Direct Cardiac Reprogramming for Cardiovascular Regeneration and Differentiation

Abstract

Cardiovascular disease is the leading cause of death worldwide. Cardiomyocytes have limited regenerative capacity; consequently, regenerative therapies are in high demand. There are currently several potential strategies for heart regeneration, with one approach involving in situ generation of new cardiomyocytes from endogenous cell sources. Direct cardiac reprogramming has emerged as a novel therapeutic approach to regenerating the damaged heart by directly converting endogenous cardiac fibroblasts into cardiomyocyte-like cells. Following our first report of direct cardiac reprogramming, significant advances have elucidated the molecular mechanisms associated with cardiac reprogramming. These advances have also improved cardiac-reprogramming efficiency by enabling direct in vivo cardiac reprogramming. Moreover, progress has been made in cardiac reprogramming of human fibroblasts. Although basic research has supported substantial progress in this field, numerous challenges remain in terms of clinical application. Here, we review the current state of cardiac reprogramming as a new technology for understanding and treating cardiovascular diseases.

Introduction

Cardiovascular diseases are the leading causes of death and disability worldwide. Adult cardiomyocytes have little regenerative capacity. Dead cardiomyocytes are replaced by fibroblasts, leading to the formation of fibrosis, cardiac remodeling, and heart failure, which is associated with high mortality.^1,2 Despite advances in medical devices and pharmacotherapy, the mortality rate of patients with severe heart failure remains high. Moreover, cardiomyocytes are considered to be in a terminally differentiated state, and the regenerative capacity of human cardiomyocytes is insufficient to completely regenerate the lost myocardium.³ Therefore, cardiac regeneration has attracted attention as a novel therapeutic approach for patients with heart disease. Numerous studies have attempted to induce cardiac regeneration by the addition of exogenous cells to stimulate cardiac repair and improve cardiac function;⁴ however, the results of clinical trials have mostly not been positive because of problems related to safety and low engraftment rates. Another promising approach to repairing the injured heart is to use cardiomyocytes derived from allogeneic pluripotent stem cells (PSCs), such as embryonic stem cells and induced PSCs (iPSCs). Much progress has been made in these fields, and the first-in-man clinical trial using iPSC-derived cardiomyocytes is currently planned by Japanese research groups. However, PSC-based therapies raise concerns that need to be addressed before their practical use in patients. These concerns include poor engraftment rates and the potential risk of tumorigenesis.⁵ To overcome the major issues that arise from the use of PSCs, we followed a novel approach to generate additional cardiomyocytes in the heart. This led to a heart repair technique that uses direct cardiac reprogramming: by applying the transduction of cardiac-specific transcription factors, resident cardiac fibroblasts (CFs) are converted to induced cardiomyocyte-like cells (iCMs) without reverting to PSCs. In this review, we summarize recent advances in cardiac reprogramming and discuss the perspectives and challenges for future clinical application.

Overview of Reprogramming by the Forced Expression of Transcription Factors

The generation of desired cell types from abundant and accessible cells holds great promise for regenerative medicine. Previous pioneering studies provided a foundation for the advent of this developing field by establishing that the stability of the differentiated state is not the result of irreversible changes that occur during the process of cell differentiation. In the 1960s, Gurdon demonstrated the first example of cellular reprogramming of a differentiated cell state into an undifferentiated embryonic cell state by somatic cell nuclear transfer, a technique in which the nucleus of a somatic cell is transferred into an enucleated oocyte.^6,7 In the late 1980s, Blau et al.^8,9 demonstrated that cell-fusion experiments of fibroblasts–myocytes resulted in heterokaryons capable of activating muscle-specific genes. These results suggested the existence of one or more reprogramming factors capable of erasing somatic memory. Indeed, Davis et al.¹⁰ provided further evidence of the existence of reprogramming factors and also demsontrated the direct fate conversion of mammalian cells through overexpression of a single transcription factor. In their experiments, overexpression of MyoD, a master regulator of skeletal muscle genes, was sufficient to convert fibroblasts into contracting myocytes in vitro. Moreover, Takahashi and Yamanaka¹¹ achieved a breakthrough in this field by overexpressing four transcription factors [Oct4, Sox2, Klf4, and c-Myc (OSKM)] in mouse fibroblasts, thereby inducing a pluripotent state; in 2006, these cells were named iPSCs. The discovery of iPSCs inspired a new approach that allows the generation of desired cell types without the need for them to pass through a stem cell stage. This was achieved by introducing combinations of multiple lineage-specific factors in a process referred to as direct reprogramming.

