PerspectiveStem Cells

# Human Stem Cells for Modeling Heart Disease and for Drug Discovery

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Science Translational Medicine  04 Jun 2014:
Vol. 6, Issue 239, pp. 239ps6
DOI: 10.1126/scitranslmed.3008921

## Abstract

A major research focus in the field of cardiovascular medicine is the prospect of using stem cells and progenitor cells for cardiac regeneration. With the advent of induced pluripotent stem cell (iPSC) technology, major efforts are also underway to use iPSCs to model heart disease, to screen for new drugs, and to test candidate drugs for cardiotoxicity. Here, we discuss recent advances in the exciting fields of stem cells and cardiovascular disease.

Cardiovascular disease is a leading cause of morbidity and mortality worldwide, with an estimated 17.3 million deaths per year (1). As this number is expected to surpass 23 million deaths annually by 2030, there is an urgent need for better treatment options (2). Current drug therapies alleviate symptoms for only 50 to 70% of cardiovascular disease patients, often with unwanted side effects (3). The availability of human stem and progenitor cells with cardiac regenerative potential has opened the door to regenerative medicine strategies for cellular cardiomyogenesis and neovascularization. Induced pluripotent stem cells (iPSCs) derived from adult somatic cells are yielding novel insights into the molecular mechanisms of heart disease, making it possible to deliver new patient-specific pharmacological, genetic, and cellular therapies for cardiovascular disease. The cardiovascular field has the potential to progress from “population medicine” toward “personalized medicine” with a new armamentarium of therapeutic and preventive strategies. Here, we highlight research progress toward the use of stem cells and progenitor cells in disease modeling, drug discovery, and cardiac regeneration.

## CARDIOVASCULAR STEM CELLS FOR CARDIAC REPAIR

Adult Stem Cells and Progenitors. Multipotent adult stem cells and progenitor cells capable of cardiac repair reside within numerous human adult tissues, including the bone marrow, skeletal muscle, adipose tissue, peripheral blood, and the heart (Fig. 1). Adult stem cells are considered reliable and renewable cellular sources for cardiac regeneration. They have demonstrated an in vitro and in vivo ability to express cardiomyocyte-specific markers or even to differentiate toward functional cardiomyocytes, albeit at very low efficiencies (4). Multipotent adult stem cells residing in the heart include c-Kit (CD117)+, stem cell antigen 1 (Sca-1)+, and side population (Hoechst 33342, CD34–/low, c-Kit+, and Sca-1+) cardiac stem cells, as well as second heart field ISL1+ progenitor cells (5). Specialized culture techniques have also enabled Sca-1+ and c-Kit+ cardiosphere-derived cell isolation from the adult human heart (6). c-Kit+ hematopoietic stem cells do reside within the bone marrow as well as the heart but have been shown to represent a more committed cell population (7). Nevertheless, the bone marrow hosts a plethora of multipotent adult stem cells, including side population cells, mesenchymal stem cells, and mononuclear stem cells, all of which are being investigated as potential cellular therapies for cardiac regeneration (8) [see Review by Lin and Pu (9)]. CD34+ cells from human peripheral blood, CD31+ circulating endothelial progenitor cells, and adipose-derived stem cells have also demonstrated cardiac regeneration abilities.

Clinical trials for treatment of post-infarct patients using multipotent adult stem cells are ongoing but with mixed results for their short-term efficacy (8), and there are no current reports about their long-term efficacy. A common drawback has been the poor survival of implanted cells, irrespective of the delivery route, immunosuppression strategy, or timing. This raises questions as to the mechanisms by which adult stem cell delivery has resulted in post-infarct functional recovery. Thus far, suggested mechanisms focus on cardiac regeneration by differentiation to cardiomyocytes, fusion with endogenous cardiomyocytes, production of exosomes that might promote endogenous adult stem cell activation (10), or secretion of paracrine factors (growth factors, cytokines, or other signaling molecules) that promote neovascularization (11).

Direct Transdifferentiation to Cardiomyocytes and Progenitors. The ability to induce transdifferentiation of adult skin or cardiac fibroblasts toward functional cardiomyocytes either in vitro or in vivo was first described in 2010 (Fig. 1) (12). Transdifferentiation was achieved by viral overexpression of cardiac transcription factors (Gata4, Mef2c, and Tbx5), resulting in the formation of induced cardiomyocytes that activate expression of sarcomeric markers and exhibit cardiomyocyte-like electrophysiological and calcium handling properties. However, transdifferentiation protocols remain elaborate and time consuming, often requiring coculture with rodent myocytes (13, 14). Some studies have disputed the ability of lineage-committed fibroblasts to generate induced cardiomyocytes (15), suggesting that experimental artifacts such as incomplete transgene silencing or cell fusion events might explain induced cardiomyocyte formation. Further work is needed to validate the direct transdifferentiation technology and make protocols more amenable for future application in cardiac regeneration.

