Instructor, Cardiovascular Institute
- A transcriptomic atlas of drug-induced endothelial dysfunction in human endothelial cells. Journal of molecular and cellular cardiology 2022; 173: 115-117
Contractility and Calcium Transient Maturation in the Human iPSC-Derived Cardiac Microfibers.
ACS applied materials & interfaces
Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are considered immature in the sarcomere organization, contractile machinery, calcium transient, and transcriptome profile, which prevent them from further applications in modeling and studying cardiac development and disease. To improve the maturity of hiPSC-CMs, here, we engineered the hiPSC-CMs into cardiac microfibers (iCMFs) by a stencil-based micropatterning method, which enables the hiPSC-CMs to be aligned in an end-to-end connection for prolonged culture on the hydrogel of physiological stiffness. A series of characterization approaches were performed to evaluate the maturation in iCMFs on both structural and functional levels, including immunohistochemistry, calcium transient, reverse-transcription quantitative PCR, cardiac contractility, and electrical pacing analysis. Our results demonstrate an improved cardiac maturation of hiPSC-CMs in iCMFs compared to micropatterned or random single hiPSC-CMs and hiPSC-CMs in a random cluster at the same cell number of iCMFs. We found an increased sarcomere length, better regularity and alignment of sarcomeres, enhanced contractility, matured calcium transient, and T-tubule formation and improved adherens junction and gap junction formation. The hiPSC-CMs in iCMFs showed a robust calcium cycling in response to the programmed and continuous electrical pacing from 0.5 to 7 Hz. Moreover, we generated the iCMFs with hiPSC-CMs with mutations in myosin-binding protein C (MYBPC3) to have a proof-of-concept of iCMFs in modeling cardiac hypertrophic phenotype. These findings suggest that the multipatterned iCMF connection of hiPSC-CMs boosts the cardiac maturation structurally and functionally, which will reveal the full potential of the application of hiPSC-CM models in disease modeling of cardiomyopathy and cardiac regenerative medicine.
View details for DOI 10.1021/acsami.2c07326
View details for PubMedID 35901275
Human Induced Pluripotent Stem Cells for Studying Mitochondrial Diseases in the Heart.
Mitochondrial dysfunction is known to contribute to a range of diseases, and primary mitochondrial defects strongly impact high-energy organs such as the heart. Platforms for high-throughput and human-relevant assessment of mitochondrial diseases are currently lacking, hindering the development of targeted therapies. In the past decade, human induced pluripotent stem cells (iPSCs) have become a promising technology for drug discovery in basic and clinical research. In particular, human iPSC-derived cardiomyocytes (iPSC-CMs) offer a unique tool to study a wide range of mitochondrial functions and possess the potential to become a key translational asset for mitochondrial drug development. This review summarizes mitochondrial functions and recent therapeutic discoveries, advancements, and limitations of using iPSC-CMs to study mitochondrial diseases of the heart with an emphasis on cardiac applications.
View details for DOI 10.1002/1873-3468.14444
View details for PubMedID 35788991
Protocol to measure contraction, calcium, and action potential in human-induced pluripotent stem cell-derived cardiomyocytes.
2021; 2 (4): 100859
Multiple strategies have been developed to efficiently differentiate human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Here, we describe a protocol for measuring three key functional parameters of hiPSC-CMs, including contractile function, calcium (Ca2+) handling, and action potential. For complete details on the use and execution of this protocol, please refer to Zhang etal. (2021).
View details for DOI 10.1016/j.xpro.2021.100859
View details for PubMedID 34746854
- Human Induced Pluripotent Stem Cells as a Screening Platform for Drug-Induced Vascular Toxicity FRONTIERS IN PHARMACOLOGY 2021; 12
Human Induced Pluripotent Stem Cells as a Screening Platform for Drug-Induced Vascular Toxicity.
