Somatic Cell Reprogramming into Cardiovascular Lineages

Abstract

Ischemic cardiac disease is the leading cause of death in the developed world. The inability of the adult mammalian heart to adequately repair itself has motivated stem cell researchers to explore various strategies to regenerate cardiomyocytes after myocardial infarction. Over the past century, progressive gains in our knowledge about the cellular mechanisms governing fate determination have led to recent advances in cellular reprogramming. The identification of specific factors capable of inducing pluripotent phenotype in somatic cells as well as factors that can directly reprogram somatic cells into cardiomyocytes suggests the potential for these approaches to translate into clinical therapies in the future. Although conceptually appealing, the field of cell lineage reprogramming is in its infancy, and further research will be needed to improve the efficiency of the reprogramming process and the fidelity of the reprogrammed cells to their in vivo counterpart.

Introduction

Although lower vertebrates such as zebra-fish are able to regenerate cardiac tissue after injury, ^{1 -5} the adult mammalian heart shows very little potential to regenerate and instead undergoes a fibrotic response. ^6,7 Thus, the human heart recovers inefficiently from myocardial infarction where as many as 1 billion cardiomyocytes are lost due to complete coronary vessel occlusion. ⁸ Hence, ischemic cardiac disease remains the leading cause of death in developed nations, accounting for over 400, 000 deaths in the United States each year. ⁹ The only cure for ischemic heart failure is whole organ transplantation that is limited by the number of donor hearts (approximately 2000 each year in the United States) and complicated by infections and immune rejection. The tremendous burden of ischemic heart disease has motivated the exploration of a number of stem cell-based strategies to treat this devastating disease.

Cellular Differentiation and Lineage Programming

The generation of therapeutically important cells like cardiomyocytes using readily available cell types remains a considerable challenge for biologists. Pluripotent embryonic stem cells (ESCs) can either self-renew or differentiate in what was long thought to be a unidirectional manner toward increasingly specialized cell types of the 3 embryonic germ layers. The latter process is often represented by Conrad Waddington’s description of an epigenetic landscape of differentiation. In this model, more potent cells sit at the peaks of a landscape before rolling irreversibly downward toward deeper valleys representing more differentiated states as the genome activates and silences fate-specific epigenetic markers. As we currently understand it, there are exceptions to this central dogma that may be exploited for the development of cell-based medical treatments. These technologies have arisen in light of a series of fundamental questions scientists have asked in the last century regarding the processes and the mechanisms of cellular differentiation.

Original hypotheses in the late 1800s advocated that cellular differentiation occurs through permanent losses of hereditary information. ¹⁰ However, German embryologists Hans Dreisch and Hans Spemann found that separation of the early blastomeres of recently fertilized animal eggs generates 2 fully formed animals. ¹¹ These “twinning” experiments challenged the hypothesis that cells permanently lose developmental potential as they become more differentiated. After Avery et al demonstrated that nuclear DNA—rather than RNA or protein—was the cellular component responsible for bacterial transformations in the early 1940s, ¹² Briggs and King successfully pioneered the technique of somatic cell nuclear transfer (SCNT) to determine whether irreversible changes in DNA occur during differentiation. ¹³ Somatic cell nuclear transfer is a process by which the nucleus of a somatic cell—a cell that is neither a germ cell nor pluripotent—is transferred into an enucleated-activated oocyte. Using the fertilized eggs of Rana pipiens, Briggs and King transplanted blastula nuclei into enucleated oocytes and successfully generated swimming larvae. ¹⁴ Although this suggested that more differentiated cells retain the genetic material necessary to direct the development of an embryo, experiments using nuclei from later stages of development revealed that as the nuclear donor somatic cell becomes more differentiated, it becomes increasingly difficult to generate clones. Briggs and King concluded that genetic potential must ultimately decline as cells differentiate.

