation of both ventricular and atrial CM subtypes within a single hiPSC line. These tools provide a platform for chamber-specific hiPSC-derived CM purification and analysis in the context of atrial- or ventricular-specific disease and therapeutic opportunities.
View details for DOI 10.1038/s41598-021-81860-x
View details for PubMedID 33542270
Myocardial Disease and Long-Distance Space Travel: Solving the Radiation Problem.
Frontiers in cardiovascular medicine
2021; 8: 631985
Radiation-induced cardiovascular disease is a well-known complication of radiation exposure. Over the last few years, planning for deep space missions has increased interest in the effects of space radiation on the cardiovascular system, as an increasing number of astronauts will be exposed to space radiation for longer periods of time. Research has shown that exposure to different types of particles found in space radiation can lead to the development of diverse cardiovascular disease via fibrotic myocardial remodeling, accelerated atherosclerosis and microvascular damage. Several underlying mechanisms for radiation-induced cardiovascular disease have been identified, but many aspects of the pathophysiology remain unclear. Existing pharmacological compounds have been evaluated to protect the cardiovascular system from space radiation-induced damage, but currently no radioprotective compounds have been approved. This review critically analyzes the effects of space radiation on the cardiovascular system, the underlying mechanisms and potential countermeasures to space radiation-induced cardiovascular disease.
View details for DOI 10.3389/fcvm.2021.631985
View details for PubMedID 33644136
Purification of Pluripotent Stem Cell-Derived Cardiomyocytes Using CRISPR/Cas9-Mediated Integration of Fluorescent Reporters.
Methods in molecular biology (Clifton, N.J.)
2021; 2158: 223–40
Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes have become critically important for the detailed study of cardiac development, disease modeling, and drug screening. However, directed differentiation of hiPSCs into cardiomyocytes often results in mixed populations of cardiomyocytes and other cell types, which may confound experiments that require pure populations of cardiomyocytes. Here, we detail the use of a CRISPR/Cas9 genome editing strategy to develop cardiomyocyte-specific reporters that allow for the isolation of hiPSC-derived cardiomyocytes and chamber-specific myocytes. Moreover, we describe a cardiac differentiation protocol to derive cardiomyocytes from hiPSCs, as well as a strategy to use fluorescence-activated cell sorting to isolate pure populations of fluorescently labeled cardiomyocytes for downstream applications.
View details for DOI 10.1007/978-1-0716-0668-1_17
View details for PubMedID 32857377
Single-Cell RNA-seq Unveils Unique Transcriptomic Signatures of Organ-Specific Endothelial Cells.
Background: Endothelial cells (ECs) display considerable functional heterogeneity depending on the vessel and tissue in which they are located. While these functional differences are presumably imprinted in the transcriptome, the pathways and networks which sustain EC heterogeneity have not been fully delineated. Methods: To investigate the transcriptomic basis of EC specificity, we analyzed single-cell RNA-sequencing (scRNA-seq) data from tissue-specific mouse ECs generated by the Tabula Muris consortium. We employed a number of bioinformatics tools to uncover markers and sources of EC heterogeneity from scRNA-seq data. Results: We found a strong correlation between tissue-specific EC transcriptomic measurements generated by either scRNA-seq or bulk RNA-seq, thus validating the approach. Using a graph-based clustering algorithm, we found that certain tissue-specific ECs cluster strongly by tissue (e.g. liver, brain) whereas others (i.e. adipose, heart) have considerable transcriptomic overlap with ECs from other tissues. We identified novel markers of tissue-specific ECs and signaling pathways that may be involved in maintaining their identity. Sex was a considerable source of heterogeneity in the endothelial transcriptome and we discovered Lars2 to be a gene that is highly enriched in ECs from male mice. In addition, we found that markers of heart and lung ECs in mice were conserved in human fetal heart and lung ECs. Finally, we identified potential angiocrine interactions between tissue-specific ECs and other cell types by analyzing ligand and receptor expression patterns. Conclusions: In summary, we use scRNA-seq data generated by the Tabula Muris consortium to uncover transcriptional networks that maintain tissue-specific EC identity and to identify novel angiocrine and functional relationships between tissue-specific ECs.
View details for DOI 10.1161/CIRCULATIONAHA.119.041433
View details for PubMedID 32929989
4HNE Impairs Myocardial Bioenergetics in Congenital Heart DiseaseInduced Right Ventricular Failure.
Background: In patients with complex congenital heart disease, such as those with tetralogy of Fallot, the right ventricle (RV) is subject to pressure overload stress, leading to RV hypertrophy and eventually RV failure. The role of lipid peroxidation, a potent form of oxidative stress, in mediating RV hypertrophy and failure in congenital heart disease is unknown. Methods: Lipid peroxidation and mitochondrial function and structure were assessed in RV myocardium collected from patients with RV hypertrophy with normal RV systolic function (RV FAC 47.3±3.8%) and in patients with RV failure showing decreased RV systolic function (RV FAC 26.6±3.1%). The mechanism of the effect of lipid peroxidation, mediated by 4-hydroxynonenal (4HNE; a byproduct of lipid peroxidation) on mitochondrial function and structure was assessed in HL1 murine cardiomyocytes and human induced pluripotent stem cellderived cardiomyocytes. Results: RV failure was characterized by an increase in 4HNE adduction of metabolic and mitochondrial proteins (16/27 identified proteins), in particular electron transport chain proteins. Sarcomeric (myosin) and cytoskeletal proteins (desmin, tubulin) also underwent 4HNEadduction. RV failure showed lower oxidative phosphorylation [moderate RV hypertrophy 287.6±19.75 vs. RV failure 137.8±11.57 pmol/(sec*ml), p=0.0004], and mitochondrial structural damage. Using a cell model, we show that 4HNE decreases cell number and oxidative phosphorylation (control 388.1±23.54 vs. 4HNE 143.7±11.64 pmol/(sec*ml), p<0.0001). Carvedilol, a known antioxidant did not decrease 4HNE adduction of metabolic and mitochondrial proteins and did not improve oxidative phosphorylation. Conclusions: Metabolic, mitochondrial, sarcomeric and cytoskeletal proteins are susceptible to 4HNE-adduction in patients with RV failure. 4HNE decreases mitochondrial oxygen consumption by inhibiting electron transport chain complexes. Carvedilol did not improve the 4HNE-mediated decrease in oxygen consumption. Strategies to decrease lipid peroxidation could improve mitochondrial energy generation and cardiomyocyte survival and improve RV failure in patients with congenital heart disease.
View details for DOI 10.1161/CIRCULATIONAHA.120.045470
View details for PubMedID 32806952
- Immune Profiling and Causal Antigen Discovery in Mouse and Human Models of Immune Checkpoint Inhibitor-induced Myocarditis LIPPINCOTT WILLIAMS & WILKINS. 2020
Proceedings From the 2019 Stanford Single Ventricle Scientific Summit: Advancing Science for Single Ventricle Patients: From Discovery to Clinical Applications.
Journal of the American Heart Association
2020; 9 (7): e015871
Abstracts Because of remarkable advances in survival over the past 40years, the worldwide population of individuals with single ventricle heart disease living with Fontan circulation has grown to 70000, with nearly half aged >18years. Survival to at least 30years of age is now achievable for 75% of Fontan patients. On the other hand, single ventricle patients account for the largest group of the 6000 to 8000 children hospitalized with circulation failure, with or without heart failure annually in the United States, with the highest in-hospital mortality. Because there is little understanding of the underlying mechanisms of heart failure, arrhythmias, pulmonary and lymphatic vascular abnormalities, and other morbidities, there are no specific treatments to maintain long-term myocardial performance or to optimize overall patient outcomes.
View details for DOI 10.1161/JAHA.119.015871
View details for PubMedID 32188306
Immune Checkpoint Inhibitor Cardiotoxicity: Understanding Basic Mechanisms and Clinical Characteristics and Finding a Cure.
Annual review of pharmacology and toxicology
Immune checkpoint inhibitors (ICIs) attenuate mechanisms of self-tolerance in the immune system, enabling T cell responses to cancerous tissues and revolutionizing care for cancer patients. However, by lowering barriers against self-reactivity, ICIs often result in varying degrees of autoimmunity. Cardiovascular complications, particularly myocarditis but also arrhythmias, pericarditis, and vasculitis, have emerged as significant complications associated with ICIs. In this review, we examine the clinical aspects and basic science principles that underlie ICI-associated myocarditis and other cardiovascular toxicities. In addition, we discuss current therapeutic approaches. We believe a better mechanistic understanding of ICI-associated toxicities can lead to improved patient outcomes by reducing treatment-related morbidity. Expected final online publication date for the Annual Review of Pharmacology and Toxicology, Volume 61 is January 8, 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
View details for DOI 10.1146/annurev-pharmtox-010919-023451
View details for PubMedID 32776859
Cardiovascular Complications in Patients with COVID-19: Consequences of Viral Toxicities and Host Immune Response
Curr Cardiol Rep
2020; 22 (5)
View details for DOI 10.1007/s11886-020-01292-3
Simple Lithography-Free Single Cell Micropatterning using Laser-Cut Stencils.
Journal of visualized experiments : JoVE
Micropatterning techniques have been widely used in cell biology to study effects of controlling cell shape and size on cell fate determination at single cell resolution. Current state-of-the-art single cell micropatterning techniques involve soft lithography and micro-contact printing, which is a powerful technology, but requires trained engineering skills and certain facility support in microfabrication. These limitations require a more accessible technique. Here, we describe a simple alternative lithography-free method: stencil-based single cell patterning. We provide step-by-step procedures including stencil design, polyacrylamide hydrogel fabrication, stencil-based protein incorporation, and cell plating and culture. This simple method can be used to pattern an array of as many as 2,000 cells. We demonstrate the patterning of cardiomyocytes derived from single human induced pluripotent stem cells (hiPSC) with distinct cell shapes, from a 1:1 square to a 7:1 adult cardiomyocyte-like rectangle. This stencil-based single cell patterning is lithography-free, technically robust, convenient, inexpensive, and most importantly accessible to those with a limited bioengineering background.
View details for DOI 10.3791/60888
View details for PubMedID 32310234
Levitating Cells to Sort the Fit and the Fat.
Density is a core material property and varies between different cell types, mainly based on differences in their lipid content. Sorting based on density enables various biomedical applications such as multi-omics in precision medicine and regenerative repair in medicine. However, a significant challenge is sorting cells of the same type based on density differences. Here, a new method for real-time monitoring and sorting of single cells based on their inherent levitation profiles driven by their lipid content is reported. As a model system, human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) from a patient with neutral lipid storage disease (NLSD) due to loss of function of adipose triglyceride lipase (ATGL) resulting in abnormal lipid storage in cardiac muscle are used. This levitation-based strategy detects subpopulations within ATGL-deficient hiPSC-CMs with heterogenous lipid content, equilibrating at different levitation heights due to small density differences. In addition, sorting of these differentially levitating subpopulations are monitored in real time. Using this approach, sorted healthy and diseased hiPSC-CMs maintain viability and function. Pixel-tracking technologies show differences in contraction between NLSD and healthy hiPSC-CMs. Overall, this is a unique approach to separate diseased cell populations based on their intracellular lipid content that cannot be achieved using traditional flow cytometry techniques.
View details for DOI 10.1002/adbi.201900300
View details for PubMedID 32352239
Cardiovascular Risks in Patients with COVID-19: Potential Mechanisms and Areas of Uncertainty.
Current cardiology reports
2020; 22 (5): 34
COronaVirus Disease 2019 (COVID-19) has spread at unprecedented speed and scale into a global pandemic with cardiovascular risk factors and complications emerging as important disease modifiers. We aim to review available clinical and biomedical literature on cardiovascular risks of COVID-19.SARS-CoV2, the virus responsible for COVID-19, enters the cell via ACE2 expressed in select organs. Emerging epidemiological evidence suggest cardiovascular risk factors are associated with increased disease severity and mortality in COVID-19 patients. Patients with a more severe form of COVID-19 are also more likely to develop cardiac complications such as myocardial injury and arrhythmia. The true incidence of and mechanism underlying these events remain elusive. Cardiovascular diseases appear intricately linked with COVID-19, with cardiac complications contributing to the elevated morbidity/mortality of COVID-19. Robust epidemiologic and biologic studies are urgently needed to better understand the mechanism underlying these associations to develop better therapies.
View details for DOI 10.1007/s11886-020-01293-2
View details for PubMedID 32350632
Effects of Spaceflight on Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Structure and Function.