Direct Reprogramming of Fibroblasts to Cardiomyocytes by Defined Factors

Discovery of direct cardiac reprogramming by Gata4, Mef2c, and Tbx5

The discovery of iPSCs cleared the way for the establishment of direct cardiac reprogramming. CFs constitute approximately 10% of all cardiac cells and are abundant in the heart;¹² CFs therefore represent a potential source of cardiomyocytes for cardiac regeneration. When heart disease causes cardiac damage, resident CFs are activated and increase in number, subsequently contributing to scar formation and poor prognosis.^13,14,15 Therefore, converting CFs to cardiomyocytes represents a promising approach to heart regeneration and reducing the level of cardiac fibrosis. We hypothesized that reprogramming factors could be identified by their specific expression during cardiac development, and that combinations of key developmental cardiac regulators could directly convert resident CFs into iCMs. To test this hypothesis, we first generated α-myosin heavy chain promoter-driven enhanced green fluorescent protein (αMHC-GFP) transgenic mice so that we could use fluorescence-activated cell sorting to detect cells harboring activated cardiac programs and expressing GFP. Following retroviral expression of candidate genes in CFs, we found that a combination of three transcription factors (Gata4, Mef2c, and Tbx5; GMT), could directly convert CFs into iCMs.¹⁶

Lineage-tracing experiments confirmed that cardiac-progenitor genes were not activated during the reprogramming process, suggesting that fibroblasts were converted directly into iCMs without passing through a progenitor-cell state. Moreover, the iCMs exhibited cardiomyocyte-like characteristics, such as well-organized sarcomeric structures and similar global gene-expression profiles. Although transduction of GMT activates αMHC-GFP in approximately 20% of CFs, only 0.01–0.1% of the original CFs were converted into spontaneously beating iCMs. This finding suggests that the majority of αMHC-GFP-positive cells became partially reprogrammed iCMs. This low efficiency and immature reprogramming might have been caused by the stoichiometry of GMT in CFs transduced using separate vectors. Previous studies generated a polycistronic retrovirus expressing GMT at near equimolar levels from the same promoter using “self-cleaving” 2A peptides.^17,18 Additionally, Wang et al.¹⁷ generated six polycistronic constructs in which each possible combination of GMT was constructed in a single transgene. They found that the order of the genes affected the subsequent protein levels, with the highest protein level observed for the factor in the first position. These findings showed that two polycistronic vectors, MGT and MTG, which expressed a high level of Mef2c and low levels of Gata4 and Tbx5, promoted efficient cardiac reprogramming, whereas the others resulted in reduced reprogramming efficiency. This revealed that elevated Mef2c levels and subsequent transcriptional activity was critical for successful cardiac reprogramming. These results suggested that reprogramming could be improved by optimizing the reprogramming factors and culture conditions, similar to other cell-reprogramming strategies (Table 1).^19,20

Table 1.Summary of direct cardiac reprogramming of mouse fibroblasts.

Reprogramming factors	In vitro/In vivo	References
Gata4, Mef2c, Tbx5 (GMT)	Both	16, 41, 46
Gata4, Hand 2, Mef2c, Tbx5 (GHMT)	Both	22, 23
miR-1, miR-133, miR-208, miR-499 (miR combo)	Both	27
GMT, miR-1/miR-133	In vitro	29
GMHT + DAPT (Notch inhibitor)	In vitro	31
GMHT, Nkx2.5 + SB431542 (TGF-β inhibitor)	In vitro	33
GMT/GMHT+ FGF2, FGF10, VEGF	In vitro	53
GMT + SB431542, XAV939 (Wnt inhibitor)	In vitro	34
GMT/GMHT+ Y-27632 (ROCK inhibitor), A83-01(TGF-β inhibitor)	In vitro	32
GMT, Bmi1	In vitro	37
GHMT, Akt1 (AGHMT)	In vitro	54
AGHMT + Znf281	In vitro	39