Conflicting hypotheses arose, however, when in 1958 Gurdon and colleagues manipulated the cells of frog species Xenopus laevis to show that transplanting nuclei from mature intestinal cells into enucleated oocytes could generate fully developed clones. ¹⁵ The debate as to whether terminally differentiated cells contained the potential to generate fully formed organisms remained unresolved until fairly recently, when in 1996 Dolly the sheep was cloned by SCNT from mammary epithelial cells. ¹⁶ In the past decade, more conclusive answers were provided in studies that cloned mice from the nuclei of definitively differentiated cell types such as adult lymphocytes, which rearrange specific parts of their genomes during differentiation, and postmitotic neurons. ^17,18

The SCNT experiments established that the genomes of differentiating cells are not irreversibly altered, with the exception of a few types of specialized cells such as lymphocytes, which alter specific parts of their genomes to perform their immunologic functions. As a result, researchers became more interested in the mechanisms that bring about changes that distinguish cells of 1 lineage from another, even as they share the same genome. This interest in epigenetics, defined as “the study of stable alterations in gene expression potential that arise during development and cell proliferation” ^19(p245) has steadily gained greater interest from the scientific community over the past 40 years. Epigenetic alterations such as DNA methylation ^{20 -24} and histone and nucleosome modifications ^{25 -27} underlie the variegated display of cell lineages seen in nature. The SCNT experiments further corroborated the idea that cellular phenotypes could be altered by specific epigenetic changes in the nucleus induced by, in this case, the introduction of an oocyte cytoplasmic environment. ²⁸

Although SCNT was critical in establishing a foundational understanding of cell fate, it has been a difficult tool to use. The efficiency of cloning remains very low and most clones exhibit phenotypic and gene expression abnormalities, indicating that SCNT often fails to fully reprogram somatic nuclei. ^{29 -31} Thus, although SCNT could theoretically be employed to derive pluripotent stem cells from which patient- or disease-specific cell types can be generated for scientific investigation or even cell transplantation, these technical obstacles render SCNT an inefficient tool to harness for therapeutic purposes. ³²

As SCNT is not well suited for genetic and biochemical studies, a novel approach to studying reprogramming on the molecular level was found in fusion hybrids. After major technical breakthroughs in isolation and culturing strategies for embryonic carcinoma cells and ESCs, ^{33 -36} it was demonstrated that the fusion of these pluripotent cells with somatic cells leads to the acquisition of a pluripotent cell phenotype in the somatic cell nucleus. ^{37 -39} These results corroborated the findings from SCNT studies and further suggested that there are specific cytoplasmic transacting factors in pluripotent cell types that can reprogram somatic nuclei.

The hypothesis that specific factors are responsible for nuclear reprogramming was bolstered by experiments that revealed the cell fate-determining role of “master regulatory” genes. ⁴⁰ In 1987, Davis et al identified a single complementary DNA encoding a gene called MyoD that, when transfected into fibroblasts, could stably convert fibroblasts into skeletal myoblasts. ⁴¹ This experiment represented the first documented attempt to deliberately reprogram cells between differentiated lineages in vitro by an overexpression of a transcription factor. Soon after, others showed that infection with retroviruses overexpressing MyoD could convert a variety of differentiated cell types into myoblasts. ⁴² These successes were followed by work from Xie et al in 2005 showing that overexpression of myeloid transcription factor C/enhancer-binding protein α could convert primary B and T cells into macrophages. ⁴³

Reprogramming of Somatic Cells to Pluripotent Stem Cells

Prior to 2006, reports of successful reprogramming events in adult mammalian cells were rare and often limited to those that could be accomplished using a single “master” transcription factor. Takahashi and Yamanaka then undertook a novel shotgun approach to combinatorially screen 24 candidate genes that were known to be expressed in ESCs and suspected to play important roles in maintaining pluripotency. ⁴⁴ From these, they found that forced expression of only 4 genes (ie, the Yamanaka factors—Oct4, Sox2, c-Myc, and Klf4) conferred a pluripotent phenotype in murine fibroblasts. These so-called induced pluripotent stem cells (iPSCs) could generate teratomas containing cells of all 3 germ layers upon subcutaneous transplantation into nude mice.