Stem cell reports
With extended stays aboard the International Space Station (ISS) becoming commonplace, there is a need to better understand the effects of microgravity on cardiac function. We utilized human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to study the effects of microgravity on cell-level cardiac function and gene expression. The hiPSC-CMs were cultured aboard the ISS for 5.5weeks and their gene expression, structure, and functions were compared with ground control hiPSC-CMs. Exposure to microgravity on the ISS caused alterations in hiPSC-CM calcium handling. RNA-sequencing analysis demonstrated that 2,635 genes were differentially expressed among flight, post-flight, and ground control samples, including genes involved in mitochondrial metabolism. This study represents the first use of hiPSC technology to model the effects of spaceflight on human cardiomyocyte structure and function.
View details for DOI 10.1016/j.stemcr.2019.10.006
View details for PubMedID 31708475
Myopathy Causing Bag3P209L Protein Leads to Restrictive Cardiomyopathy Caused by Aggregate Formation and Sarcomere Disruption in Cardiomyocytes
LIPPINCOTT WILLIAMS & WILKINS. 2019
View details for Web of Science ID 000511467800268
Hypertrophic Cardiomyopathy Mutations With Opposite Effects on [latin sharp s]-myosin Biomechanics Show Similar Structural and Biomechanical Phenotypes in Human Induced Pluripotent Stem Cell Derived Cardiomyocytes (hipsc-cms)
LIPPINCOTT WILLIAMS & WILKINS. 2019
View details for Web of Science ID 000511467800091
Single cell expression analysis reveals anatomical and cell cycle-dependent transcriptional shifts during heart development.
Development (Cambridge, England)
The heart is a complex organ composed of multiple cell and tissue types. Cardiac cells from different regions of the growing embryonic heart exhibit distinct patterns of gene expression, which are thought to contribute to heart development and morphogenesis. Single cell RNA sequencing allows genome-wide analysis of gene expression at the single cell level. Here, we analyzed cardiac cells derived from early stage developing hearts by single cell RNA-seq and identified cell cycle gene expression as a major determinant of transcriptional variation. Within cell cycle stage-matched CMs from a given heart chamber, we found that CMs in the G2/M phase downregulated sarcomeric and cytoskeletal markers. We also identified cell location-specific signaling molecules that may influence the proliferation of other nearby cell types. Our data highlight how variations in cell cycle activity selectively promote cardiac chamber growth during development, reveal profound chamber-specific cell cycle-linked transcriptional shifts, and open the way to deeper understanding of pathogenesis of congenital heart disease.
View details for DOI 10.1242/dev.173476
View details for PubMedID 31142541
Single Cell Analysis of Endothelial Cells Identified Organ-Specific Molecular Signatures and Heart-Specific Cell Populations and Molecular Features.
Frontiers in cardiovascular medicine
2019; 6: 165
Endothelial cells line the inner surface of vasculature and play an important role in normal physiology and disease progression. Although most tissue is known to have a heterogeneous population of endothelial cells, transcriptional differences in organ specific endothelial cells have not been systematically analyzed at the single cell level. The Tabula Muris project profiled mouse single cells from 20 organs. We found 10 of the organs profiled by this Consortium have endothelial cells. Unsupervised analysis of these endothelial cells revealed that they were mainly grouped by organs, and organ-specific cells were further partially correlated by germ layers. Unexpectedly, we found all lymphatic endothelial cells grouped together regardless of their resident organs. To further understand the cellular heterogeneity in organ-specific endothelial cells, we used the heart as an example. As a pump of the circulation system, the heart has multiple types of endothelial cells. Detailed analysis of these cells identified an endocardial endothelial cell population, a coronary vascular endothelial cell population, and an aorta-specific cell population. Through integrated analysis of the single cell data from another two studies analyzing the aorta, we identified conserved cell populations and molecular markers across the datasets. In summary, by reanalyzing the existing endothelial cell single-cell data, we identified organ-specific molecular signatures and heart-specific subpopulations and molecular markers. We expect these findings will pave the way for a deeper understanding of vascular biology and endothelial cell-related diseases.
View details for DOI 10.3389/fcvm.2019.00165
View details for PubMedID 31850371
View details for PubMedCentralID PMC6901932
- Cardiovascular Regenerative Medicine: Challenges, Perspectives, and Future Directions Cardiovasscular Regenerative Medicine Springer Nature. 2019: 223–225
Cardiovascular Regenerative Medicine
edited by Serpooshan, V., Wu, S. M.
Springer Nature. 2019; null (null)
View details for DOI 10.1007/9783030200473
Bioprinting Approaches to Engineering Vascularized 3D Cardiac Tissues.
Current cardiology reports
2019; 21 (9): 90
3D bioprinting technologies hold significant promise for the generation of engineered cardiac tissue and translational applications in medicine. To generate a clinically relevant sized tissue, the provisioning of a perfusable vascular network that provides nutrients to cells in the tissue is a major challenge. This review summarizes the recent vascularization strategies for engineering 3D cardiac tissues.Considerable steps towards the generation of macroscopic sizes for engineered cardiac tissue with efficient vascular networks have been made within the past few years. Achieving a compact tissue with enough cardiomyocytes to provide functionality remains a challenging task. Achieving perfusion in engineered constructs with media that contain oxygen and nutrients at a clinically relevant tissue sizes remains the next frontier in tissue engineering. The provisioning of a functional vasculature is necessary for maintaining a high cell viability and functionality in engineered cardiac tissues. Several recent studies have shown the ability to generate tissues up to a centimeter scale with a perfusable vascular network. Future challenges include improving cell density and tissue size. This requires the close collaboration of a multidisciplinary teams of investigators to overcome complex challenges in order to achieve success.
View details for DOI 10.1007/s11886-019-1179-8
View details for PubMedID 31352612
- Modelling inherited cardiac disease using human induced pluripotent stem cell-derived cardiomyocytes: progress, pitfalls, and potential CARDIOVASCULAR RESEARCH 2018; 114 (14): 1828–42
- Large-Scale Single-Cell RNA-Seq Reveals Molecular Signatures of Heterogeneous Populations of Human Induced Pluripotent Stem Cell-Derived Endothelial Cells CIRCULATION RESEARCH 2018; 123 (4): 443–50
- Fates Aligned: Origins and Mechanisms of Ventricular Conduction System and Ventricular Wall Development SPRINGER. 2018: 1090–98
- Reassessment of c-Kit in Cardiac Cells A Complex Interplay Between Expression, Fate, and Function CIRCULATION RESEARCH 2018; 123 (1): 9–11
Reactivation of the Nkx2.5 cardiac enhancer after myocardial infarction does not presage myogenesis.
The contribution of resident stem or progenitor cells to cardiomyocyte renewal after injury in adult mammalian hearts remains a matter of considerable debate. We evaluated a cell population in the adult mouse heart induced by myocardial infarction (MI) and characterized by an activated Nkx2.5 enhancer element that is specific for multipotent cardiac progenitor cells during embryonic development. We hypothesized that these MI induced cells (MICs) harbor cardiomyogenic properties similar to their embryonic counterparts.MICs reside in the heart and mainly localize to the infarction area and border zone. Interestingly, gene expression profiling of purified MICs one week after infarction revealed increased expression of stem cell markers and embryonic cardiac transcription factors in these cells as compared to the non-mycoyte cell fraction of adult hearts. A subsequent global transcriptome comparison with embryonic cardiac progenitor cells and fibroblasts and in vitro culture of MICs unveiled that (myo-) fibroblastic features predominated and that cardiac transcription factors were only expressed at background levels.Adult injury induced reactivation of a cardiac-specific Nkx2.5 enhancer element known to specifically mark myocardial progenitor cells during embryonic development does not reflect hypothesized embryonic cardiomyogenic properties. Our data suggest a decreasing plasticity of cardiac progenitor (-like) cell populations with increasing age. A re-expression of embryonic, stem or progenitor cell features in the adult heart must be interpreted very carefully with respect to the definition of cardiac resident progenitor cells. Albeit, the abundance of scar formation after cardiac injury suggests a potential to target predestinated activated profibrotic cells to push them towards cardiomyogenic differentiation to improve regeneration.
View details for PubMedID 29579159
Cardiovascular tissue bioprinting: Physical and chemical processes.
Applied physics reviews
2018; 5 (4): 041106
Three-dimensional (3D) cardiac tissue bioprinting occupies a critical crossroads position between the fields of materials engineering, cardiovascular biology, 3D printing, and rational organ replacement design. This complex area of research therefore requires expertise from all those disciplines and it poses some unique considerations that must be accounted for. One of the chief hurdles is that there is a relatively limited systematic organization of the physical and chemical characteristics of bioinks that would make them applicable to cardiac bioprinting. This is of great significance, as heart tissue is functionally complex and the in vivo extracellular niche is under stringent controls with little room for variability before a cardiomyopathy manifests. This review explores the critical parameters that are necessary for biologically relevant bioinks to successfully be leveraged for functional cardiac tissue engineering, which can have applications in in vitro heart tissue models, cardiotoxicity studies, and implantable constructs that can be used to treat a range of cardiomyopathies, or in regenerative medicine.
View details for DOI 10.1063/1.5048807
View details for PubMedID 32550960
View details for PubMedCentralID PMC7187889
Genome Editing Redefines Precision Medicine in the Cardiovascular Field.
Stem cells international
2018; 2018: 4136473
Genome editing is a powerful tool to study the function of specific genes and proteins important for development or disease. Recent technologies, especially CRISPR/Cas9 which is characterized by convenient handling and high precision, revolutionized the field of genome editing. Such tools have enormous potential for basic science as well as for regenerative medicine. Nevertheless, there are still several hurdles that have to be overcome, but patient-tailored therapies, termed precision medicine, seem to be within reach. In this review, we focus on the achievements and limitations of genome editing in the cardiovascular field. We explore different areas of cardiac research and highlight the most important developments: (1) the potential of genome editing in human pluripotent stem cells in basic research for disease modelling, drug screening, or reprogramming approaches and (2) the potential and remaining challenges of genome editing for regenerative therapies. Finally, we discuss social and ethical implications of these new technologies.
View details for PubMedID 29731778
View details for PubMedCentralID PMC5872631
- 4D Printing of Actuating Cardiac Tissue 3D PRINTING APPLICATIONS IN CARDIOVASCULAR MEDICINE 2018: 153–62
- Big bottlenecks in cardiovascular tissue engineering COMMUNICATIONS BIOLOGY 2018; 1
Stage-specific Effects of Bioactive Lipids on Human iPSC Cardiac Differentiation and Cardiomyocyte Proliferation.
2018; 8 (1): 6618
Bioactive lipids such as sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) regulate diverse processes including cell proliferation, differentiation, and migration. However, their roles in cardiac differentiation and cardiomyocyte proliferation have not been explored. Using a 96-well differentiation platform for generating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) we found that S1P and LPA can independently enhance cardiomyocyte generation when administered at an early stage of differentiation. We showed that the combined S1P and LPA treatment of undifferentiated hiPSCs resulted in increased nuclear accumulation of β-catenin, the canonical Wnt signaling pathway mediator, and synergized with CHIR99021, a glycogen synthase kinase 3 beta inhibitor, to enhance mesodermal induction and subsequent cardiac differentiation. At later stages of cardiac differentiation, the addition of S1P and LPA resulted in cell cycle initiation in hiPSC-CMs, an effect mediated through increased ERK signaling. Although the addition of S1P and LPA alone was insufficient to induce cell division, it was able to enhance β-catenin-mediated hiPSC-CM proliferation. In summary, we demonstrated a developmental stage-specific effect of bioactive lipids to enhance hiPSC-CM differentiation and proliferation via modulating the effect of canonical Wnt/β-catenin and ERK signaling. These findings may improve hiPSC-CM generation for cardiac disease modeling, precision medicine, and regenerative therapies.
View details for PubMedID 29700394
- Myocardial Development Reference Modules in Biomedical Sciences Elsevier. 2018; 1
Bioengineering of vascular myocardial tissue; a 3D bioprinting approach
MARY ANN LIEBERT, INC. 2017: S158–S159
View details for Web of Science ID 000416247300609
Bioacoustic-enabled patterning of human iPSC-derived cardiomyocytes into 3D cardiac tissue
2017; 131: 47-57
The creation of physiologically-relevant human cardiac tissue with defined cell structure and function is essential for a wide variety of therapeutic, diagnostic, and drug screening applications. Here we report a new scalable method using Faraday waves to enable rapid aggregation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) into predefined 3D constructs. At packing densities that approximate native myocardium (10(8)-10(9) cells/ml), these hiPSC-CM-derived 3D tissues demonstrate significantly improved cell viability, metabolic activity, and intercellular connection when compared to constructs with random cell distribution. Moreover, the patterned hiPSC-CMs within the constructs exhibit significantly greater levels of contractile stress, beat frequency, and contraction-relaxation rates, suggesting their improved maturation. Our results demonstrate a novel application of Faraday waves to create stem cell-derived 3D cardiac tissue that resembles the cellular architecture of a native heart tissue for diverse basic research and clinical applications.