Development of alternative reprogramming cocktails

Progress in other fields utilizing direct-reprogramming methods, such as those for iPSCs, indicated that the addition or modification of transcription factors might promote reprogramming efficiency and maturation.²¹ The first alternative reprogramming cocktail was discovered by Song et al.²² in 2012. They investigated the optimal combination of six conserved cardiac-lineage transcription factors, including GMT, for reprogramming adult CFs and tail-tip fibroblasts (TTFs) into beating iCMs. They demonstrated that GMT factors plus Hand2 (GHMT) resulted in more efficient reprogramming of CFs into beating iCMs than the use of GMT alone. GHMT induced an approximately 3-fold increase in cTnT/αMHC-GFP double-positive iCMs relative to those generated by GMT after 1 week. This finding suggested that Hand2 enhances cardiac reprogramming, at least in part, by increasing cardiac-marker-expressing cells at the early stage.^22,23 Other studies analyzed the properties of each reprogramming factor in more detail. Hirai et al.²⁴ found that the transcriptional activity of Mef2c is also critical for successful reprogramming with GHMT to attain mature iCMs, which was consistent with the results reported by Wang et al.^17,24 Hirai et al.²⁴ fused the MyoD transactivation domain to GHMT and transduced these genes in fibroblasts. This resulted in increased Mef2c activation along with the wild-type forms of the other three genes and enhanced the reprogramming efficiency and the generation of beating iCMs. Reprogramming cocktails can contain not only transcription factors but also microRNAs (miRs), which are short noncoding RNAs that regulate gene expression post-transcriptionally. miRs target numerous genes related to signaling pathways, transcription factors, and epigenetic regulators and play important roles in cell fate and embryonic cardiac development.^25,26 Jayawardena et al.^27,28 introduced a combination of muscle-specific miRs (miR-1, miR-133, miR-208, and miR-499) into neonatal CFs to successfully create functional iCMs. To investigate the possibility of synergistic effects between transcription factors and miRs in cardiac reprogramming, we demonstrated that the addition of miR-133 to GMT promoted cardiac-reprogramming efficiency and maturation, resulting in the generation of a 7-fold increase of beating iCMs compared with GMT treatment alone.²⁹ Additionally, this treatment also shortened the time required to induce beating iCMs to one-third of that using GMT. The molecular mechanisms associated with the reprogramming process are discussed below.

Barriers to Cardiac Reprogramming

A better understanding of the molecular mechanisms involved in direct cardiac reprogramming enables the development of novel approaches for improving reprogramming efficiency. The optimization of reprogramming cocktails, the modification of signaling pathways, and epigenetic alterations represent potential strategies for overcoming the challenges associated with direct reprogramming. Fibroblast signatures are a major hurdle for efficient and successful cardiac reprogramming and need to be suppressed to allow successful conversion. For example, miRs bind to the 3′ untranslated region of their target mRNAs to repress protein translation. We showed that miR-133 plus GMT improves cardiac reprogramming because miR-133 directly targets Snai1, a master regulator of the epithelial-to-mesenchymal transition. Snai1 knockdown subsequently suppresses the expression of fibroblast genes and upregulates cardiac gene expression, whereas Snai1 overexpression maintains fibroblast gene expression and suppresses the generation of beating iCMs.²⁹

During direct cardiac reprogramming, a variety of signaling pathways, including those associated with transforming growth factor-β (TGF-β), Wnt, Notch, and Akt, interact with each other.^30,31,32 Notably, the TGF-β and Wnt pathways play a key role in fibroblast activation. Ifkovits et al.³³ demonstrated that inhibition of TGF-β signaling increased cardiac reprogramming by silencing fibroblast signatures. Subsequently, Mohamed et al.³⁴ confirmed that inhibition of TGF-β and Wnt signaling enhanced cardiac-reprogramming efficiency and maturation. Therefore, overcoming these mechanistic hurdles allowed successful reprogramming of efficient and functionally mature iCMs.

Because the epigenetic state of genes controls their transcription, the pre-existing chromatin states also potentially adversely impact the direct cardiac-reprogramming process.³⁵ Epigenetic conversion of iCMs to CMs was discovered through epigenetic analyses that revealed histone modifications and DNA methylation at cardiac-specific gene promoters.^16,36 A previous study showed that during cardiac direct reprogramming, trimethylation at lysine 27 of histone 3, which results in a closed chromatin structure, increased at fibroblast-specific gene promoters and decreased at cardiac-specific gene promoters. In contrast, trimethylation at lysine 4 of histone 3, which results in an open chromatin structure, is enriched at cardiac promoters early in GMT-cocktail reprogramming.³⁶

To achieve successful reprogramming, reprogramming transcription factors require access to genes that are developmentally silenced and possibly inappropriate for expression during early cell maturation; consequently, epigenetic modifiers might affect reprogramming efficiency. Recently, Zhou et al.³⁷ identified Bmi1 as a critical epigenetic hurdle to direct cardiac reprogramming through its regulation of key cardiogenic genes. Bmi1 binds to specific loci associated with fibroblast genes, and Bmi1 inhibition promotes an open-chromatin state. These results suggest that epigenetic regulation is involved in orchestrating the reprogramming process.