Shortly after, it was found that the ability of the 4 Yamanaka factors to reprogram fibroblast into iPSC is conserved in a number of different species, including humans. ^{45 -49} Furthermore, a variety of somatic cell types can be reprogrammed into pluripotent stem cells. ^{50 -54} However, many of the first attempts to generate iPSCs resulted in cells that were only partially reprogrammed as they showed differential gene expression and gene methylation patterns when compared to bona fide ESCs. Furthermore, these cells failed to contribute to postnatal chimeras or show germline transmission when injected into developing blastocysts. ^{44,55 -57}

More recently, investigators have uncovered that a rare number of iPSC lines are truly pluripotent and can generate live born mice by tetraploid complementation. ^{58 -60} However, not all iPSC lines are entirely equivalent to ESCs. ^59,61,62 Despite this, the discovery of iPSCs has spurred the generation of patient-specific iPSCs for disease modeling, drug screening, and therapeutic application. ³²

The ability to generate patient-specific cell types could circumvent the difficulties of allogeneic organ transplantation where immune challenges continue to plaque long-term outcomes. Furthermore, the genetic etiology of disease could be corrected ex vivo using tools of modern genetics. ³² For example, genetically corrected iPSCs have been used to treat a mouse model of sickle cell anemia. ⁶³ Nevertheless, there remain significant obstacles in applying autologous iPSCs to correct diseases of many organ systems.

One obstacle that has been enthusiastically addressed by investigators is the use of integrating viruses in protocols to create iPSCs. These viruses are known to increase the risk of tumors, making iPSCs and their derivatives incompatible for patient use. Gene therapy trials in patients with X-linked severe combined immunodeficiency showed that integrating viruses activated proto-oncogene LMO2 and resulted in leukemia. ⁶⁴ Although it may possible to screen all iPSCs for their viral integration sites, ⁶⁵ the use of c-Myc, a known oncogene, as 1 of the 4 reprogramming factors increases the risks of transgene reactivation. ⁶⁶ Many groups have since shown that iPSCs can be generated without permanent viral integration using adenoviruses, ⁶⁷ plasmids, ⁶⁸ modified RNA, ⁶⁹ or transgene cassettes expressing the 4 factors that can be removed via methods like Cre-mediated excision. ⁷⁰ Even so, there is a risk of iPSCs acquiring other de novo genetic mutations as they are manipulated and proliferated in vitro; therefore, it may be necessary to sequence the genomes of iPSC clones to ensure that there are no unexpected integrations of viral or exogenous plasmid fragments that might cause spontaneous mutations. ⁶⁵

A second obstacle for those attempting to derive specific cell types from ESCs or iPSCs for direct transplantation is the ability to generate pure populations of desired cell types. Studies of embryonic development have led to the identification of key factors and culture conditions that aid the directed differentiation of ESCs or iPSCs into different lineages, ⁷¹ but efficient generation of therapeutically useful cells that faithfully reproduce their functions in vivo remains challenging. ⁷² Differentiation of cardiomyocytes from human ESCs or iPSCs using embryoid bodies produces heterogenous cell populations with variable levels of efficiency depending on the iPSC line. ⁷³ Modifications to this protocol may increase the efficiency of differentiation, ⁷⁴ and a recent report suggests that an alternative monolayer platform can generate greater than 30% cardiomyocytes in some iPSC lines. ⁷⁵ Furthermore, the use of small-molecule activators and inhibitors of the Wnt pathway have been shown to enhance iPSC differentiation to reliably generate greater than 90% cardiomyocytes from each preparation. Indeed, the ability to efficiently generate and purify populations of usable cardiomyocytes is critical to the progress of iPSC-derived cardiac therapies. Remaining issues, such as epigenetic “memory” of the iPSCs and in vitro culture-induced chromosomal mutation, may pose additional barriers to translation of these cells into human therapy. Thus, source of starting cells for reprogramming may influence the efficiency of iPSCs differentiation toward a desired lineage. ^{65,76 -80}