View details for DOI 10.1016/j.biomaterials.2017.03.037
View details for PubMedID 28376365
Contractile force generation by 3D hiPSC-derived cardiac tissues is enhanced by rapid establishment of cellular interconnection in matrix with muscle-mimicking stiffness
2017; 131: 111-120
Engineering 3D human cardiac tissues is of great importance for therapeutic and pharmaceutical applications. As cardiac tissue substitutes, extracellular matrix-derived hydrogels have been widely explored. However, they exhibit premature degradation and their stiffness is often orders of magnitude lower than that of native cardiac tissue. There are no reports on establishing interconnected cardiomyocytes in 3D hydrogels at physiologically-relevant cell density and matrix stiffness. Here we bioengineer human cardiac microtissues by encapsulating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in chemically-crosslinked gelatin hydrogels (1.25 × 10(8)/mL) with tunable stiffness and degradation. In comparison to the cells in high stiffness (16 kPa)/slow degrading hydrogels, hiPSC-CMs in low stiffness (2 kPa)/fast degrading and intermediate stiffness (9 kPa)/intermediate degrading hydrogels exhibit increased intercellular network formation, α-actinin and connexin-43 expression, and contraction velocity. Only the 9 kPa microtissues exhibit organized sarcomeric structure and significantly increased contractile stress. This demonstrates that muscle-mimicking stiffness together with robust cellular interconnection contributes to enhancement in sarcomeric organization and contractile function of the engineered cardiac tissue. This study highlights the importance of intercellular connectivity, physiologically-relevant cell density, and matrix stiffness to best support 3D cardiac tissue engineering.
View details for DOI 10.1016/j.biomaterials.2017.03.039
View details for PubMedID 28384492
YY1 Expression is Sufficient for the Maintenance of Cardiac Progenitor Cell State.
During cardiac development, DNA binding transcription factors and epigenetic modifiers regulate gene expression in cardiac progenitor cells (CPCs). We have previously shown that Yin Yang 1 (YY1) is essential for the commitment of mesodermal precursors into CPCs. However, the role of YY1 in the maintenance of CPC phenotype and their differentiation into cardiomyocytes is unknown. In this study, we found, by genome-wide transcriptional profiling and phenotypic assays, that YY1 overexpression prevents cardiomyogenic differentiation and maintains the proliferative capacity of CPCs. We show further that the ability of YY1 to regulate CPC phenotype is associated with its ability to modulate histone modifications specifically at a developmentally critical enhancer of Nkx2-5 and other key cardiac transcription factor such as Tbx5. Specifically, YY1 overexpression helps to maintain markers of gene activation such as the acetylation of histone H3 at lysine 9 (H3K9Ac) and lysine 27 (H3K27Ac) as well as trimethylation at lysine 4 (H3K4Me3) at the Nkx2-5 cardiac enhancer. Furthermore, transcription factors associated proteins such as PoIII, p300, and Brg1 are also enriched at the Nkx2-5 enhancer with YY1 overexpression. The biological activities of YY1 in CPCs appear to be cell autonomous, based coculture assays in differentiating embryonic stem cells. Altogether, these results demonstrate that YY1 overexpression is sufficient to maintain a CPC phenotype through its ability to sustain the presence of activating epigenetic/chromatin marks at key cardiac enhancers. Stem Cells 2017.
View details for DOI 10.1002/stem.2646
View details for PubMedID 28580685
Untangling the Biology of Genetic Cardiomyopathies with Pluripotent Stem Cell Disease Models
CURRENT CARDIOLOGY REPORTS
2017; 19 (4)
Recently, the discovery of strategies to reprogram somatic cells into induced pluripotent stem (iPS) cells has led to a major paradigm change in developmental and stem cell biology. The application of iPS cells and their cardiac progeny has opened novel directions to study cardiomyopathies at a cellular and molecular level. This review discusses approaches currently undertaken to unravel known inherited cardiomyopathies in a dish.With improved efficiency for mutation correction by genome editing, human iPS cells have now provided a platform to untangle the biology of cardiomyopathies. Multiple studies have derived pluripotent stem cells lines from patients with genetic heart diseases. The generation of cardiomyocytes from these cells lines has, for the first time, enable the study of cardiomyopathies using cardiomyocytes harboring patient-specific mutations and their corrected isogenic counterpart. The molecular analyses, functional assays, and drug tests of these lines have led to new molecular insights in the early pathophysiology of left ventricular non-compaction cardiomyopathy (LVNC), hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), arrhythmogenic right ventricular cardiomyopathy (ARVC), and others. The advent of iPS cells offers an exceptional opportunity for creating disease-specific cellular models to investigate their underlying mechanisms and to optimize future therapy through drug and toxicity screening. Thus far, the iPS cell model has improved our understanding of the genetic and molecular pathophysiology of patients with various genetic cardiomyopathies. It is hoped that the new discoveries arising from using these novel platforms for cardiomyopathy research will lead to new diagnostic and therapeutic approaches to prevent and treat these diseases.
View details for DOI 10.1007/s11886-017-0842-1
View details for Web of Science ID 000399238000003
View details for PubMedID 28315121
Partial Reprogramming of Pluripotent Stem Cell-Derived Cardiomyocytes into Neurons
Direct reprogramming of somatic cells has been demonstrated, however, it is unknown whether electrophysiologically-active somatic cells derived from separate germ layers can be interconverted. We demonstrate that partial direct reprogramming of mesoderm-derived cardiomyocytes into neurons is feasible, generating cells exhibiting structural and electrophysiological properties of both cardiomyocytes and neurons. Human and mouse pluripotent stem cell-derived CMs (PSC-CMs) were transduced with the neurogenic transcription factors Brn2, Ascl1, Myt1l and NeuroD. We found that CMs adopted neuronal morphologies as early as day 3 post-transduction while still retaining a CM gene expression profile. At week 1 post-transduction, we found that reprogrammed CMs expressed neuronal markers such as Tuj1, Map2, and NCAM. At week 3 post-transduction, mature neuronal markers such as vGlut and synapsin were observed. With single-cell qPCR, we temporally examined CM gene expression and observed increased expression of neuronal markers Dcx, Map2, and Tubb3. Patch-clamp analysis confirmed the neuron-like electrophysiological profile of reprogrammed CMs. This study demonstrates that PSC-CMs are amenable to partial neuronal conversion, yielding a population of cells exhibiting features of both neurons and CMs.
View details for DOI 10.1038/srep44840
View details for Web of Science ID 000396983300001
View details for PubMedID 28327614
View details for PubMedCentralID PMC5361100
Cardiac Regeneration Lessons From Development
2017; 120 (6): 941-959
Palliative surgery for congenital heart disease has allowed patients with previously lethal heart malformations to survive and, in most cases, to thrive. However, these procedures often place pressure and volume loads on the heart, and over time, these chronic loads can cause heart failure. Current therapeutic options for initial surgery and chronic heart failure that results from failed palliation are limited, in part, by the mammalian heart's low inherent capacity to form new cardiomyocytes. Surmounting the heart regeneration barrier would transform the treatment of congenital, as well as acquired, heart disease and likewise would enable development of personalized, in vitro cardiac disease models. Although these remain distant goals, studies of heart development are illuminating the path forward and suggest unique opportunities for heart regeneration, particularly in fetal and neonatal periods. Here, we review major lessons from heart development that inform current and future studies directed at enhancing cardiac regeneration.
View details for DOI 10.1161/CIRCRESAHA.116.309040
View details for Web of Science ID 000397330700007
View details for PubMedID 28302741
Strategies for the acquisition of transcriptional and epigenetic information in single cells.
Journal of thoracic disease
2017; 9: S9-S16
As the basic unit of living organisms, each single cell has unique molecular signatures and functions. Our ability to uncover the transcriptional and epigenetic signature of single cells has been hampered by the lack of tools to explore this area of research. The advent of microfluidic single cell technology along with single cell genome-wide DNA amplification methods had greatly improved our understanding of the expression variation in single cells. Transcriptional expression profile by multiplex qPCR or genome-wide RNA sequencing has enabled us to examine genes expression in single cells in different tissues. With the new tools, the identification of new cellular heterogeneity, novel marker genes, unique subpopulations, and spatial locations of each single cell can be acquired successfully. Epigenetic modifications for each single cell can also be obtained via similar methods. Based on single cell genome sequencing, single cell epigenetic information including histone modifications, DNA methylation, and chromatin accessibility have been explored and provided valuable insights regarding gene regulation and disease prognosis. In this article, we review the development of strategies to obtain single cell transcriptional and epigenetic data. Furthermore, we discuss ways in which single cell studies may help to provide greater understanding of the mechanisms of basic cardiovascular biology that will eventually lead to improvement in our ability to diagnose disease and develop new therapies.
View details for DOI 10.21037/jtd.2016.08.17
View details for PubMedID 28446964
- Mammalian Heart Regeneration: The Race to the Finish Line. Circulation research 2017; 120 (4): 630-632
High-throughput screening of tyrosine kinase inhibitor cardiotoxicity with human induced pluripotent stem cells.
Science translational medicine
2017; 9 (377)
Tyrosine kinase inhibitors (TKIs), despite their efficacy as anticancer therapeutics, are associated with cardiovascular side effects ranging from induced arrhythmias to heart failure. We used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), generated from 11 healthy individuals and 2 patients receiving cancer treatment, to screen U.S. Food and Drug Administration-approved TKIs for cardiotoxicities by measuring alterations in cardiomyocyte viability, contractility, electrophysiology, calcium handling, and signaling. With these data, we generated a "cardiac safety index" to reflect the cardiotoxicities of existing TKIs. TKIs with low cardiac safety indices exhibit cardiotoxicity in patients. We also derived endothelial cells (hiPSC-ECs) and cardiac fibroblasts (hiPSC-CFs) to examine cell type-specific cardiotoxicities. Using high-throughput screening, we determined that vascular endothelial growth factor receptor 2 (VEGFR2)/platelet-derived growth factor receptor (PDGFR)-inhibiting TKIs caused cardiotoxicity in hiPSC-CMs, hiPSC-ECs, and hiPSC-CFs. With phosphoprotein analysis, we determined that VEGFR2/PDGFR-inhibiting TKIs led to a compensatory increase in cardioprotective insulin and insulin-like growth factor (IGF) signaling in hiPSC-CMs. Up-regulating cardioprotective signaling with exogenous insulin or IGF1 improved hiPSC-CM viability during cotreatment with cardiotoxic VEGFR2/PDGFR-inhibiting TKIs. Thus, hiPSC-CMs can be used to screen for cardiovascular toxicities associated with anticancer TKIs, and the results correlate with clinical phenotypes. This approach provides unexpected insights, as illustrated by our finding that toxicity can be alleviated via cardioprotective insulin/IGF signaling.
View details for DOI 10.1126/scitranslmed.aaf2584
View details for PubMedID 28202772
The relationship between cardiac endothelium and fibroblasts: it's complicated.
The Journal of clinical investigation
Coronary revascularization is an effective means of treating ischemic heart disease; however, current therapeutic revascularization strategies are limited to large caliber vessels. Because the mammalian heart scars following cardiac injury, recent work showing that cardiac fibroblasts can transdifferentiate into new coronary endothelium raises a new and exciting approach to promoting endogenous revascularization following cardiac injury. In this issue of the JCI, He et al. report on their employment of a battery of lineage-tracing tools to address the developmental origins of fibroblasts that give rise to new endothelial cells. Surprisingly, cardiac fibroblasts did not appear to contribute appreciably to regeneration of cardiac endothelium. Instead, cardiac endothelial cells were likely to proliferate and generate new endothelium following injury. As these conclusions diverge from prior findings, additional work will be required to understand the sources that generate cardiac endothelium in new blood vessels after injury. Clarification of the origins of coronary endothelial cells during cardiac repair is essential for identifying improved approaches to revascularizing damaged myocardium in patients with ischemic heart disease.
View details for PubMedID 28650344
Bioengineering cardiac constructs using 3D printing
Journal of 3D Printing in Medicine
2017; 1 (2): 1-8
View details for DOI 10.2217/3dp-2016-0009
Tissue Engineering of 3D Organotypic Microtissues by Acoustic Assembly.
Methods in molecular biology (Clifton, N.J.)
There is a rapidly growing interest in generation of 3D organotypic microtissues with human physiologically relevant structure, function, and cell population in a wide range of applications including drug screening, in vitro physiological/pathological models, and regenerative medicine. Here, we provide a detailed procedure to generate structurally defined 3D organotypic microtissues from cells or cell spheroids using acoustic waves as a biocompatible and scaffold-free tissue engineering tool.
View details for PubMedID 28921421
In vivo rescue of the hematopoietic niche by pluripotent stem cell complementation of defective osteoblast compartments.
