Ellen Poon

Physical developmental cues for the maturation of human pluripotent stem cell-derived cardiomyocytes.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are the most promising source of cardiomyocytes (CMs) for experimental and clinical applications, but their use is largely limited by a structurally and functionally immature phenotype that most closely resembles embryonic or fetal heart cells. The application of physical stimuli to influence hPSC-CMs through mechanical and bioelectrical transduction offers a powerful strategy for promoting more developmentally mature CMs. Here we summarize the major events associated with in vivo heart maturation and structural development. We then review the developmental state of in vitro derived hPSC-CMs, while focusing on physical (electrical and mechanical) stimuli and contributory (metabolic and hypertrophic) factors that are actively involved in structural and functional adaptations of hPSC-CMs. Finally, we highlight areas for possible future investigation that should provide a better understanding of how physical stimuli may promote in vitro development and lead to mechanistic insights. Advances in the use of physical stimuli to promote developmental maturation will be required to overcome current limitations and significantly advance research of hPSC-CMs for cardiac disease modeling, in vitro drug screening, cardiotoxicity analysis and therapeutic applications.

Human Pluripotent Stem Cell-Based Approaches for Myocardial Repair: From the Electrophysiological Perspective

Heart diseases are a leading cause of mortality worldwide. Terminally differentiated adult cardiomyocytes (CMs) lack the innate ability to regenerate. Their malfunction or significant loss can lead to conditions from cardiac arrhythmias to heart failure. For myocardial repair, cell- and gene-based therapies offer promising alternatives to donor organ transplantation. Human embryonic stem cells (hESCs) can self-renew while maintaining their pluripotency. Direct reprogramming of adult somatic cells to become pluripotent hES-like cells (also known as induced pluripotent stem cells or iPSCs) has been achieved. Both hESCs and iPSCs have been successfully differentiated into genuine human CMs. In this review, we describe our current knowledge of the structure-function properties of hESC/iPSC-CMs, with an emphasis on their electrophysiology and Ca(2+) handling, along with the hurdles faced and potential solutions for translating into clinical and other applications (e.g., disease modeling, cardiotoxicity and drug screening).

Transcriptome-Guided Functional Analyses Reveal Novel Biological Properties and Regulatory Hierarchy of Human Embryonic Stem Cell-Derived Ventricular Cardiomyocytes Crucial for Maturation

Abstract Human (h) embryonic stem cells (ESC) represent an unlimited source of cardiomyocytes (CMs); however, these differentiated cells are immature. Thus far, gene profiling studies have been performed with non-purified or non-chamber specific CMs. Here we took a combinatorial approach of using systems biology to guide functional discoveries of novel biological properties of purified hESC-derived ventricular (V) CMs. We profiled the transcriptomes of hESCs, hESC-, fetal (hF) and adult (hA) VCMs, and showed that hESC-VCMs displayed a unique transcriptomic signature. Not only did a detailed comparison between hESC-VCMs and hF-VCMs confirm known expression changes in metabolic and contractile genes, it further revealed novel differences in genes associated with reactive oxygen species (ROS) metabolism, migration and cell cycle, as well as potassium and calcium ion transport. Following these guides, we functionally confirmed that hESC-VCMs expressed IKATP with immature properties, and were accordingly vulnerable to hypoxia/reoxygenation-induced apoptosis. For mechanistic insights, our coexpression and promoter analyses uncovered a novel transcriptional hierarchy involving select transcription factors (GATA4, HAND1, NKX2.5, PPARGC1A and TCF8), and genes involved in contraction, calcium homeostasis and metabolism. These data highlight novel expression and functional differences between hESC-VCMs and their fetal counterparts, and offer insights into the underlying cell developmental state. These findings may lead to mechanism-based methods for in vitro driven maturation.

Nitric Oxide‐cGMP‐PKG Pathway Acts on Orai1 to Inhibit the Hypertrophy of Human Embryonic Stem Cell‐Derived Cardiomyocytes