Third, in addition to the challenge of efficiently generating pure populations of many therapeutically useful cell types, there remain significant concerns that hidden within a heterogeneous population of differentiating cells are a few inclined to divide uncontrollably. ^{81 -83} Fortunately, methods have been reported that allow for the selective removal of undifferentiated cells; Tang et al recently used an antibody against stage-specific embryonic antigen 5 glycan and 2 other PSC surface markers to cause a loss of teratoma-forming potential in differentiated human ESC cultures. ⁸⁴ Further research will improve these strategies to remove undesirable cells thereby increasing the safety of ESC/iPSC-derived cell therapies.

Finally, for all iPSC-based therapies, the challenges of cellular transplantation remain daunting. Factors that may contribute to the poor engraftment efficiency in the damaged heart include ischemia-, anoikis-, and inflammation-related processes. Laflamme et al report, for example, that a cocktail of prosurvival factors may enhance cellular engraftment in the rat heart. ⁸⁵ However, the overall engraftment rate remains less than 5% of the starting cell population. Hence, future studies should focus on optimizing the transplantation process (eg, cell harvesting, storage, and injection) to enhance engraftment and survival in order to bring iPSC- and ESC-based therapies closer to the bedside.

Direct Cell Lineage Reprogramming

Although improvements in methods and technologies may eventually eliminate the risks of teratoma formation in ESC- or iPSC-based therapies, many of the aforementioned safety obstacles for this technology remain unresolved. Meanwhile, alternative approaches to generating therapeutically valuable cells are being explored. Somatic stem cells and tissues, for example, are attractive cell sources for cell-based therapies as they are readily available in the body and do not have the potential to form teratomas-like pluripotent stem cells.

A strategy that has received significant attention is the reprogramming of 1 somatic cell type to another without passing through a pluripotent state. This approach, termed “transdifferentiation” or “direct reprogramming,” was inspired by the shotgun approach use by Takahashi et al to generate the first iPSCs. Different research groups have screened combinatorial sets of transcription factors to find those that can induce cells to change from one somatic cell type to another. Zhou et al first reported the direct reprogramming of adult pancreatic exocrine cells to β cells in vivo by the viral overexpression of 3 transcription factors, isolated from a candidate pool of 30. ⁸⁶

Other successes include the generation of functional neurons, ^{87 -89} neural progenitors, ⁹⁰ blood progenitors, ⁹¹ endothelial cells, ⁹² and hepatocyte-like cells ⁹³ from mouse fibroblasts using a small number of overexpressed transcription factors. Since fibroblasts represent a readily available cell population, it is not surprising that many of the studies described thus far have employed fibroblasts as a starting cell source (Figure 1). As one would expect, the resulting cell types of interest in these early direct reprogramming efforts are often ones that are therapeutically valuable. As such, the direct reprogramming of dopaminergic neurons from both murine and human fibroblasts in vitro via the overexpression of Mash1, Nurr1, and Lmx1a ⁸⁹ was likely inspired by the finding that transplantation of ESC-derived dopaminergic neurons improves motor symptoms in a mouse model of Parkinson disease. ^94,95

Figure 1.

Three principal strategies to achieve somatic cell reprogramming and the relative quality of the final reprogrammed cell type. Therapeutically important cells may be generated via somatic cell nuclear transfer (SCNT), in vitro differentiation of induced pluripotent stem cells (iPSCs), and direct reprogramming of somatic cells without an intermediate pluripotent state.