Stem cells (Dayton, Ohio)
Bone-forming osteoblasts play critical roles in supporting bone marrow hematopoiesis. Pluripotent stem cells (PSCs), including embryonic stem (ES) cells and induced pluripotent stem (iPS) cells, are capable of differentiating into osteoblasts. To determine the capacity of stem cells needed to rescue aberrant skeletal development and bone marrow hematopoiesis in vivo, we employed a skeletal complementation model. Mice deficient in Runx2, a master transcription factor for osteoblastogenesis, fail to form a mineralized skeleton and bone marrow. Wild-type GFP(+) ES and YFP(+) iPS cells were introduced into Runx2-null blastocyst-stage embryos. We assessed GFP/YFP(+) cell contribution by whole-mount fluorescence and histological analysis and found that the proportion of PSCs in the resulting chimeric embryos is directly correlated with the degree of mineralization in the skull. Moreover, PSC contribution to long bones successfully restored bone marrow hematopoiesis. We validated this finding in a separate model with diphtheria toxin A-mediated ablation of hypertrophic chondrocytes and osteoblasts. Remarkably, chimeric embryos harboring as little as 37.5% wild-type PSCs revealed grossly normal skeletal morphology, suggesting a near-complete rescue of skeletogenesis. In summary, we demonstrate that fractional contribution of PSCs in vivo is sufficient to complement and reconstitute an osteoblast-deficient skeleton and hematopoietic marrow. Further investigation using genetically modified PSCs with conditional loss of gene function in osteoblasts will enable us to address the specific roles of signaling mediators to regulate bone formation and hematopoietic niches in vivo. This article is protected by copyright. All rights reserved.
View details for PubMedID 28741855
Nkx2.5+ Cardiomyoblasts Contribute to Cardiomyogenesis in the Neonatal Heart.
2017; 7 (1): 12590
During normal lifespan, the mammalian heart undergoes limited renewal of cardiomyocytes. While the exact mechanism for this renewal remains unclear, two possibilities have been proposed: differentiated myocyte replication and progenitor/immature cell differentiation. This study aimed to characterize a population of cardiomyocyte precursors in the neonatal heart and to determine their requirement for cardiac development. By tracking the expression of an embryonic Nkx2.5 cardiac enhancer, we identified cardiomyoblasts capable of differentiation into striated cardiomyocytes in vitro. Genome-wide expression profile of neonatal Nkx2.5+ cardiomyoblasts showed the absence of sarcomeric gene and the presence of cardiac transcription factors. To determine the lineage contribution of the Nkx2.5+ cardiomyoblasts, we generated a doxycycline suppressible Cre transgenic mouse under the regulation of the Nkx2.5 enhancer and showed that neonatal Nkx2.5+ cardiomyoblasts mature into cardiomyocytes in vivo. Ablation of neonatal cardiomyoblasts resulted in ventricular hypertrophy and dilation, supporting a functional requirement of the Nkx2.5+ cardiomyoblasts. This study provides direct lineage tracing evidence that a cardiomyoblast population contributes to cardiogenesis in the neonatal heart. The cell population identified here may serve as a promising therapeutic for pediatric cardiac regeneration.
View details for PubMedID 28974782
Identification of a hybrid myocardial zone in the mammalian heart after birth.
2017; 8 (1): 87
Noncompaction cardiomyopathy is characterized by the presence of extensive trabeculations, which could lead to heart failure and malignant arrhythmias. How trabeculations resolve to form compact myocardium is poorly understood. Elucidation of this process is critical to understanding the pathophysiology of noncompaction disease. Here we use genetic lineage tracing to mark the Nppa(+) or Hey2(+) cardiomyocytes as trabecular and compact components of the ventricular wall. We find that Nppa(+) and Hey2(+) cardiomyocytes, respectively, from the endocardial and epicardial zones of the ventricular wall postnatally. Interposed between these two postnatal layers is a hybrid zone, which is composed of cells derived from both the Nppa(+) and Hey2(+) populations. Inhibition of the fetal Hey2(+) cell contribution to the hybrid zone results in persistence of excessive trabeculations in postnatal heart. Our findings indicate that the expansion of Hey2(+) fetal compact component, and its contribution to the hybrid myocardial zone, are essential for normal formation of the ventricular walls.Fetal trabecular muscles in the heart undergo a poorly described morphogenetic process that results into a solidified compact myocardium after birth. Tian et al. show that cardiomyocytes in the fetal compact layer also contribute to this process, forming a hybrid myocardial zone that is composed of cells derived from both trabecular and compact layers.
View details for PubMedID 28729659
Integrative Analysis of PRKAG2 Cardiomyopathy iPS and Microtissue Models Identifies AMPK as a Regulator of Metabolism, Survival, and Fibrosis
2016; 17 (12): 3292-3304
AMP-activated protein kinase (AMPK) is a metabolic enzyme that can be activated by nutrient stress or genetic mutations. Missense mutations in the regulatory subunit, PRKAG2, activate AMPK and cause left ventricular hypertrophy, glycogen accumulation, and ventricular pre-excitation. Using human iPS cell models combined with three-dimensional cardiac microtissues, we show that activating PRKAG2 mutations increase microtissue twitch force by enhancing myocyte survival. Integrating RNA sequencing with metabolomics, PRKAG2 mutations that activate AMPK remodeled global metabolism by regulating RNA transcripts to favor glycogen storage and oxidative metabolism instead of glycolysis. As in patients with PRKAG2 cardiomyopathy, iPS cell and mouse models are protected from cardiac fibrosis, and we define a crosstalk between AMPK and post-transcriptional regulation of TGFβ isoform signaling that has implications in fibrotic forms of cardiomyopathy. Our results establish critical connections among metabolic sensing, myocyte survival, and TGFβ signaling.
View details for DOI 10.1016/j.celrep.2016.11.066
View details for Web of Science ID 000390895600019
View details for PubMedID 28009297
Inhibition of Apoptosis Overcomes Stage-Related Compatibility Barriers to Chimera Formation in Mouse Embryos.
Cell stem cell
2016; 19 (5): 587-592
Cell types more advanced in development than embryonic stem cells, such as EpiSCs, fail to contribute to chimeras when injected into pre-implantation-stage blastocysts, apparently because the injected cells undergo apoptosis. Here we show that transient promotion of cell survival through expression of the anti-apoptotic gene BCL2 enables EpiSCs and Sox17(+) endoderm progenitors to integrate into blastocysts and contribute to chimeric embryos. Upon injection into blastocyst, BCL2-expressing EpiSCs contributed to all bodily tissues in chimeric animals while Sox17(+) endoderm progenitors specifically contributed in a region-specific fashion to endodermal tissues. In addition, BCL2 expression enabled rat EpiSCs to contribute to mouse embryonic chimeras, thereby forming interspecies chimeras that could survive to adulthood. Our system therefore provides a method to overcome cellular compatibility issues that typically restrict chimera formation. Application of this type of approach could broaden the use of embryonic chimeras, including region-specific chimeras, for basic developmental biology research and regenerative medicine.
View details for DOI 10.1016/j.stem.2016.10.013
View details for PubMedID 27814480
iPSC-derived cardiomyocytes reveal abnormal TGF-ß signalling in left ventricular non-compaction cardiomyopathy.
Nature cell biology
2016; 18 (10): 1031-1042
Left ventricular non-compaction (LVNC) is the third most prevalent cardiomyopathy in children and its pathogenesis has been associated with the developmental defect of the embryonic myocardium. We show that patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) generated from LVNC patients carrying a mutation in the cardiac transcription factor TBX20 recapitulate a key aspect of the pathological phenotype at the single-cell level and this was associated with perturbed transforming growth factor beta (TGF-β) signalling. LVNC iPSC-CMs have decreased proliferative capacity due to abnormal activation of TGF-β signalling. TBX20 regulates the expression of TGF-β signalling modifiers including one known to be a genetic cause of LVNC, PRDM16, and genome editing of PRDM16 caused proliferation defects in iPSC-CMs. Inhibition of TGF-β signalling and genome correction of the TBX20 mutation were sufficient to reverse the disease phenotype. Our study demonstrates that iPSC-CMs are a useful tool for the exploration of pathological mechanisms underlying poorly understood cardiomyopathies including LVNC.
View details for DOI 10.1038/ncb3411
View details for PubMedID 27642787
Endocardium Minimally Contributes to Coronary Endothelium in the Embryonic Ventricular Free Walls
2016; 118 (12): 1880-?
There is persistent uncertainty regarding the developmental origins of coronary vessels, with two principal sources suggested as ventricular endocardium or sinus venosus (SV). These two proposed origins implicate fundamentally distinct mechanisms of vessel formation. Resolution of this controversy is critical for deciphering the programs that result in the formation of coronary vessels, and has implications for research on therapeutic angiogenesis.To resolve the controversy over the developmental origin of coronary vessels.We first generatedNfatc1-CreandNfatc1-Drelineage tracers for endocardium labeling. We found that Nfatc1 recombinases also label a significant portion of SV endothelial cells in addition to endocardium. Therefore, restricted endocardial lineage tracing requires a specific marker that distinguishes endocardium from SV. By single cell gene expression analysis, we identified a novel endocardial gene natriuretic peptide receptor 3 (Npr3). Npr3 is expressed in the entirety of the endocardium but not in the SV. Genetic lineage tracing based onNpr3-CreERshowed that endocardium contributes to a minority of coronary vessels in the free walls of embryonic heart. Intersectional genetic lineage tracing experiments demonstrated that endocardium minimally contributes to coronary endothelium in the embryonic ventricular free walls.Our study suggested that SV, but not endocardium, is the major origin for coronary endothelium in the embryonic ventricular free walls. This work thus resolves the recent controversy over the developmental origin of coronary endothelium, providing the basis for studying coronary vessel formation and regeneration after injury.
View details for DOI 10.1161/CIRCRESAHA.116.308749
View details for Web of Science ID 000377885100009
View details for PubMedID 27056912
Distilling complexity to advance cardiac tissue engineering
SCIENCE TRANSLATIONAL MEDICINE
2016; 8 (342)
The promise of cardiac tissue engineering is in the ability to recapitulate in vitro the functional aspects of a healthy heart and disease pathology as well as to design replacement muscle for clinical therapy. Parts of this promise have been realized; others have not. In a meeting of scientists in this field, five central challenges or "big questions" were articulated that, if addressed, could substantially advance the current state of the art in modeling heart disease and realizing heart repair.
View details for DOI 10.1126/scitranslmed.aad2304
View details for Web of Science ID 000377443800001
View details for PubMedID 27280684
Regenerative Medicine: Potential Mechanisms of Cardiac Recovery in Takotsubo Cardiomyopathy.
Current treatment options in cardiovascular medicine
2016; 18 (3): 20-?
Takotsubo cardiomyopathy is an increasingly reported cause of acute chest pain and acute heart failure and is often associated with significant hemodynamic compromise. The illness is remarkable for the reversibility in systolic dysfunction seen in the disease course. While the pathophysiology of takotsubo syndrome is not completely elucidated, research suggests the presence of a cytoprotective process that allows the myocardium to recover following the inciting insult. Here, we summarize molecular and histologic studies exploring the response to injury in takotsubo disease and provide some discussion on how they may contribute to further investigations in cardiac recovery and regeneration.
View details for DOI 10.1007/s11936-016-0443-0
View details for PubMedID 26874708
Cardioprotective Actions of TGF beta RI Inhibition Through Stimulating Autocrine/Paracrine of Survivin and Inhibiting Wnt in Cardiac Progenitors
2016; 34 (2): 445-455
Heart failure due to myocardial infarction (MI) is a major cause of morbidity and mortality in the world. We found previously that A83-01, a TGFβRI inhibitor, could facilitate cardiac repair in post-MI mice and induce the expansion of a Nkx2.5+ cardiomyoblast population. The present study aimed to investigate the key autocrine/paracrine factors regulated by A83-01 in the injured heart and the mechanism of cardioprotection by this molecule. Using a previously described transgenic Nkx2.5 enhancer-GFP reporter mice, we isolated cardiac progenitor cells (CPC) including Nkx2.5-GFP+ (Nkx2.5+), sca1+ and Nkx2.5+/sca1+ cells. A83-01 was found to induce proliferation of these three subpopulations mainly through increasing Birc5 expression in the MEK/ERK-dependent pathway. Survivin, encoded by Birc5, could also directly proliferate Nkx2.5+ cells and enhance cultured cardiomyocytes viability. A83-01 could also reverse the down-regulation of Birc5 in post-injured mice hearts (n=6) to expand CPCs. Moreover, the increased Wnt3a in post-injured hearts could decrease CPCs, which could be reversed by A83-01 via inhibiting Fzd6 and WISP1 expressions in CPCs. Next, we used inducible αMHC-cre/mTmG mice to label cardiomyocytes with GFP and non-myocytes with RFP. We found A83-01 preserved more GFP+ myocytes (68.6±3.1% vs 80.9±3.0%; P<0.05, n=6) and fewer renewed RFP+ myocytes (0.026±0.005 vs 0.062±0.008%; P<0.05, n=6) in parallel with less cardiac fibrosis in isoprenaline-injected mice treated with A83-01. TGFβRI inhibition in an injured adult heart could both stimulate the autocrine/paracrine activity of survivin and inhibit Wnt in CPCs to mediate cardioprotection and improve cardiac function. This article is protected by copyright. All rights reserved.