Frontiers in pharmacology
2021; 12: 613837
Evaluation of potential vascular injury is an essential part of the safety study during pharmaceutical development. Vascular liability issues are important causes of drug termination during preclinical investigations. Currently, preclinical assessment of vascular toxicity primarily relies on the use of animal models. However, accumulating evidence indicates a significant discrepancy between animal toxicity and human toxicity, casting doubt on the clinical relevance of animal models for such safety studies. While the causes of this discrepancy are expected to be multifactorial, species differences are likely a key factor. Consequently, a human-based model is a desirable solution to this problem, which has been made possible by the advent of human induced pluripotent stem cells (iPSCs). In particular, recent advances in the field now allow the efficient generation of a variety of vascular cells (e.g., endothelial cells, smooth muscle cells, and pericytes) from iPSCs. Using these cells, different vascular models have been established, ranging from simple 2D cultures to highly sophisticated vascular organoids and microfluidic devices. Toxicity testing using these models can recapitulate key aspects of vascular pathology on molecular (e.g., secretion of proinflammatory cytokines), cellular (e.g., cell apoptosis), and in some cases, tissue (e.g., endothelium barrier dysfunction) levels. These encouraging data provide the rationale for continuing efforts in the exploration, optimization, and validation of the iPSC technology in vascular toxicology.
View details for DOI 10.3389/fphar.2021.613837
View details for PubMedID 33790786
View details for PubMedCentralID PMC8006367
Transcriptome Analysis of Non-Human Primate Induced Pluripotent Stem Cell-Derived Cardiomyocytes in 2D Monolayer Culture versus 3D Engineered Heart Tissue.
Stem cell therapy has shown promise for treating myocardial infarction (MI) via re-muscularization and paracrine signaling in both small and large animals. Non-human primates (NHPs), such as rhesus macaques (Macaca mulatta), are primarily utilized in preclinical trials due to their similarity to humans, both genetically and physiologically. Currently, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are delivered into the infarcted myocardium by either direct cell injection or an engineered tissue patch. Although both approaches have advantages in terms of sample preparation, cell-host interaction, and engraftment, how the iPSC-CMs respond to ischemic conditions in the infarcted heart under these two different delivery approaches remain unclear. Here we aim to gain a better understanding of the effects of hypoxia on iPSC-CMs at the transcriptome level.NHP iPSC-CMs in both monolayer culture (2 D) and engineered heart tissue (EHT) (3 D) format were exposed to hypoxic conditions to serve as surrogates of direct cell injection and tissue implantation in vivo, respectively. Outcomes were compared at the transcriptome level. We found the 3 D EHT model was more sensitive to ischemic conditions and similar to the native in vivo myocardium in terms of cell-extracellular matrix/cell-cell interactions, energy metabolism, and paracrine signaling.By exposing NHP iPSC-CMs to different culture conditions, transcriptome profiling improves our understanding of the mechanism of ischemic injury.Stem cell therapy has shown promise for treating ischemic heart tissue. However, how stem cells respond following different delivery method is unclear. Here hypoxic conditioning was applied to non-human primate iPSC-CMs in 2 D monolayer culture and 3 D engineered heart tissue to model cell injection versus patch implantation, respectively, in an ischemic milieu. The differential transcriptome of hypoxic effects on iPSC-CMs show upregulation of ECM-cell/cell-cell interactions (COL9A1, ITGB6, CTSV, and EPHA1), energy metabolism/hypoxia (ALDOC, ENO2, PFKFB4, CA3, and CA9), and paracrine signaling (WNT, PDGF, FGFR, EGFR, PI3K, and VEGF) in the 3 D format, which suggest engineered heart tissue as more suitable model for evaluating cardiac regenerative therapy.
View details for DOI 10.1093/cvr/cvaa281
View details for PubMedID 33002105
- Improving the engraftment and integration of cell transplantation for cardiac regeneration. Cardiovascular research 2019
Generation of Quiescent Cardiac Fibroblasts from Human Induced Pluripotent Stem Cells for In Vitro Modeling of Cardiac Fibrosis.