Even among successful reports of direct reprogramming, there remain significant concerns regarding the efficiency of reprogramming and the fidelity of the resulting phenotypes to their gold standards in vivo. Since reprogrammed cells may no longer be proliferative, the efficiency of reprogramming must be also high enough for this approach to be considered for therapeutic uses.

Direct Reprogramming of Fibroblasts into Cardiomyocytes

Although many groups continue to explore cell therapy-based treatments for cardiac diseases using in vitro ESC- or iPSC-derived cardiomyocytes, the efficient deployment of these cardiomyocytes for therapy remains challenging. This has led to recent efforts to directly reprogram somatic cells to cardiomyocytes. One of the first reports of this effort was published by Efe et al in which they reprogrammed murine fibroblasts into cardiomyocytes using iPSC-generating factors without allowing these cells to pass through a pluripotent state (Figure 1). ⁹⁶ After infecting fibroblasts with Oct4, Sox2 and Klf4, cells were maintained in a LIF-free culture condition to prevent the induction or maintenance of a pluripotent cell phenotype. Subsequently, growth factors and small molecules were introduced to redirect partially reprogrammed cells into cardiomyocytes. They showed that this procedure is significantly more efficient and faster than the generation of cardiomyocytes by iPSCs in vitro differentiation. Interestingly, this method appears to generate mostly atrial cardiomyocytes, which may be therapeutically less useful than ventricular cardiomyocytes.

Another reprogramming method to generate cardiomyocytes from somatic cells has been recently studied in the developing embryo. Takeuchi and Bruneau found that mouse mesoderm can be directed into becoming contracting cardiomyocytes through the overexpression of Gata4, Tbx5, and BAF60c (a cardiac-specific subunit of BAF chromatin remodeling complexes that modulates the transcription of genes). ⁹⁷ Given the need for embryonic tissues as the starting cell source for this reprogramming strategy, it is unclear whether this combination of factors will be useful in adult somatic cell reprogramming.

Following the work of Takeuchi and Bruneau, Ieda et al examined whether mouse cardiac and tail tip fibroblasts can be directly reprogrammed into cardiomyocytes by virally mediated overexpression of Gata4, Mef2c, and Tbx5 (GMT; Figure 1). ⁹⁸ These authors found that GMT induces murine cardiac and tail tip fibroblasts into becoming cardiomyocyte-like cells that express cardiac troponin T and alpha-myosin heavy chain; the resulting cells could be further triggered to generate spontaneous action potentials in vitro. These “induced cardiomyocytes” (iCMs) are reportedly adult cardiomyocyte-like in that they lack an expression history of precardiac mesoderm and cardiac progenitor cell markers such as Mesp1 or Isl-1, respectively. Furthermore, fibroblasts infected in vitro are reportedly able to transdifferentiate in vivo into cardiomyocytes after transplantation into immunocompromised murine hearts. The epigenetic conversion of fibroblasts into cardiomyocyte-like cells was supported by the loss of repressive histone methylations and activation of permissive histone markings in promoter regions of cardiac genes.

Given the difficulty of translating ESC/iPSC-derived cardiomyocytes into therapy where cardiomyocyte enrichment, elimination of partially differentiated cells, and improvements in cell survival after transplantation remain unresolved issues, there is a growing enthusiasm for direct reprogramming as a novel approach to generate cardiomyocytes. This report from Ieda et al suggests a potential paradigm shift in our current approach to cardiac regenerative therapy as it promises the ability to inject transcription factors to reprogram endogenous cardiac fibroblasts at or near the site of ischemia–infarction. ⁹⁸ However, many questions remain as to whether the efficiency of this reprogramming method is sufficient to generate a large number of cardiomyocytes for therapeutic application, ⁹⁹ particularly when only a small percentage of fibroblasts can be reprogrammed in vitro into cardiomyocyte-like cells with low proliferative ability. To address the issue of efficiency, other groups have demonstrated that the addition of other transcription factors to the reprogramming cocktails, such as Myocardin, ¹⁰⁰ Hand2, ¹⁰¹ or microRNAs, ¹⁰² may improve the efficiency of reprogramming (Table 1). Moreover, Qian et al report that in vivo reprogramming through direct myocardial injection of GMT factor can achieve greater efficiency of fibroblast conversion into cardiomyocyte-like cells than reprogramming in vitro, leading to attenuation of infarct size and improvement in cardiac function. ¹⁰³

Table 1.