View details for DOI 10.1002/stem.2216
View details for Web of Science ID 000370353200017
View details for PubMedID 26418219
Harnessing the Induction of Cardiomyocyte Proliferation for Cardiac Regenerative Medicine.
Current treatment options in cardiovascular medicine
2015; 17 (10): 404-?
Adult human cardiomyocytes are terminally differentiated and have limited capacity for cell division. Hence, they are not naturally replaced following ischemic injury to the heart. As such, cardiac function is often permanently compromised after an event such as myocardial infarction. In recent years, investigators have focused intensively on ways to reactivate cardiomyocyte mitotic activity in both in vitro cell culture systems and in vivo animal models. In parallel, advances in stem cell biology have allowed for the mass production of patient-specific human cardiomyocytes from human-induced pluripotent stem cells. These cells can be produced via chemically defined differentiation of human pluripotent stem cells in a matter of weeks and could theoretically be utilized directly for therapeutic purposes to replace damaged myocardium. However, stem cell-derived cardiomyocytes, like their adult counterparts, are post-mitotic and incapable of large-scale expansion after reaching a certain stage of in vitro differentiation. Due to this shared characteristic, these stem cell-derived cardiomyocytes may provide a platform for studying genes, pathways, and small molecules that induce cell cycle reentry and proliferation of human cardiomyocytes. Ultimately, the discovery of novel mechanisms or pathways to induce human cardiomyocyte proliferation should improve our ability to regenerate adult cardiomyocytes and help restore cardiac function following injury.
View details for DOI 10.1007/s11936-015-0404-z
View details for PubMedID 26324824
Members Only: Hypoxia-Induced Cell-Cycle Activation in Cardiomyocytes.
2015; 22 (3): 365-366
A low level of cardiomyocyte turnover occurs in the adult mammalian heart, but the source of these new cells remains unknown. Kimura et al., 2015 utilized a novel hypoxia-induced fate mapping system to identify a rare population of adult cardiomyocytes undergoing cell-cycle entry and expansion in healthy adult hearts and following ischemic injury.
View details for DOI 10.1016/j.cmet.2015.08.004
View details for PubMedID 26331604
Integrin Based Isolation Enables Purification of Murine Lineage Committed Cardiomyocytes
2015; 10 (8)
In contrast to mature cardiomyocytes which have limited regenerative capacity, pluripotent stem cells represent a promising source for the generation of new cardiomyocytes. The tendency of pluripotent stem cells to form teratomas and the heterogeneity from various differentiation stages and cardiomyocyte cell sub-types, however, are major obstacles to overcome before this type of therapy could be applied in a clinical setting. Thus, the identification of extracellular markers for specific cardiomyocyte progenitors and mature subpopulations is of particular importance. The delineation of cardiomyocyte surface marker patterns not only serves as a means to derive homogeneous cell populations by FACS, but is also an essential tool to understand cardiac development. By using single-cell expression profiling in early mouse embryonic hearts, we found that a combination of integrin alpha-1, alpha-5, alpha-6 and N-cadherin enables isolation of lineage committed murine cardiomyocytes. Additionally, we were able to separate trabecular cardiomyocytes from solid ventricular myocardium and atrial murine cells. These cells exhibit expected subtype specific phenotype confirmed by electrophysiological analysis. We show that integrin expression can be used for the isolation of living, functional and lineage-specific murine cardiomyocytes.
View details for DOI 10.1371/journal.pone.0135880
View details for Web of Science ID 000360435500010
Fetal Mammalian Heart Generates a Robust Compensatory Response to Cell Loss.
2015; 132 (2): 109-121
-Heart development is tightly regulated by signaling events acting upon a defined number of progenitor and differentiated cardiac cells. While loss-of-function of these signaling pathways leads to congenital malformation, the consequences of cardiac progenitor cell (CPC) or embryonic cardiomyocyte loss are less clear. In this study, we tested the hypothesis that embryonic mouse hearts exhibit a robust mechanism for regeneration following extensive cell loss.-By combining a conditional cell ablation approach with a novel blastocyst complementation strategy, we generated murine embryos that exhibit a full spectrum of CPC or cardiomyocyte ablation. Remarkably, ablation of up to 60% of CPCs at embryonic day 7.5 was well-tolerated and permitted embryo survival. Ablation of embryonic cardiomyocytes to a similar degree (50-60%) at embryonic day 9.0 could be fully rescued by residual myocytes with no obvious adult cardiac functional deficit. In both ablation models, an increase in cardiomyocyte proliferation rate was detected and accounted for at least some of the rapid recovery of myocardial cellularity and heart size.-Our study defines the threshold for cell loss in the embryonic mammalian heart and reveals a robust cardiomyocyte compensatory response that sustains normal fetal development.
View details for DOI 10.1161/CIRCULATIONAHA.114.011490
View details for PubMedID 25995316
Identification of cardiovascular lineage descendants at single-cell resolution.
2015; 142 (5): 846-857
The transcriptional profiles of cardiac cells derived from murine embryos and from mouse embryonic stem cells (mESCs) have primarily been studied within a cell population. However, the characterization of gene expression in these cells at a single-cell level might demonstrate unique variations that cannot be appreciated within a cell pool. In this study, we aimed to establish a single-cell quantitative PCR platform and perform side-by-side comparison between cardiac progenitor cells (CPCs) and cardiomyocytes (CMs) derived from mESCs and mouse embryos. We first generated a reference map for cardiovascular single cells through quantifying lineage-defining genes for CPCs, CMs, smooth muscle cells (SMCs), endothelial cells (EDCs), fibroblasts and mESCs. This panel was then applied against single embryonic day 10.5 heart cells to demonstrate its ability to identify each endocardial cell and chamber-specific CM. In addition, we compared the gene expression profile of embryo- and mESC-derived CPCs and CMs at different developmental stages and showed that mESC-derived CMs are phenotypically similar to embryo-derived CMs up to the neonatal stage. Furthermore, we showed that single-cell expression assays coupled with time-lapse microscopy can resolve the identity and the lineage relationships between progenies of single cultured CPCs. With this approach, we found that mESC-derived Nkx2-5(+) CPCs preferentially become SMCs or CMs, whereas single embryo-derived Nkx2-5(+) CPCs represent two phenotypically distinct subpopulations that can become either EDCs or CMs. These results demonstrate that multiplex gene expression analysis in single cells is a powerful tool for examining the unique behaviors of individual embryo- or mESC-derived cardiac cells.
View details for DOI 10.1242/dev.116897
View details for PubMedID 25633351
- Small RNAs make big impact in cardiac repair. Circulation research 2015; 116 (3): 393-395
Molecular Regulation of Cardiomyocyte Differentiation
2015; 116 (2): 341-353
The heart is the first organ to form during embryonic development. Given the complex nature of cardiac differentiation and morphogenesis, it is not surprising that some form of congenital heart disease is present in ≈1 percent of newborns. The molecular determinants of heart development have received much attention over the past several decades. This has been driven in large part by an interest in understanding the causes of congenital heart disease coupled with the potential of using knowledge from developmental biology to generate functional cells and tissues that could be used for regenerative medicine purposes. In this review, we highlight the critical signaling pathways and transcription factor networks that regulate cardiomyocyte lineage specification in both in vivo and in vitro models. Special focus will be given to epigenetic regulators that drive the commitment of cardiomyogenic cells from nascent mesoderm and their differentiation into chamber-specific myocytes, as well as regulation of myocardial trabeculation.
View details for DOI 10.1161/CIRCRESAHA.116.302752
View details for Web of Science ID 000347939000019
View details for PubMedCentralID PMC4299877
Pharmacological inhibition of TGFß receptor improves Nkx2.5 cardiomyoblast-mediated regeneration.
2015; 105 (1): 44-54
Our previous study found that A83-01, a small molecule type 1 TGFβ receptor inhibitor, could induce proliferation of postnatal Nkx2.5(+) cardiomyoblasts in vitro and enhance their cardiomyogenic differentiation. The present study addresses whether A83-01 treatment in vivo could increase cardiomyogenesis and improve cardiac function after myocardial infarction through an Nkx2.5(+) cardiomyoblast-dependent process.To determine the effect of A83-01 on the number of Nkx2.5(+) cardiomyoblasts in the heart after myocardial injury, we treated transgenic Nkx2.5 enhancer-GFP reporter mice for 7 days with either A83-01 or DMSO and measured the number of GFP(+) cardiomyoblasts in the heart at 1 week after injury by flow cytometry. To determine the degree of new cardiomyocyte formation after myocardial injury and the effect of A83-01 in this process, we employed inducible Nkx2.5 enhancer-Cre transgenic mice to lineage label postnatal Nkx2.5(+) cardiomyoblasts and their differentiated progenies after myocardial injury. We also examined the cardiac function of each animal by intracardiac haemodynamic measurements. We found that A83-01 treatment significantly increased the number of Nkx2.5(+) cardiomyoblasts at baseline and after myocardial injury, resulting in an increase in newly formed cardiomyocytes. Finally, we showed that A83-01 treatment significantly improved ventricular elastance and stroke work, leading to improved contractility after injury.Pharmacological inhibition of TGFβ signalling improved cardiac function in injured mice and promoted the expansion and cardiomyogenic differentiation of Nkx2.5(+) cardiomyoblasts. Direct modulation of resident cardiomyoblasts in vivo may be a promising strategy to enhance therapeutic cardiac regeneration.
View details for DOI 10.1093/cvr/cvu229
View details for PubMedID 25362681
Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation.
Journal of visualized experiments : JoVE
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes available for basic research and translational applications. To differentiate hiPSCs into cardiomyocytes, various protocols including embryoid body (EB)-based differentiation and growth factor induction have been developed. However, these protocols are inefficient and highly variable in their ability to generate purified cardiomyocytes. Recently, a small molecule-based protocol utilizing modulation of Wnt/β-Catenin signaling was shown to promote cardiac differentiation with high efficiency. With this protocol, greater than 50%-60% of differentiated cells were cardiac troponin-positive cardiomyocytes were consistently observed. To further increase cardiomyocyte purity, the differentiated cells were subjected to glucose starvation to specifically eliminate non-cardiomyocytes based on the metabolic differences between cardiomyocytes and non-cardiomyocytes. Using this selection strategy, we consistently obtained a greater than 30% increase in the ratio of cardiomyocytes to non-cardiomyocytes in a population of differentiated cells. These highly purified cardiomyocytes should enhance the reliability of results from human iPSC-based in vitro disease modeling studies and drug screening assays.
View details for DOI 10.3791/52628
View details for PubMedID 25867738
Comparing mouse and human pluripotentstem cell derived cardiac cells: both systemshave advantages for pharmacological and toxicological screening.
Journal of pharmacological and toxicological methods
Pluripotent stem cells offer an unparalleled opportunity to investigate cardiac physiology, pharmacology, toxicology and pathophysiology. In this paper we describe the use of both mouse (Nkx2-5(eGFP/w)) and human (NKX2-5(eGFP/w)) pluripotent stem cell reporter lines, differentiated toward cardiac lineage, for live single cell high acquisition rate calcium imaging. We also assess the potential of NKX2-5(eGFP/w) cardiac lineage cells for use toxicological screening as well as establish their sensitivity to a shift between low and high oxygen environments. Differentiated mouse Nkx2-5(eGFP/w) cells demonstrated a wide range of spontaneous oscillation rates that could be reduced by ryanodine (10 μM), thapsigargin (1 μM) and ZD7288 (10μM). In contrast human NKX2-5(eGFP/w) cell activity was only reduced by thapsigargin (1 μM). Human cells were also sensitive to the addition of trastuzumab and doxorubicin as well as a low oxygen environment. We suggest that the human NKX2-5(eGFP/w) cells are less suitable for studies of compounds affecting cardiac pacemaker activity than mouse Nkx2-5(eGFP/w) cells, but are very suitable for cardiac toxicity studies.
View details for PubMedID 25957031
Patching up broken hearts: cardiac cell therapy gets a bioengineered boost.
Cell stem cell
2014; 15 (6): 671-673
Preclinical and clinical studies for cardiac cell therapy have only seen moderate success due to poor engraftment and survival of transplanted cells. In this issue of Cell Stem Cell, Ye et al. (2014) employ a growth-factor-loaded fibrin patch and show improved cardiovascular cell survival after cell transplantation into a porcine model of ischemia reperfusion.