RATIONALE: Activated fibroblasts are the major cell type that secrete excessive extracellular matrix in response to injury, contributing to pathological fibrosis and leading to organ failure. Effective anti-fibrotic therapeutic solutions, however, are not available due to the poorly defined characteristics and unavailability of tissue-specific fibroblasts. Recent advances in single-cell RNA-sequencing (scRNA-seq) fill such gaps of knowledge by enabling delineation of the developmental trajectories and identification of regulatory pathways of tissue-specific fibroblasts among different organs.OBJECTIVE: This study aims to define the transcriptome profiles of tissue-specific fibroblasts using recently reported mouse scRNA-seq atlas, and to develop a robust chemically defined protocol to derive cardiac fibroblasts (CFs) from human induced pluripotent stem cells (iPSCs) for in vitro modeling of cardiac fibrosis and drug screening.METHODS AND RESULTS: By analyzing the single-cell transcriptome profiles of fibroblasts from 10 selected mouse tissues, we identified distinct tissue-specific signature genes, including transcription factors that define the identities of fibroblasts in the heart, lungs, trachea, and bladder. We also determined that CFs in large are of the epicardial lineage. We thus developed a robust chemically-defined protocol that generates CFs from human iPSCs. Functional studies confirmed that iPSC-derived CFs preserved a quiescent phenotype and highly resembled primary CFs at the transcriptional, cellular, and functional levels. We demonstrated that this cell-based platform is sensitive to both pro- and anti-fibrosis drugs. Finally, we showed that crosstalk between cardiomyocytes and CFs via the atrial/brain natriuretic peptide-natriuretic peptide receptor 1 pathway is implicated in suppressing fibrogenesis.CONCLUSIONS: This study uncovers unique gene signatures that define tissue-specific identities of fibroblasts. The bona fide quiescent CFs derived from human iPSCs can serve as a faithful in vitro platform to better understand the underlying mechanisms of cardiac fibrosis and to screen anti-fibrotic drugs.
View details for DOI 10.1161/CIRCRESAHA.119.315491
View details for PubMedID 31288631
Commonly used thiol-containing antioxidants reduce cardiac differentiation and alter gene expression ratios of sarcomeric isoforms
EXPERIMENTAL CELL RESEARCH
2018; 370 (1): 150–59
Reactive oxygen species (ROS) scavengers such as beta-mercaptoethanol (BME) and monothiol glycerol (MTG) are extensively used in stem cell research to prevent cellular oxidative stress. However, how these antioxidant supplements impact stem cell cardiac differentiation, a process regulated by redox-signaling remains unknown. In this study, we found that removal of BME from the conventional high-glucose, serum-based differentiation medium improved cardiac differentiation efficiency by 2-3 fold. BME and MTG treatments during differentiation significantly reduced mRNA expression of cardiac progenitor markers (NKX2.5 and ISL1) as well as sarcomeric markers (MLC2A, MLC2V, TNNI3, MYH6 and MYH7), suggesting reduced cardiomyogenesis by BME or MTG. Moreover, BME and MTG altered the expression ratios between the sarcomeric isoforms. In particular, TNNI3 to TNNI1 ratio and MLC2V to MLC2A ratio were significantly lower in BME or MTG treated cells than untreated cells, implying altered cardiomyocyte phenotype and maturity. Lastly, BME and MTG treatments resulted in less frequent beating, slower contraction and relaxation velocities than untreated cells. Interestingly, none of the above-mentioned effects was observed with Trolox, a non-thiol based antioxidant, despite its strong antioxidant activity. This work demonstrates that commonly used antioxidant supplements may cause considerable changes to cellular redox state and the outcome of differentiation.