Cardiac hypertrophy is an abnormal enlargement of heart muscle. It frequently results in congestive heart failure, which is a leading cause of human death. Previous studies demonstrated that the nitric oxide (NO), cyclic GMP (cGMP), and protein kinase G (PKG) signaling pathway can inhibit cardiac hypertrophy and thus improve cardiac function. However, the underlying mechanisms are not fully understood. Here, based on the human embryonic stem cell‐derived cardiomyocyte (hESC‐CM) model system, we showed that Orai1, the pore‐forming subunit of store‐operated Ca2+ entry (SOCE), is the downstream effector of PKG. Treatment of hESC‐CMs with an α‐adrenoceptor agonist phenylephrine (PE) caused a marked hypertrophy, which was accompanied by an upregulation of Orai1. Moreover, suppression of Orai1 expression/activity using Orai1‐siRNAs or a dominant‐negative construct Orai1G98A inhibited the hypertrophy, suggesting that Orai1‐mediated SOCE is indispensable for the PE‐induced hypertrophy of hESC‐CMs. In addition, the hypertrophy was inhibited by NO and cGMP via activating PKG. Importantly, substitution of Ala for Ser34 in Orai1 abolished the antihypertrophic effects of NO, cGMP, and PKG. Furthermore, PKG could directly phosphorylate Orai1 at Ser34 and thus prevent Orai1‐mediated SOCE. Together, we conclude that NO, cGMP, and PKG inhibit the hypertrophy of hESC‐CMs via PKG‐mediated phosphorylation on Orai1‐Ser‐34. These results provide novel mechanistic insights into the action of cGMP‐PKG‐related antihypertrophic agents, such as NO donors and sildenafil. Stem Cells 2015;33:2973–2984

Proteomic Analysis of Human Pluripotent Stem Cell-Derived, Fetal, and Adult Ventricular Cardiomyocytes Reveals Pathways Crucial for Cardiac Metabolism and Maturation

Background—Differentiation of pluripotent human embryonic stem cells (hESCs) to the cardiac lineage represents a potentially unlimited source of ventricular cardiomyocytes (VCMs), but hESC-VCMs are developmentally immature. Previous attempts to profile hESC-VCMs primarily relied on transcriptomic approaches, but the global proteome has not been examined. Furthermore, most hESC-CM studies focus on pathways important for cardiac differentiation, rather than regulatory mechanisms for CM maturation. We hypothesized that gene products and pathways crucial for maturation can be identified by comparing the proteomes of hESCs, hESC-derived VCMs, human fetal and human adult ventricular and atrial CMs. Methods and Results—Using two-dimensional–differential-in-gel electrophoresis, 121 differentially expressed (>1.5-fold; P<0.05) proteins were detected. The data set implicated a role of the peroxisome proliferator–activated receptor &agr; signaling in cardiac maturation. Consistently, WY-14643, a peroxisome proliferator–activated receptor &agr; agonist, increased fatty oxidative enzyme level, hyperpolarized mitochondrial membrane potential and induced a more organized morphology. Along this line, treatment with the thyroid hormone triiodothyronine increased the dynamic tension developed in engineered human ventricular cardiac microtissue by 3-fold, signifying their maturation. Conclusions—We conclude that the peroxisome proliferator–activated receptor &agr; and thyroid hormone pathways modulate the metabolism and maturation of hESC-VCMs and their engineered tissue constructs. These results may lead to mechanism-based methods for deriving mature chamber-specific CMs.

Graphene Sheet-Induced Global Maturation of Cardiomyocytes Derived from Human Induced Pluripotent Stem Cells

Human induced pluripotent stem cells (hiPSCs) can proliferate infinitely. Their ability to differentiate into cardiomyocytes provides abundant sources for disease modeling, drug screening and regenerative medicine. However, hiPSC-derived cardiomyocytes (hiPSC-CMs) display a low degree of maturation and fetal-like properties. Current in vitro differentiation methods do not mimic the structural, mechanical, or physiological properties of the cardiogenesis niche. Recently, we present an efficient cardiac maturation platform that combines hiPSCs monolayer cardiac differentiation with graphene substrate, which is a biocompatible and superconductive material. The hiPSCs lines were successfully maintained on the graphene sheets and were able to differentiate into functional cardiomyocytes. This strategy markedly increased the myofibril ultrastructural organization, elevated the conduction velocity, and enhanced both the Ca2+ handling and electrophysiological properties in the absence of electrical stimulation. On the graphene substrate, the expression of connexin 43 increased along with the conduction velocity. Interestingly, the bone morphogenetic proteins signaling was also significantly activated during early cardiogenesis, confirmed by RNA sequencing analysis. Here, we reasoned that graphene substrate as a conductive biomimetic surface could facilitate the intrinsic electrical propagation, mimicking the microenvironment of the native heart, to further promote the global maturation of hiPSC-CMs. Our findings highlight the capability of electrically active substrates to influence cardiomyocyte development. We believe that application of graphene sheets will be useful for simple, fast, and scalable maturation of regenerated cardiomyocytes.