View details for DOI 10.1016/j.stem.2014.11.008
View details for PubMedID 25479741
Human induced pluripotent stem cell-derived cardiomyocytes as an in vitro model for coxsackievirus b3-induced myocarditis and antiviral drug screening platform.
2014; 115 (6): 556-566
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.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.hiPSC-CMs were infected with a luciferase-expressing coxsackievirus B3 strain (CVB3-Luc). Brightfield microscopy, immunofluorescence, and calcium imaging were used 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 interferonβ1, 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 interferonβ1 treatment.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
View details for PubMedCentralID PMC4149868
Somatic Cell Reprogramming into Cardiovascular Lineages
JOURNAL OF CARDIOVASCULAR PHARMACOLOGY AND THERAPEUTICS
2014; 19 (4): 340-349
Ischemic cardiac disease is the leading cause of death in the developed world. The inability of the adult mammalian heart to adequately repair itself has motivated stem cell researchers to explore various strategies to regenerate cardiomyocytes after myocardial infarction. Over the past century, progressive gains in our knowledge about the cellular mechanisms governing fate determination have led to recent advances in cellular reprogramming. The identification of specific factors capable of inducing pluripotent phenotype in somatic cells as well as factors that can directly reprogram somatic cells into cardiomyocytes suggests the potential for these approaches to translate into clinical therapies in the future. Although conceptually appealing, the field of cell lineage reprogramming is in its infancy, and further research will be needed to improve the efficiency of the reprogramming process and the fidelity of the reprogrammed cells to their in vivo counterpart.
View details for DOI 10.1177/1074248414527641
View details for Web of Science ID 000338394200003
Insulin-like growth factor promotes cardiac lineage induction in vitro by selective expansion of early mesoderm.
2014; 32 (6): 1493-1502
A thorough understanding of the developmental signals that direct pluripotent stem cells (PSCs) towards a cardiac fate is essential for translational applications in disease modeling and therapy. We screened a panel of 44 cytokines/signaling molecules for their ability to enhance Nkx2.5(+) cardiac progenitor cell (CPC) formation during in vitro embryonic stem cell (ESC) differentiation. Treatment of murine ESCs with insulin or insulin-like growth factors (IGF1/2) during early differentiation increased mesodermal cell proliferation and, consequently, CPC formation. Furthermore, we show that downstream mediators of IGF signaling (e.g. phospho-Akt and mTOR) are required for this effect. These data support a novel role for IGF family ligands to expand the developing mesoderm and promote cardiac differentiation. Insulin or IGF treatment could provide an effective strategy to increase the PSC-based generation of CPCs and cardiomyocytes for applications in regenerative medicine. Stem Cells 2014.
View details for DOI 10.1002/stem.1660
View details for PubMedID 24496962
Telocytes in human heart valves
JOURNAL OF CELLULAR AND MOLECULAR MEDICINE
2014; 18 (5): 759-765
Valve interstitial cells (VICs) are responsible for maintaining the structural integrity and dynamic behaviour of the valve. Telocytes (TCs), a peculiar type of interstitial cells, have been recently identified by Popescu's group in epicardium, myocardium and endocardium (visit www.telocytes.com). The presence of TCs has been identified in atria, ventricles and many other tissues and organ, but not yet in heart valves. We used transmission electron microscopy and immunofluorescence methods (double labelling for CD34 and c-kit, or vimentin, or PDGF Receptor-β) to provide evidence for the existence of TCs in human heart valves, including mitral valve, tricuspid valve and aortic valve. TCs are found in both apex and base of heart valves, with a similar density of 27-28 cells/mm(2) in mitral valve, tricuspid valve and aortic valve. Since TCs are known for the participation in regeneration or repair biological processes, it remains to be determined how TCs contributes to the valve attempts to re-establish normal structure and function following injury, especially a complex junction was found between TCs and a putative stem (progenitor) cell.
View details for DOI 10.1111/jcmm.12285
View details for Web of Science ID 000335861500002
View details for PubMedID 24674389
Myeloid zinc finger 1 (mzf1) differentially modulates murine cardiogenesis by interacting with an nkx2.5 cardiac enhancer.
2014; 9 (12): e113775
Vertebrate heart development is strictly regulated by temporal and spatial expression of growth and transcription factors (TFs). We analyzed nine TFs, selected by in silico analysis of an Nkx2.5 enhancer, for their ability to transactivate the respective enhancer element that drives, specifically, expression of genes in cardiac progenitor cells (CPCs). Mzf1 showed significant activity in reporter assays and bound directly to the Nkx2.5 cardiac enhancer (Nkx2.5 CE) during murine ES cell differentiation. While Mzf1 is established as a hematopoietic TF, its ability to regulate cardiogenesis is completely unknown. Mzf1 expression was significantly enriched in CPCs from in vitro differentiated ES cells and in mouse embryonic hearts. To examine the effect of Mzf1 overexpression on CPC formation, we generated a double transgenic, inducible, tetOMzf1-Nkx2.5 CE eGFP ES line. During in vitro differentiation an early and continuous Mzf1 overexpression inhibited CPC formation and cardiac gene expression. A late Mzf1 overexpression, coincident with a second physiological peak of Mzf1 expression, resulted in enhanced cardiogenesis. These findings implicate a novel, temporal-specific role of Mzf1 in embryonic heart development. Thereby we add another piece of puzzle in understanding the complex mechanisms of vertebrate cardiac development and progenitor cell differentiation. Consequently, this knowledge will be of critical importance to guide efficient cardiac regenerative strategies and to gain further insights into the molecular basis of congenital heart malformations.
View details for DOI 10.1371/journal.pone.0113775
View details for PubMedID 25436607
View details for PubMedCentralID PMC4249966
Patching Up Broken Hearts: Cardiac Cell Therapy Gets a Bioengineered Boost
Cell Stem Cell
2014; 15 (6): 671–673
Preclinical and clinical studies for cardiac cell therapy have only seen moderate success due to poor engraftment and survival of transplanted cells. In this issue of Cell Stem Cell, Ye et al. (2014) employ a growth-factor-loaded fibrin patch and show improved cardiovascular cell survival after cell transplantation into a porcine model of ischemia reperfusion.
View details for DOI 10.1016/j.stem.2014.11.008
- Induced pluripotent stem cell-derived cardiomyocytes for cardiovascular disease modeling and drug screening STEM CELL RESEARCH & THERAPY 2013; 4
Screening drug-induced arrhythmia events 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
View details for PubMedCentralID PMC3855862
Meta-Analysis of Stem Cell Therapy in Chronic Ischemic Cardiomyopathy
AMERICAN JOURNAL OF CARDIOLOGY
2013; 112 (2): 217-225
Studies investigating bone marrow stem cell therapy (BMSCT) in patients with chronic ischemic cardiomyopathy have yielded mixed results. A meta-analysis of randomized controlled trials of BMSCT in patients with chronic ischemic cardiomyopathy was undertaken to assess its efficacy and the best route of administration. The MEDLINE, Embase, Cumulative Index to Nursing & Allied Health Literature, and Cochrane Library databases were searched through April 2012 for randomized controlled trials that investigated the impact of BMSCT and its route of administration on left ventricular (LV) function in patients with chronic ischemic cardiomyopathy and systolic dysfunction. Of the 226 reports identified, 10 randomized controlled trials including 519 patients (average LV ejection fraction [LVEF] at baseline 32 ± 7%) were included in the analysis. On the basis of a random-effects model, BMSCT improved the LVEF at 6 months by 4.48% (95% confidence interval [CI] 2.43% to 6.53%, p = 0.0001). A greater improvement in the LVEF was seen with intramyocardial injection compared with intracoronary infusion (5.13% [95% CI 3.17% to 7.10%], p <0.00001, vs 2.32% [95% CI -2.06% to 6.70%], p = 0.30). Overall, there were reductions in LV end-systolic volume of -20.64 ml (95% CI -33.21 to -8.07, p = 0.001) and LV end-diastolic volume of -16.71 ml (95% CI -31.36 to -2.06, p = 0.03). In conclusion, stem cell therapy may improve LVEF and favorably remodel the heart in patients with chronic ischemic cardiomyopathy. On the basis of current limited data, intramyocardial injection may be superior to intracoronary infusion in patients with LV systolic dysfunction.
View details for DOI 10.1016/j.amjcard.2013.03.021
View details for Web of Science ID 000322206500013
View details for PubMedID 23623290
A83-01, a TGF beta RI inhibitor, can proliferate adult cardiac progenitor cells and improve cardiac contractility of myocardial infarcted mice
ACTA PHARMACOLOGICA SINICA. 2013: 57–57
View details for Web of Science ID 000322051500249
- Autophagy - the friendly fire in endothelial cell regeneration. Focus on "Autophagy in endothelial progenitor cells is cytoprotective in hypoxic conditions" AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY 2013; 304 (7): C614-C616
- At a Crossroad Cell Therapy for Cardiac Repair CIRCULATION RESEARCH 2013; 112 (6): 884-890
Essential and Unexpected Role of Yin Yang 1 to Promote Mesodermal Cardiac Differentiation
2013; 112 (6): 900-U104
Cardiogenesis is regulated by a complex interplay between transcription factors. However, little is known about how these interactions regulate the transition from mesodermal precursors to cardiac progenitor cells (CPCs). Objective: To identify novel regulators of mesodermal cardiac lineage commitment.We performed a bioinformatic-based transcription factor binding site analysis on upstream promoter regions of genes that are enriched in embryonic stem cell-derived CPCs. From 32 candidate transcription factors screened, we found that Yin Yang 1 (YY1), a repressor of sarcomeric gene expression, is present in CPCs in vivo. Interestingly, we uncovered the ability of YY1 to transcriptionally activate Nkx2.5, a key marker of early cardiogenic commitment. YY1 regulates Nkx2.5 expression via a 2.1-kb cardiac-specific enhancer as demonstrated by in vitro luciferase-based assays, in vivo chromatin immunoprecipitation, and genome-wide sequencing analysis. Furthermore, the ability of YY1 to activate Nkx2.5 expression depends on its cooperative interaction with Gata4 at a nearby chromatin. Cardiac mesoderm-specific loss-of-function of YY1 resulted in early embryonic lethality. This was corroborated in vitro by embryonic stem cell-based assays in which we showed that the overexpression of YY1 enhanced the cardiogenic differentiation of embryonic stem cells into CPCs.These results demonstrate an essential and unexpected role for YY1 to promote cardiogenesis as a transcriptional activator of Nkx2.5 and other CPC-enriched genes.
View details for DOI 10.1161/CIRCRESAHA.113.259259
View details for Web of Science ID 000316189900007
View details for PubMedID 23307821
View details for PubMedCentralID PMC3629954
- Of fish and men: clonal lineage analysis identifies divergence in myocardial development. Circulation research 2013; 112 (4): 583-585
iPS Cell Modeling of Cardiometabolic Diseases
JOURNAL OF CARDIOVASCULAR TRANSLATIONAL RESEARCH
2013; 6 (1): 46-53
Cardiometabolic diseases encompass simple monogenic enzyme deficiencies with well-established pathogenesis and clinical outcomes to complex polygenic diseases such as the cardiometabolic syndrome. The limited availability of relevant human cell types such as cardiomyocytes has hampered our ability to adequately model and study pathways or drugs relevant to these diseases in the heart. The recent discovery of induced pluripotent stem (iPS) cell technology now offers a powerful opportunity to establish translational platforms for cardiac disease modeling, drug discovery, and pre-clinical testing. In this article, we discuss the excitement and challenges of modeling cardiometabolic diseases using iPS cell and their potential to revolutionize translational research.
View details for DOI 10.1007/s12265-012-9413-4
View details for Web of Science ID 000313657700006
View details for PubMedID 23070616
View details for PubMedCentralID PMC3547131
Early cardiac development: a view from stem cells to embryos
2012; 96 (3): 352-362
From the 1920s, early cardiac development has been studied in chick and, later, in mouse embryos in order to understand the first cell fate decisions that drive specification and determination of the endocardium, myocardium, and epicardium. More recently, mouse and human embryonic stem cells (ESCs) have demonstrated faithful recapitulation of early cardiogenesis and have contributed significantly to this research over the past few decades. Derived almost 15 years ago, human ESCs have provided a unique developmental model for understanding the genetic and epigenetic regulation of early human cardiogenesis. Here, we review the biological concepts underlying cell fate decisions during early cardiogenesis in model organisms and ESCs. We draw upon both pioneering and recent studies and highlight the continued role for in vitro stem cells in cardiac developmental biology.
