Sean M. Wu

Epicardial progenitors contribute to the cardiomyocyte lineage in the developing heart

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.

Developmental Origin of a Bipotential Myocardial and Smooth Muscle Cell Precursor in the Mammalian Heart

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.

Harnessing the potential of induced pluripotent stem cells for regenerative medicine

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.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.

Generation of Functional Ventricular Heart Muscle from Mouse Ventricular Progenitor Cells

A Fix to the Heart Regenerative cardiovascular medicine is a promising avenue for therapeutic application in advanced heart failure. Although clinical trials have suggested some limited benefits in cell transplantation therapy, robust cardiac muscle formation is lacking. Domian et al. (p. 426) examined the developmental processes in normal mature cardiac muscle. A two-color murine reporter system was used to isolate committed ventricular progenitors, which were then used to build functional force-generating cardiac tissue. Such combinations of tissue engineering and stem cell biology may eventually lead to cardiac regenerative therapy. A combination of tissue engineering and stem cell biology is used to build functional force-generating mouse cardiac tissue. 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.

Screening Drug-Induced Arrhythmia Events Using Human Induced Pluripotent Stem Cell–Derived Cardiomyocytes and Low-Impedance Microelectrode Arrays

Background— 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. Methods and Results— 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. Conclusions— 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.

Inefficient Reprogramming of Fibroblasts into Cardiomyocytes Using Gata4, Mef2c, and Tbx5

Rationale: Direct reprogramming of fibroblasts into cardiomyocytes is a novel strategy for cardiac regeneration. However, the key determinants involved in this process are unknown. Objective: To assess the efficiency of direct fibroblast reprogramming via viral overexpression of GATA4, Mef2c, and Tbx5 (GMT). Methods and Results: 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. Conclusions: 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.

Origins and Fates of Cardiovascular Progenitor Cells

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.

Molecular Regulation of Cardiomyocyte Differentiation

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.

Human Induced Pluripotent Stem Cell–Derived Cardiomyocytes as an In Vitro Model for Coxsackievirus B3–Induced Myocarditis and Antiviral Drug Screening Platform

Rationale: Viral myocarditis is a life-threatening illness that may lead to heart failure or cardiac arrhythmias. A major causative agent for viral myocarditis is the B3 strain of coxsackievirus, a positive-sense RNA enterovirus. However, human cardiac tissues are difficult to procure in sufficient enough quantities for studying the mechanisms of cardiac-specific viral infection. Objective: This study examined whether human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) could be used to model the pathogenic processes of coxsackievirus-induced viral myocarditis and to screen antiviral therapeutics for efficacy. Methods and Results: 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 interferon&bgr;1, 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 interferon&bgr;1 treatment. Conclusions: This study demonstrates that hiPSC-CMs express the coxsackievirus and adenovirus receptor, are susceptible to coxsackievirus infection, and can be used to predict antiviral drug efficacy. Our results suggest that the hiPSC-CM/CVB3-Luc assay is a sensitive platform that can screen novel antiviral therapeutics for their effectiveness in a high-throughput fashion.

Developmental and Regenerative Biology of Multipotent Cardiovascular Progenitor Cells

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.