Summary of iCM Direct Reprogramming Studies in Murine Tissues.

	Mouse
	In Vitro			In Vivo
	Embryonic Fibroblasts	Cardiac Fibroblasts	Tail Tip Fibroblasts	Cardiac Fibroblasts, Infected In Vitro, Transplanted In Vivo	Cardiac Fibroblasts, Direct Infection
Efe et al 96,a Oct4, Sox2, Klf4, c-Myc, JAK inhibitor 1, BMP4	X	X
Ieda et al 98 GMT		X	X	X
Chen et al 100 GMT		X	X	X
Protze et al 101 Mef2c, Tbox 5, Myocd	X	X	X
Song et al 102,a GMT, Hand2		X	X		X
Jayawardena et al 103 miR-1, miR-133, miR-208, miR-499	X	X	X		X
Qian et al 104,a GMT + TGF-β					X

Abbreviations: GMT, Gata4, Mef2c, and Tbx5; iCM, induced cardiomyocyte; TGF-β, transforming growth factor β.

^aStudy used retroviruses. Studies used lentiviral vectors unless otherwise indicated.

Although these studies appear quite promising, questions remain regarding some of the discrepancies noted between the efficiency of reprogramming in vitro versus in vivo. The efficiency of in vivo reprogramming is remarkably higher (10%-20% cell conversion) than the efficiency of in vitro reprogramming (1%-5% or less). The most likely explanation for this difference is that the in vivo microenvironment provides soluble or contact factors that enable rapid phenotypic conversion of fibroblasts into cardiomyocytes. ¹⁰³ In addition, it is possible that the injected virus is able to concentrate itself within the relatively small extracellular compartment in the adult heart to achieve a much greater biological effect locally. Nevertheless, the Cre/LoxP technology used to identify iCM from reprogrammed fibroblast remains fraught with confounding issues (eg, reactivated Cre expression in the setting of myocardial injury, direct promotion of Cre expression by rare integration of lentiviruses into existing cardiomyocytes, leaky Cre expression in cardiomyocytes in the absence of viral infection, etc). Confirmation of the increased reprogramming efficiency in vivo by different laboratories using distinct methodologies will be key to sustain the interest in this technology from the cardiovascular research community.

Intriguingly, the factors used to reprogram murine and human fibroblasts appear somewhat distinct (Table 2). Although GMT factors were not sufficient for the direct reprogramming of human cardiac fibroblasts, the addition of Mesp1 and Myocd successfully generated more cardiac-like cells in vitro. ¹⁰⁴ Others have reported that a combination of transcription factors (Gata4, Tbx5, Hand2, myocardin, and Mesp1) and microRNAs (miR-1 and miR-133) among other factors can more effectively reprogram human fibroblasts toward cardiac phenotypes. ^105,106

Table 2.

Summary of iCM Direct Reprogramming Studies in Human Tissues.

	Human
	In Vitro
	Embryonic Fibroblasts	Neonatal Dermal Fibroblasts	Adult Dermal Fibroblasts	Cardiac Fibroblasts
Wada et al 105,a GMT, Mesp1, Myocd		X		X Adult
Nam et al106, b Gata4, Tbx5, Hand2, Myocd, miR-1, MiR-133		X	X	X Adult
Fu et al107, b GMT, ESRRG, Mesp1, Myocd, ZFPM2, TGF-β	X Differentiated from hESC	X		X Fetal

Abbreviations: GMT, Gata4, Mef2c, and Tbx5; hESC, human embryonic stem cell; iCM, induced cardiomyocyte; TGF-β, transforming growth factor β.