View details for DOI 10.1093/cvr/cvs270
View details for Web of Science ID 000311306800006
View details for PubMedID 22893679
- Reprogramming the Beat Kicking It Up a Notch CIRCULATION 2012; 126 (9): 1009-1011
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
View details for PubMedCentralID PMC3390172
Small molecule regulators of postnatal Nkx2.5 cardiomyoblast proliferation and differentiation
JOURNAL OF CELLULAR AND MOLECULAR MEDICINE
2012; 16 (5): 961-965
While recent data have supported the capacity for a neonatal heart to undergo cardiomyogenesis, it is unclear whether these new cardiomyocytes arise from an immature cardiomyoblast population or from the division of mature cardiomyocytes. By following the expression of enhanced Green Fluorescent Protein (eGFP) in an Nkx2.5 enhancer-eGFP transgenic mice, we have identified a population of immature cells that can undergo cardiomyogenic as well as smooth muscle cell differentiation in the neonatal heart. Here, we examined growth factors and small molecule regulators that potentially regulate the proliferation and cardiomyogenic versus smooth muscle cell differentiation of neonatal Nkx2.5-GFP (+) cells in vitro. We found that A83-01 (A83), an inhibitor of TGF-βRI, was able to induce an expansion of neonatal Nkx2.5-eGFP (+) cells. In addition, the ability of A83 to expand eGFP (+) cells in culture was dependent on signalling from the mitogen-activated protein kinase kinase (MEK) as treatment with a MEK inhibitor, PD0325901, abolished this effect. On the other hand, activation of neonatal Nkx2.5-eGFP (+) cells with TGF-β1, but not activin A nor BMP2, led to smooth muscle cell differentiation, an effect that can be reversed by treatment with A83. In summary, small molecule inhibition of TGF-β signalling may be a promising strategy to induce the expansion of a rare population of postnatal cardiomyoblasts.
View details for DOI 10.1111/j.1582-4934.2011.01513.x
View details for Web of Science ID 000303239500002
View details for PubMedID 22212626
- Putting the Pieces Together: Stem Cells and The Quest to Heal A Broken Heart CardioSource World News 2012; 12 (null): 22-27
Induced pluripotent stem cell modeling of complex genetic diseases.
Drug discovery today. Disease models
2012; 9 (4): e147–e152
The study of complex disease genetics by genome-wide association studies (GWAS) has led to hundreds of genomic loci associated with disease traits in humans. However, the functional consequences of most loci are largely undefined. We discuss here the potential for human induced pluripotent stem (iPS) cells to bridge the gap between genetic variant and mechanisms of complex disease. We also highlight specific diseases and the roadblocks that must be overcome before iPS cell technology can be widely adopted for complex disease modeling.
View details for DOI 10.1016/j.ddmod.2012.04.002
View details for PubMedID 23690830
View details for PubMedCentralID PMC3653342
- A Brief Primer on the Development of the Heart Heart Failure, 2nd Ed. 2012: Chapter 1
Epigenetic mechanisms in cardiac development and disease
ACTA BIOCHIMICA ET BIOPHYSICA SINICA
2012; 44 (1): 92-102
During mammalian development, cardiac specification and ultimately lineage commitment to a specific cardiac cell type is accomplished by the action of specific transcription factors (TFs) and their meticulous control on an epigenetic level. In this review, we detail how cardiac-specific TFs function in concert with nucleosome remodeling and histone-modifying enzymes to regulate a diverse network of genes required for processes such as cell growth and proliferation, or epithelial to mesenchymal transition (EMT), for instance. We provide examples of how several cardiac TFs, such as Nkx2.5, WHSC1, Tbx5, and Tbx1, which are associated with developmental and congenital heart defects, are required for the recruitment of histone modifiers, such as Jarid2, p300, and Ash2l, and components of ATP-dependent remodeling enzymes like Brg1, Baf60c, and Baf180. Binding of these TFs to their respective sites at cardiac genes coincides with a distinct pattern of histone marks, indicating that the precise regulation of cardiac gene networks is orchestrated by interactions between TFs and epigenetic modifiers. Furthermore, we speculate that an epigenetic signature, comprised of TF occupancy, histone modifications, and overall chromatin organization, is an underlying mechanism that governs cardiac morphogenesis and disease.
View details for DOI 10.1093/abbs/gmr090
View details for Web of Science ID 000298386700010
View details for PubMedID 22194017
View details for PubMedCentralID PMC3244653
Reprogramming of mouse, rat, pig, and human fibroblasts into iPS cells.
Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]
2012; Chapter 23: Unit 23 15-?
The induction of pluripotency in somatic cells by transcription-factor overexpression has been widely regarded as one of the major breakthroughs in stem cell biology within this decade. The generation of these induced pluripotent stem cells (iPSCs) has enabled investigators to develop in vitro disease models for biological discovery and drug screening, and in the future, patient-specific therapy for tissue or organ regeneration. While new technologies for reprogramming are continually being discovered, the availability of iPSCs from different species is also increasing rapidly. Comparison of iPSCs across species may provide new insights into key aspects of pluripotency and early embryonic development. iPSCs from large animals may enable the generation of genetically modified large animal models or potentially transplantable donor tissues or organs. This unit describes the procedure for the generation of iPSCs from mouse, rat, pig and human fibroblasts.
View details for DOI 10.1002/0471142727.mb2315s97
View details for PubMedID 22237859
Developmental and Regenerative Biology of Multipotent Cardiovascular Progenitor Cells
2011; 108 (3): 353-364
Our limited ability to improve the survival of patients with heart failure is attributable, in part, to the inability of the mammalian heart to meaningfully regenerate itself. The recent identification of distinct families of multipotent cardiovascular progenitor cells from endogenous, as well as exogenous, sources, such as embryonic and induced pluripotent stem cells, has raised much hope that therapeutic manipulation of these cells may lead to regression of many forms of cardiovascular disease. Although the exact source and cell type remains to be clarified, our greater understanding of the scientific underpinning behind developmental cardiovascular progenitor cell biology has helped to clarify the origin and properties of diverse cells with putative cardiogenic potential. In this review, we highlight recent advances in the understanding of cardiovascular progenitor cell biology from embryogenesis to adulthood and their implications for therapeutic cardiac regeneration. We believe that a detailed understanding of cardiogenesis will inform future applications of cardiovascular progenitor cells in heart failure therapy and regenerative medicine.
View details for DOI 10.1161/CIRCRESAHA.110.227066
View details for Web of Science ID 000286930500012
View details for PubMedID 21293007
View details for PubMedCentralID PMC3073355
- Regenerative strategies for cardiac disease In: Stem Cells and Regenerative Medicine. Humana Press. 2011; 1: 579-593
Origin of Cardiac Progenitor Cells in the Developing and Postnatal Heart
JOURNAL OF CELLULAR PHYSIOLOGY
2010; 225 (2): 321-325
The mammalian heart lacks the capacity to replace the large numbers of cardiomyocytes lost due to cardiac injury. Several different cell-based routes to myocardial regeneration have been explored, including transplantation of cardiac progenitors and cardiomyocytes into injured myocardium. As seen with cell-based therapies in other solid organ systems, inherent limitations, such as host immune response, cell death and long-term graft instability have hampered meaningful cardiac regeneration. An understanding of the cell biology of cardiac progenitors, including their developmental origin, lineage markers, renewal pathways, differentiation triggers, microenvironmental niche, and mechanisms of homing and migration to the site of injury, will enable further refinement of therapeutic strategies to enhance clinically meaningful cardiac repair.
View details for DOI 10.1002/jcp.22281
View details for Web of Science ID 000283003400007
View details for PubMedID 20568226
Isolation and functional characterization of pluripotent stem cell-derived cardiac progenitor cells.
Current protocols in stem cell biology
2010; Chapter 1: Unit 1F 10-?
The use of transgenic markers in pluripotent stem cells allows the facile isolation of transient cell populations that appear at certain phases of embryonic development. Here, we describe a procedure for deriving cardiac progenitors from mouse pluripotent stem cells carrying a GFP reporter under the control of an Nkx2.5 enhancer sequence. The cells are propagated under standard conditions and are differentiated using the hanging-droplet method with medium optimized for commitment to the cardiac lineage. Cardiac progenitors are isolated from the differentiation culture using fluorescence-activated cell sorting (FACS) and can be cultured further for functional characterization and experimentation. The protocols described here can be applied to both embryonic and induced pluripotent stem cells and can easily be adapted to transgenic lines carrying other cardiac cell lineage reporters.
View details for DOI 10.1002/9780470151808.sc01f10s14
View details for PubMedID 20814937
View details for PubMedCentralID PMC2947085
Promises and pitfalls in cell replacement therapy for heart failure.
Drug discovery today. Disease mechanisms
2010; 7 (2): e109-e115
Symptomatic heart failure is a complex clinical syndrome with a poor prognosis. Many efforts have been made to develop new therapeutic strategies to improve prognosis associated with heart failure. In this context, different stem cell populations for cardiac regenerative therapy have been examined recently. Here we discuss the potential strategies for using stem cells in cardiac regenerative therapy and the barriers that remain before an effective cell-based cardiac regenerative therapy can be employed clinically.
View details for PubMedID 21180399
- Cardiac progenitor cells: from embryonic to the aging heart. Aging Health 2010; 6 (6): 679-686
The integrative aspects of cardiac physiology and their implications for cell-based therapy
Ananlysis of Cardiac Development: From Embryo to Old Age
Annals of the New York Academy of Sciences. 2010; 1188: 7–14
View details for DOI 10.1111/j.1749-6632.2009.05077.x
Myocardial Injury Induces the Expansion and Cardiomyogenic Differentiation of Postnatal Nkx2.5 Progenitor Cells via Inflammatory Signals
82nd National Conference and Exhibitions and Annual Scientific Session of the American-Heart-Association
LIPPINCOTT WILLIAMS & WILKINS. 2009: S756–S756
View details for Web of Science ID 000271831502235
- VISIONS: the art of science. Molecular reproduction and development 2009; 76 (6): 525-?
Committed Ventricular Progenitors in the Islet-1 Lineage Expand and Assemble Into Functional Ventricular Heart Muscle
58th Annual Scientific Session of the American-College-of-Cardiology
ELSEVIER SCIENCE INC. 2009: A468–A468
View details for Web of Science ID 000263864201940
- Platypnea-orthodeoxia syndrome in two previously healthy adults: a case-based review Clinical Medicine Insights: Cardiology 2009; 3: 37-43
Derivation and Functional Characterization of Nkx2.5+Cardiac Progenitor Cells from Mouse Induced Pluripotent Stem Cells
81st Annual Scientific Session of the American-Heart-Association
LIPPINCOTT WILLIAMS & WILKINS. 2008: S428–S428
View details for Web of Science ID 000262104500714
Mesp1 at the heart of mesoderm lineage specification
CELL STEM CELL
2008; 3 (1): 1-2
Stem cell-based cardiac regeneration requires a detailed understanding of the factors that induce cardiac lineage commitment. In this issue of Cell Stem Cell, Lindsley et al. (2008) and Bondue et al. (2008) use embryonic stem cells to identify a key role for Mesp1 in this process.
View details for DOI 10.1016/j.stem.2008.06.017
View details for Web of Science ID 000257622300001
View details for PubMedID 18593549
Multipotent stem cells in cardiac regenerative therapy
2008; 3 (2): 189-198
The potential for stem cells to ameliorate or cure heart diseases has galvanized a cadre of cardiovascular translational and clinical scientists to take a 'first-in-man' approach using autologous stem cells from a variety of tissues. However, recent clinical trial data show that when these cells are given by intracoronary infusion or direct myocardial injection, limited improvement in heart function occurs with no evidence of cardiomyogenesis. These studies illustrate the great need to understand the logic of cell-lineage commitment and the principles of cardiac differentiation. Recent identification of stem/progenitor cells of embryological origin with intrinsic competence to differentiate into multiple lineages within the heart offers new possibilities for cardiac regeneration. When combined with developments in nuclear reprogramming and provided that tumor risks and other challenges of embryonic cell transplantation can be overcome, the prospect of achieving autologous, cardiomyogenic, stem cell-based therapy may be within reach.
View details for DOI 10.2217/174607184.108.40.206
View details for Web of Science ID 000257995000013
View details for PubMedID 18307403
Cardiovascular Stem Cells in Regenerative Medicine: Ready for Prime Time?
Drug discovery today. Therapeutic strategies
2008; 5 (4): 201-207
Restoration of cardiovascular function is the ultimate goal of stem cell-based therapy. In principle, cardiovascular stem cells can improve cardiac function via de novo cardiomyogenesis, enhanced myocardial neovascularization, and prevention of post-infarct remodeling. Stem cell transplantation to improve cardiac function has received mixed results in human clinical trials. These early data suggest that a critical reassessment of the scientific basis to stem cell-based therapy is needed in order to bring this highly promising treatment modality to mainstream clinical care.
