Kuppusamy Rajarajan

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.

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.

Fetal Mammalian Heart Generates a Robust Compensatory Response to Cell Loss

Background— Heart development is tightly regulated by signaling events acting on a defined number of progenitor and differentiated cardiac cells. Although loss of function of these signaling pathways leads to congenital malformation, the consequences of cardiac progenitor cell or embryonic cardiomyocyte loss are less clear. In this study, we tested the hypothesis that embryonic mouse hearts exhibit a robust mechanism for regeneration after extensive cell loss. Methods and Results— By combining a conditional cell ablation approach with a novel blastocyst complementation strategy, we generated murine embryos that exhibit a full spectrum of cardiac progenitor cell or cardiomyocyte ablation. Remarkably, ablation of up to 60% of cardiac progenitor cells at embryonic day 7.5 was well tolerated and permitted embryo survival. Ablation of embryonic cardiomyocytes to a similar degree (50% to 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. Conclusion— 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.

Reprogramming of Mouse, Rat, Pig, and Human Fibroblasts into iPS Cells

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. Curr. Protoc. Mol. Biol. 97:23.15.1‐23.15.32.

Derivation of highly purified cardiomyocytes from human induced pluripotent stem cells using small molecule-modulated differentiation and subsequent glucose starvation.

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.

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.

IGF promotes Cardiac Lineage Induction In Vitro by Selective Expansion of Early Mesoderm

A thorough understanding of the developmental signals that direct pluripotent stem cells (PSCs) toward 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;32:1493–1502

Insulin-Like Growth Factor Promotes Cardiac Lineage Induction In Vitro by Selective Expansion of Early Mesoderm: IGF Induces Early Mesoderm Expansion

A thorough understanding of the developmental signals that direct pluripotent stem cells (PSCs) toward 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;32:1493–1502