An alternative approach toward cardiac transdifferentiation has been described more recently, in which fibroblasts first undergo partial reprogramming by expression of exogenously supplied pluripotency-associated genes (Oct4, Sox2, and Klf4) for a 4- to 6-day period, followed by differentiation to cardiomyocytes without formation of pluripotent intermediates (16). The possibility that partially reprogrammed cells could undergo proliferation in vitro has raised hopes that sufficiently large numbers of cells primed for cardiac differentiation could be created for regenerative medicine applications.

Pluripotent Stem Cell–Derived Cardiomyocytes and Cardiac Progenitors. Cardiovascular precursor cells have also been derived from pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and iPSCs. Both of these PSC types have the ability to generate cell lineages from all three embryonic germ layers, but whereas ESC isolation requires donation of human embryos, iPSCs can be derived from adult tissue (e.g., skin, adipose cells, keratinocytes, or peripheral blood) by overexpressing key pluripotency-associated genes (OCT4, SOX2, KLF4, or c-MYC), thus alleviating the need for human embryonic tissue (17). PSC-derived cardiovascular progenitors include human HCN4+ populations that later become specified as first heart field cardiomyocytes (18). A population of human ESC-derived ROR2+/CD13+/KDR+/PDGFRα+ cells has also been identified to generate cardiomyocytes, endothelial cells, and vascular smooth muscle cells in vitro (19). Aside from isolation of precursor cells based on surface marker expression, PSC-derived cardiovascular precursor cells have been generated by treatment with small molecules such as nicotinamide, which was sufficient to induce cardiac mesoderm specification and trigger progression to beating cardioblasts from ESCs with high efficiency (20). In a separate study, a combination of bone morphogenetic protein 4 (BMP4), the glycogen synthase kinase 3 inhibitor CHIR99021, and ascorbic acid was also sufficient to rapidly convert human PSCs into cardiovascular precursor cells (21). A key advantage of cardiovascular precursor cells is their ability to self-renew and to retain the potential for efficient in vitro specification toward cardiovascular lineages. Therefore, this cell population represents a powerful means for understanding the mechanisms of early cardiovascular development and may provide a new source of cells for cardiac regenerative medicine.

Contracting cardiomyocytes can also be generated directly from human PSCs by using one of three methods: (i) coculture with END2 mouse endoderm-like cells, (ii) embryoid body formation, or (iii) monolayer culture. END2 coculture is restricted by its reliance on animal cells. Methods to make embryoid bodies involve the suspension of PSC colonies in fetal bovine serum, enabling the formation of three-dimensional (3D) aggregates that are thought to recapitulate the growth factor gradients and cell-cell interactions that normally occur in the human embryo (22). Although initial protocols produced contracting embryoid bodies with only 5 to 15% efficiency (23), subsequent optimization with the timely addition of growth factors (such as BMP4, fibroblast growth factor 2, Activin A, and WNT3A) during early differentiation (day 0 to day 4), coupled with WNT inhibition during later stages of differentiation (day 4 to day 8), could improve efficiency to >70% (24). Monolayer differentiation (25), which involves simple serum-free and scalable protocols, has largely replaced embryoid body formation. Meanwhile, Activin A and BMP4 growth factors have been replaced by CHIR99021, a small molecule that can be reproduced more reliably than growth factors. Maintaining undifferentiated PSCs in a defined biologics-free culture system that allows faithful expansion and controllable direct differentiation will be crucial for their therapeutic application.

## REFERENCES

1. Acknowledgments: We thank J. Gold and B. Wu for critical reading of the manuscript and funding support from Leducq Foundation; National Institutes of Health (NIH) R01 HL113006, NIH U01 HL099776, NIH R24 HL117756; California Institute for Regenerative Medicine (CIRM) TR3-05556 and CIRM DR2-05394 (J.C.W.); the NIH Progenitor Cell Biology Jump Start Award (E.M.); and the American Heart Association Postdoctoral Fellowship (P.W.B.).
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