View details for PubMedID 29920245
- Strategies for Improving the Maturity of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes CIRCULATION RESEARCH 2018; 123 (5): 512–14
- Strategies for Improving the Maturity of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes. Circulation research 2018; 123 (5): 512-514
Glycogen synthase kinase-3 inhibition sensitizes human induced pluripotent stem cells to thiol-containing antioxidants induced apoptosis
STEM CELL RESEARCH
2017; 23: 182–87
Inhibition of glycogen synthase kinase 3 (GSK3) is an extensively used strategy to activate Wnt pathway for pluripotent stem cell (PSC) differentiation. However, the effects of such inhibition on PSCs, besides upregulating the Wnt pathway, have rarely been investigated despite that GSK3 is broadly involved in other cellular activities such as insulin signaling and cell growth/survival regulation. Here we describe a previously unknown synergistic effect between GSK3 inhibition (e.g., Chir99021 and LY2090314) and various normally non-toxic thiol-containing antioxidants (e.g., N-acetylcysteine, NAC) on the induction of apoptosis in human induced pluripotent stem cells (iPSCs). Neither Chir99021 nor the antioxidants individually induced significant apoptosis, whereas their combined treatment resulted in rapid and extensive apoptosis, with substantial caspase 3 activity observed within 3h and over 90% decrease in cell viability after 24h. We confirmed the generality of this phenomenon with multiple independent iPSCs lines, various thiol-based antioxidants and distinct GSK3 inhibitors. Mechanistically, we demonstrated that rapamycin treatment could substantially reduce cell death, suggesting the critical role of mammalian target of rapamycin (mTOR). Akt dysregulation was also found to partially contribute to cell apoptosis but was not the primary cause. Further, this coordinated proapoptotic effect was not detected in mouse ESCs but was present in another human cells line: a breast cancer cell line (MDA-MB-231). Given the wide use of GSK3 inhibition in biomedical research: from iPSC differentiation to cancer intervention and the treatment of neuronal diseases, researchers can potentially take advantage of or avoid this synergistic effect for improved experimental or clinical outcome.
View details for PubMedID 28772167
Monitoring protein synthesis in single live cancer cells
2016; 8 (5): 645–53
Protein synthesis is generally under sophisticated and dynamic regulation to meet the ever-changing demands of a cell. Global up or down-regulation of protein synthesis and the shift of protein synthesis location (as shown, for example, during cellular stress or viral infection) are recognized as cellular responses to environmental changes such as nutrient/oxygen deprivation or to alterations such as pathological mutations in cancer cells. Monitoring protein synthesis in single live cells can be a powerful tool for cancer research. Here we employed a microfluidic platform to perform high throughput delivery of fluorescent labeled tRNAs into multiple myeloma cells with high transfection efficiency (∼45%) and high viability (>80%). We show that the delivered tRNAs were actively recruited to the ER for protein synthesis and that treatment with puromycin effectively disrupted this process. Interestingly, we observed the scattered distribution of tRNAs in cells undergoing mitosis, which has not been previously reported. Fluorescence lifetime analysis detected extensive FRET signals generated from tRNAs labeled as FRET pairs, further confirming that the delivered tRNAs were used by active ribosomes for protein translation. Our work demonstrates that the microfluidic delivery of FRET labeled tRNAs into living cancer cells can provide new insights into basic cancer metabolism and has the potential to serve as a platform for drug screening, diagnostics, or personalized medication.
View details for PubMedID 26956582
Nanoscale Strategies: Treatment for Peripheral Vascular Disease and Critical Limb Ischemia
2015; 9 (4): 3436-3452
Peripheral vascular disease (PVD) is one of the most prevalent vascular diseases in the U.S. afflicting an estimated 8 million people. Obstruction of peripheral arteries leads to insufficient nutrients and oxygen supply to extremities, which, if not treated properly, can potentially give rise to a severe condition called critical limb ischemia (CLI). CLI is associated with extremely high morbidities and mortalities. Conventional treatments such as angioplasty, atherectomy, stent implantation and bypass surgery have achieved some success in treating localized macrovascular disease but are limited by their invasiveness. An emerging alternative is the use of growth factor (delivered as genes or proteins) and cell therapy for PVD treatment. By delivering growth factors or cells to the ischemic tissue, one can stimulate the regeneration of functional vasculature network locally, re-perfuse the ischemic tissue, and thus salvage the limb. Here we review recent advance in nanomaterials, and discuss how their application can improve and facilitate growth factor or cell therapies. Specifically, nanoparticles (NPs) can serve as drug carrier and target to ischemic tissues and achieve localized and sustained release of pro-angiogenic proteins. As nonviral vectors, NPs can greatly enhance the transfection of target cells with pro-angiogenic genes with relatively fewer safety concern. Further, NPs may also be used in combination with cell therapy to enhance cell retention, cell survival and secretion of angiogenic factors. Lastly, nano/micro fibrous vascular grafts can be engineered to better mimic the structure and composition of native vessels, and hopefully overcome many complications/limitations associated with conventional synthetic grafts.
View details for DOI 10.1021/nn507269g
View details for PubMedID 25844518