^aStudy used retroviruses and lentiviruses.

^bStudy used retroviruses.

As the direct cardiac reprogramming field is relatively new, an in-depth analysis of the iCM phenotypes created by each cocktail of factors has not been published. Thus far, no study has performed a direct side-by-side comparison of the phenotypes of iCMs generated from the direct differentiation of iPSCs with cardiomyocyte-like cells generated from direct reprogramming or a bona fide cardiomyocyte from adult heart. Although it has been demonstrated that iPSCs/ESCs can be differentiated into both atrial and ventricular cardiac phenotypes, ^{107 -110} most studies of direct reprogramming have yet to fully characterize the electrophysiological and contractile parameters of their resultant cell populations. Similarly, iCMs from direct reprogramming may also bear different epigenetic modifications than those differentiated from iPSCs/ESCs ⁷⁶ as the former does not pass through a progenitor state, but no study has explored this in detail. Further research on the functional phenotypes and genetic signatures of iCMs will elucidate how they compare with iPSC/ESC-derived cardiomyocytes as potential tools for cardiac therapy.

In addition to the need for more detailed understandings of the biology of iCMs, considerable improvements in the basic methods of reprogramming must be made before the therapeutic promise of this approach might be realized. First, the use of viral vectors should be minimized to prevent the risk of tumor formation. ¹¹¹ Second, the targeting of transcription factor to fibroblasts and not endothelial or smooth muscle cells should be optimized in order to minimize off-target effects. Finally, the efficiency, speed, and quality of conversion from cardiac fibroblasts to cardiomyocytes should be sufficiently high (eg, >50% of cells introduced with factors converted to fully mature cardiomyocytes within 5-10 days of transduction) for this strategy to be therapeutically relevant in the postinfarct setting to prevent adverse remodeling.

Despite these challenges, the direct reprogramming of endogenous cardiac fibroblasts to iCMs after myocardial ischemic injury remains promising as a therapeutic tool because the technique does not require ex vivo manipulation, which has been shown to potentiate genotoxicity ¹¹² or cellular transplantation, which remains difficult to optimize to achieve sufficient cell survival and engraftment. ^85,99 With the development of innovative methodologies to safely and efficaciously deliver reprogramming factors, the direct reprogramming strategy may become a viable therapeutic option for patients with cardiac disease in the future.

Conclusion/Future Perspective

The recent successes of cardiomyocyte generation from reprogramming approaches build upon half a century of basic developmental biology research. Early investigators sought to establish that the genetic content of cells do not change throughout differentiation, leading to future studies involving fusion hybrids and SCNT. Better understanding of the molecular determinants of genetic expression—from epigenetic states to “master regulator genes”—has opened the doors to the direct reprogramming of somatic cells into therapeutically valuable cell types.

In the future, the application of direct reprogramming strategy may allow a patient with a recent myocardial infarction to undergo a treatment regimen, whereby a cocktail of factors is introduced into the scar tissue at the site of infarction. This treatment would aim to reprogram the endogenous fibroblasts into bona fide cardiomyocytes, thereby diminishing the scar size and increasing the cardiomyocyte content and improving overall cardiac function. ^101,103

At the present time, however, numerous challenges of reprogramming such as efficiency, scalability, and quality of the reprogrammed cells must be overcome in order to realize such a therapeutic promise. The availability of a stringent and uniform standard for the characterization of reprogrammed cardiomyocytes will help to guide the generation of fully functional cells. ¹¹³ Concurrently, considerable progress needs to be made in order to improve the maturation of in vitro iPSC/ESC-derived or reprogrammed cardiomyocytes. With additional efforts, the transplantation of reprogrammed cardiomyocytes or the in situ reprogramming of cardiac fibroblasts directly into new cardiomyocytes may prove feasible for clinical application in the near future.