Consensus Comparative Analysis of Human Embryonic Stem Cell-Derived Cardiomyocytes

Global transcriptional analyses have been performed with human embryonic stem cells (hESC) derived cardiomyocytes (CMs) to identify molecules and pathways important for human CM differentiation, but variations in culture and profiling conditions have led to greatly divergent results among different studies. Consensus investigation to identify genes and gene sets enriched in multiple studies is important for revealing differential gene expression intrinsic to human CM differentiation independent of the above variables, but reliable methods of conducting such comparison are lacking. We examined differential gene expression between hESC and hESC-CMs from multiple microarray studies. For single gene analysis, we identified genes that were expressed at increased levels in hESC-CMs in seven datasets and which have not been previously highlighted. For gene set analysis, we developed a new algorithm, consensus comparative analysis (CSSCMP), capable of evaluating enrichment of gene sets from heterogeneous data sources. Based on both theoretical analysis and experimental validation, CSSCMP is more efficient and less susceptible to experimental variations than traditional methods. We applied CSSCMP to hESC-CM microarray data and revealed novel gene set enrichment (e.g., glucocorticoid stimulus), and also identified genes that might mediate this response. Our results provide important molecular information intrinsic to hESC-CM differentiation. Data and Matlab codes can be downloaded from S1 Data.

Are these cardiomyocytes? Protocol development reveals impact of sample preparation on the accuracy of identifying cardiomyocytes by flow cytometry

Modern differentiation protocols enable efficient, yet imperfect, differentiation of human pluripotent stem cells into cardiomyocytes (hPSC-CM). As the number of laboratories and studies implementing this technology expands, the accurate assessment of cell identity in differentiation cultures is paramount to well-defined studies that can be replicated among laboratories. While flow cytometry is apt for routine assessment, a standardized protocol for assessing cardiomyocyte identity in hPSC-CM cultures has not yet been established. To address this gap, the current study leveraged targeted mass spectrometry to confirm the presence of troponin proteins in hPSC-CM and systematically evaluated multiple anti-troponin antibodies and sample preparation protocols for their suitability in assessing cardiomyocyte identity. Results demonstrate challenges of interpreting data generated by published methods and informed the development of a robust protocol for routine assessment of hPSC-CM. Overall, the new data, workflow for evaluating fit-for-purpose use of antibodies, and standardized protocol described here should benefit investigators new to this field as well as those with expertise in hPSC-CM differentiation.

MicroRNA and Pluripotent Stem Cell-Based Heart Therapies: The Electrophysiological Perspective

MicroRNAs (miRs) are nonencoding RNAs that function as negative transcriptional regulators via degradation or inhibition by RNA interference. Recent studies have demonstrated that miRs are important regulators of cardiovascular cell differentiation, growth, proliferation, and apoptosis. Specifically, miRs modulate electrophysiological properties such as automaticity, conduction, and membrane repolarization by regulating a wide range of target genes. Not surprisingly, dysregulation of miR function can lead to cardiovascular diseases. Indeed, abnormal miR expression patterns have been detected in hypertrophy and arrhythmias. Cardiomyocytes (CMs) are terminally differentiated and cannot regenerate. Therefore, significant loss of CMs due to disease or aging can lead to cardiac arrhythmias, heart failure, and subsequently death. Human embryonic stem cells (hESCs) can self-renew while maintaining their pluripotency to differentiate into all cell types, including CMs. Therefore, hESCs may provide an unlimited ex vivo source of CMs for cell-based heart therapies. The process of deriving CMs from hESCs can be optimized by miR-directed differentiation and specification. When combined with stem cell biology, the emerging field of miR presents novel approaches for the improvement of many cardiac therapy strategies.

Human Pluripotent Stem Cells in Cardiovascular Research and Regenerative Medicine

Heart disease is one of the leading causes of mortality worldwide. Because adult cardiomyocytes (CMs) lack the ability to regenerate, malfunctions or significant loss of CMs due to disease or aging can lead to cardiac arrhythmias, heart failure, and subsequently death. Heart transplantation for patients with end stage heat failure is limited by the number of donor organs available. Cell-based therapies offer a promising alternative for myocardial repair, but there are significant challenges involved. The transplantation of human CMs, eg fetal CMs, is difficult for practical and ethical reasons, thus cells of noncardiac lineage, such as skeletal myoblasts (Murry et al., 1996; Menasche et al., 2003) and mesenchymal stem cells (Shake et al., 2002; Toma et al., 2002), have been considered as alternatives. Animal studies and clinical trials involving these cells have yielded conflicting results. Transplanted non-cardiac cells such as bone marrow-derived hematopoietic cells do not transdifferentiate into the cardiac lineage (Balsam et al., 2004; Murry et al., 2004). They also do not integrate into the host myocardium. For instance, the lack of electrical integration of skeletal myoblasts after their autologous transplantation into the myocardium resulted in the generation of malignant ventricular arrhythmias, which led to the premature termination of clinical trials involving skeletal myoblasts (Menasche et al., 2003; Smits et al., 2003). Therefore, an alternative cell source is needed.