View details for PubMedID 20054428
alpha(2)-Macraglobulin from rheumatoid arthritis synovial fluid: Functional analysis defines a role for oxidation in inflammation
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
2001; 391 (1): 119-126
A hallmark of inflammation is the release of oxidants, proteinases, and cytokines, all important mediators of the inflammatory cascade. alpha(2)-Macroglobulin (alpha(2)M) is a high-affinity, broad-specificity proteinase inhibitor that also binds and regulates the biological activities of a number of cytokines. We demonstrated recently that hypochlorite-oxidized alpha(2)M has decreased ability to inhibit proteinases and regulate cytokines in vitro. The role of oxidation in regulating alpha(2)M functions in vivo is largely unknown. To determine the extent and biological consequence of in vivo alpha(2)M oxidation, we measured the degree of oxidative alpha(2)M modification from rheumatoid arthritis (RA) synovial fluid and compared this with osteoarthritis (OA) as noninflammatory controls. We found that RA synovial fluid alpha(2)M is significantly more oxidized than that from OA. RA synovial fluid also contains a twofold higher median alpha(2)M level than OA, while having only half the alpha(2)M-proteinase inhibitory activity. Detailed biochemical analysis demonstrates proteolytically degraded alpha(2)M in RA greater than in OA synovial fluid. Additionally, the hypochlorite-mediated oxidation product, chlorotyrosine, is present in RA more than in OA or plasma alpha(2)M samples. Taken together, these findings confirm a role for oxidative regulation of inflammation by altering the functions of extracellular mediators such as alpha(2)M.
View details for Web of Science ID 000169700200015
View details for PubMedID 11414692
Differential regulation of the fibroblast growth factor (FGF) family by alpha(2)-macroglobulin: evidence for selective modulation of FGF-2-induced angiogenesis
2001; 97 (11): 3450-3457
The fibroblast growth factor (FGF) family has an important role in processes such as angiogenesis, wound healing, and development in which precise control of proteinase activity is important. The human plasma proteinase inhibitor alpha(2)-macroglobulin (alpha(2)M) regulates cellular growth by binding and modulating the activity of many cytokines and growth factors. These studies investigate the ability of native and activated alpha(2)M (alpha(2)M*) to bind to members of the FGF family. Both alpha(2)M and alpha(2)M* bind specifically and saturably to FGF-1, -2, -4, and -6, although the binding to alpha(2)M* is of significantly higher affinity. Neither alpha(2)M nor alpha(2)M* bind to FGF-5, -7, -9, or -10. FGF-2 was chosen for more extensive study in view of its important role in angiogenesis. It was demonstrated that FGF-2 binds to the previously identified TGF-beta binding site. The alpha(2)M* inhibits FGF-2-dependent fetal bovine heart endothelial cell proliferation in a dose-dependent manner. Unexpectedly, alpha(2)M* does not affect FGF-2-induced vascular tubule formation on Matrigel basement membrane matrix or collagen gels. Further studies demonstrate that FGF-2 partitions between fluid-phase alpha(2)M* and solid-phase Matrigel or collagen. These studies suggest that the ability of alpha(2)M* to modulate the activity of FGF-2 is dependent on an interplay with extracellular matrix components. (Blood. 2001;97:3450-3457)
View details for Web of Science ID 000168927900019
View details for PubMedID 11369636
The conformation-dependent interaction of alpha(2)-macroglobulin with vascular endothelial growth factor - A novel mechanism of alpha(2)-macroglobulin/growth factor binding
JOURNAL OF BIOLOGICAL CHEMISTRY
2000; 275 (35): 26806-26811
alpha(2)-Macroglobulin (alpha(2)M) is a highly conserved proteinase inhibitor present in human plasma at high concentration (2-4 mg/ml). alpha(2)M exists in two conformations, a native form and an activated, receptor-recognized form. While alpha(2)M binds to numerous cytokines and growth factors, in most cases, the nature of the alpha(2)M interaction with these factors is poorly understood. We examined in detail the interaction between alpha(2)M and vascular endothelial growth factor (VEGF) and found a novel and unexpected mechanism of interaction as demonstrated by the following observations: 1) the binding of VEGF to alpha(2)M occurs at a site distinct from the recently characterized growth factor binding site; 2) VEGF binds different forms of alpha(2)M with distinct spatial arrangement, namely to the interior of methylamine or ammonia-treated alpha(2)M and to the exterior of native and proteinase-converted alpha(2)M; and 3) VEGF (molecular mass approximately 40 kDa) can access the interior of receptor-recognized alpha(2)M in the absence of a proteinase trapped within the molecule. VEGF bound to receptor-recognized forms of alpha(2)M is internalized and degraded by macrophages via the alpha(2)M receptor, the low density lipoprotein receptor-related protein. Oxidation of both native and receptor-recognized alpha(2)M results in significant inhibition of VEGF binding. We also examined the biological significance of this interaction by studying the effect of alpha(2)M on VEGF-induced cell proliferation and VEGF-induced up-regulation of intracellular Ca(2+) levels. We demonstrate that under physiological conditions, alpha(2)M does not impact the ability of VEGF to induce cell proliferation or up-regulate Ca(2+).
View details for Web of Science ID 000089144800021
View details for PubMedID 10862607
- a-Macroglobulins/Kunins In: Hemostasis and Thrombosis: Basic Principles and Clinical Practice, 4th Ed. 2000: 367-379
Mechanism of hypochlorite-mediated inactivation of proteinase inhibition by alpha(2)-Macroglobulin
1999; 38 (42): 13983-13990
The proteinase-proteinase inhibitor balance plays an important role in mediating inflammation-associated tissue destruction. alpha 2-Macroglobulin (alpha 2M) is a high-affinity, broad-spectrum proteinase inhibitor found abundantly in plasma and interstitial fluids. Increased levels of alpha 2M and proteinase-alpha 2M complexes can be demonstrated in patients with sepsis, emphysema, peridontitis, rheumatoid arthritis, and other inflammatory diseases. Despite these increased levels, proteolysis remains a significant problem. We hypothesized that a mechanism for inactivating alpha 2M-mediated proteinase inhibition must exist and recently demonstrated that alpha 2M isolated from human rheumatoid arthritis synovial fluid is oxidized and has decreased functional activity. The oxidant responsible for alpha 2M inactivation and the mechanism of such destruction were not studied. We now report that while hypochlorite and hydroxyl radical both modify amino acid residues on alpha 2M, only hypochlorite can abolish the ability of alpha 2M to inhibit proteinases. Hydrogen peroxide, on the other hand, has no effect on alpha 2M structure or function. Protein unfolding with increased susceptibility to proteolytic cleavage appears to be involved in alpha 2M inactivation by oxidation. The in vivo relevance of this mechanism is supported by the presence of multiple cleavage fragments of alpha 2M in synovial fluid from patients with rheumatoid arthritis, where significant tissue destruction occurs, but not in patients with osteoarthritis. These results provide strong evidence that hypochlorite oxidation contributes to enhanced tissue destruction during inflammation by inactivating alpha 2M.
View details for Web of Science ID 000083288400025
View details for PubMedID 10529245
Oxidized alpha(2)-macroglobulin (alpha(2)M) differentially regulates receptor binding by cytokines growth factors: Implications for tissue injury and repair mechanisms in inflammation
JOURNAL OF IMMUNOLOGY
1998; 161 (8): 4356-4365
Alpha2M binds specifically to TNF-alpha, IL-1beta, IL-2, IL-6, IL-8, basic fibroblast growth factor (bFGF), beta-nerve growth factor (beta-NGF), platelet-derived growth factor (PDGF), and TGF-beta. Since many of these cytokines are released along with neutrophil-derived oxidants during acute inflammation, we hypothesize that oxidation alters the ability of alpha2M to bind to these cytokines, resulting in differentially regulated cytokine functions. Using hypochlorite, a neutrophil-derived oxidant, we show that oxidized alpha2M exhibits increased binding to TNF-alpha, IL-2, and IL-6 and decreased binding to beta-NGF, PDGF-BB, TGF-beta1, and TGF-beta2. Hypochlorite oxidation of methylamine-treated alpha2M (alpha2M*), an analogue of the proteinase/alpha2M complex, also results in decreased binding to bFGF, beta-NGF, PDGF-BB, TGF-beta1, and TGF-beta2. Concomitantly, we observed decreased ability to inhibit TGF-beta binding and regulation of cells by oxidized alpha2M and alpha2M*. We then isolated alpha2M from human rheumatoid arthritis synovial fluid and showed that the protein is extensively oxidized and has significantly decreased ability to bind to TGF-beta compared with alpha2M derived from plasma and osteoarthritis synovial fluid. We, therefore, propose that oxidation serves as a switch mechanism that down-regulates the progression of acute inflammation by sequestering TNF-alpha, IL-2, and IL-6, while up-regulating the development of tissue repair processes by releasing bFGF, beta-NGF, PDGF, and TGF-beta from binding to alpha2M.
View details for Web of Science ID 000076343300073
View details for PubMedID 9780213
The binding of receptor-recognized alpha(2)-macroglobulin to the low density lipoprotein receptor-related protein and the alpha(2)M signaling receptor is decoupled by oxidation
JOURNAL OF BIOLOGICAL CHEMISTRY
1997; 272 (33): 20627-20635
Receptor-recognized forms of alpha2-macroglobulin (alpha2M*) bind to two classes of cellular receptors, a high affinity site comprising approximately 1500 sites/cell and a lower affinity site comprising about 60,000 sites/cell. The latter class has been identified as the so-called low density lipoprotein receptor-related protein (LRP). Ligation of receptors distinct from LRP activates cell signaling pathways. Strong circumstantial evidence suggests that the high affinity binding sites are responsible for cell signaling induced by alpha2M*. Using sodium hypochlorite, a powerful oxidant produced by the H2O2-myeloperoxidase-Cl- system, we now demonstrate that binding to the high affinity sites correlates directly with activation of the signaling cascade. Oxidation of alpha2M* using 200 microM hypochlorite completely abolishes its binding to LRP without affecting its ability to activate the macrophage signaling cascade. Scatchard analysis shows binding to a single class of high affinity sites (Kd - 71 +/- 12 pM). Surprisingly, oxidation of native alpha2-macroglobulin (alpha2M) with 125 microM hypochlorite results in the exposure of its receptor-binding site to LRP, but the ligand is unable to induce cell signaling. Scatchard analysis shows binding to a single class of lower affinity sites (Kd - 0.7 +/- 0.15 nM). Oxidation of a cloned and expressed carboxyl-terminal 20-kDa fragment of alpha2M (RBF), which is capable of binding to both LRP and the signaling receptor, results in no significant change in its binding Kd, supporting our earlier finding that the oxidation-sensitive site is predominantly outside of RBF. Attempts to understand the mechanism responsible for the selective exposure of LRP-binding sites in oxidized native alpha2M suggest that partial protein unfolding may be the most likely mechanism. These studies provide strong evidence that the high affinity sites (Kd - 71 pM) are the alpha2M* signaling receptor.
View details for Web of Science ID A1997XR22100048
View details for PubMedID 9252378
- Crashing the Boards: A User Friendly Study Guide for the USMLE Step 1 Lippincott-Raven 1997; 1
Low-density lipoprotein receptor-related protein alpha(2)-macroglobulin receptor on murine peritoneal macrophages mediates the binding and catabolism of low-density lipoprotein
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
1996; 326 (1): 39-47
Low-density lipoprotein receptor-related protein (LRP)/alpha 2-macroglobulin receptor is a member of the low-density lipoprotein receptor family. It is known to bind a wide variety of unrelated ligands including alpha 2-macroglobulin-proteinase complexes, tissue plasminogen activator, apolipoprotein E-enriched very low density lipoprotein, lipoprotein lipase, and Pseudomonas exotoxin A. Receptor-associated protein (RAP), a protein which copurifies with LRP, can inhibit the binding and internalization of all known ligands to LRP. Recent studies have shown that some ligands can bind to more than one receptor in this family. However, the ability of low-density lipoprotein (LDL) to bind to LRP in addition to the LDL receptor has not been demonstrated consistently. In this study we demonstrate that LDL binds with high affinity to macrophage cell surface receptors at 4 degrees C (Kd = 1.8 nM) and competes for the binding of a receptor-recognized form of alpha 2-macroglobulin (alpha 2M*) (Ki = 3 nM). alpha 2M* and RAP can inhibit the binding of LDL to macrophages completely (96 and 100% inhibition, respectively), after cell surface heparin has been removed by treatment with heparinase. Using a solid-phase assay, we show that LDL binds specifically, saturably, and with high affinity to purified LRP (Kd = 5 nM). LDL can also completely inhibit the binding of alpha 2M* to purified LRP. These results indicate that LDL binds directly to LRP. The ability of LDL to cross-compete with alpha 2M* for binding to LRP suggests that LDL binds to a similar or overlapping site as alpha 2M*. In addition, the ability of alpha 2M* to inhibit most of the receptor-mediated binding of LDL to macrophages suggests that LDL receptors on murine peritoneal macrophages are predominantly LRP.
View details for Web of Science ID A1996TT88500006
View details for PubMedID 8579370