We recently revealed that inflammation acts as a barrier to direct cardiac reprogramming, particularly in aged fibroblasts.³⁸ Although adult fibroblasts are less efficient at reprogramming than embryonic fibroblasts are, the reasons for attenuated cardiac reprogramming associated with aging remain elusive. High-throughput screening of 8400 chemical libraries for compounds associated with cardiac reprogramming revealed that diclofenac sodium, a nonsteroidal anti-inflammatory drug, enhances cardiac-reprogramming efficiency and maturation in combination with GMT or GHMT in adult fibroblasts. Mechanistically, cyclooxygenase-2 (COX-2) is more strongly expressed in postnatal and adult TTFs than in embryonic fibroblasts in an age-dependent manner. Diclofenac enhances cardiac reprogramming by inhibiting signaling associated with COX-2, prostaglandin E2 receptor 4, cyclic AMP/protein kinase A, and interleukin-1β and by silencing inflammatory and fibroblast programs that are activated in postnatal and adult TTFs. This is consistent with the results of other cardiac-reprogramming studies that involved unbiased screening of human transcription factors. For example, Zhou et al.³⁹ found that Znf281 induces cardiac reprogramming by suppressing the expression of genes associated with the inflammatory response and modulating cardiac gene expression by interacting with the transcription factor Gata4. Although several studies have found ways to improve cardiac-reprogramming efficiency and maturation by modifying reprogramming cocktails and overcoming molecular hurdles, significant aspects of the molecular mechanisms associated with cardiac reprogramming remain to be addressed. Therefore, further investigation is needed to elucidate these molecular mechanisms and to facilitate the development of novel methods for cardiac reprogramming (Fig. 1).

Fig. 1.

Schematic representation of the hurdles to direct reprogramming along with proposed solutions determined through elucidation of the associated molecular mechanisms.

In Vivo Cardiac Repair and Regeneration

The ultimate goal of direct reprogramming is to provide a foundation for in vivo repair of the heart by targeting endogenous CFs with reprogramming factors. Previous in vivo cardiac regeneration involved cell-based therapies, such as the use of bone marrow-derived cells and cardiac-progenitor cells. However, the primary mechanisms associated with this approach to cardiac repair resulted in low engraftment rates following paracrine activation.⁴⁰In vivo direct reprogramming following acute myocardial infarction (MI) in mice was reported after retroviral or lentiviral delivery of reprogramming factors (GMT, GHMT, and miR combinations).^18,22,28,41 In contrast to previous in vivo cell-based therapies, the lineage-tracing approach demonstrated that these newly generated iCMs were derived from resident CFs and not from cell fusion with endogenous cardiomyocytes. Interestingly, these studies also demonstrated that iCMs generated in vivo more closely resembled endogenous cardiomyocytes than those produced in vitro and could form gap junctions with endogenous cardiomyocytes.²² Indeed, genome-wide transcriptional profiling confirmed that iCMs generated in vivo were highly similar to bona fide cardiomyocytes.⁴²

Additionally, two in vitro studies demonstrated that physiologically relevant culture conditions promoted cardiac reprogramming. Sia et al.⁴³ demonstrated that a micro-grooved substrate improved cardiac-reprogramming efficiency, and Li et al.⁴⁴ showed that a three-dimensional fibrin hydrogel culture enhanced direct cardiac reprogramming. These results indicated that the native microenvironment in vivo, which includes extracellular matrix, secreted proteins, electromechanical stimulation, and tissue stiffness, might enforce direct cardiac reprogramming.⁴⁵

Although in vivo reprogramming can improve cardiac function and reduce fibrosis after MI, current protocols, through the use of retroviral and lentiviral vectors, can potentially lead to insertional mutagenesis as a result of random genomic integration of virally overexpressed factors. Therefore, the development of a safe delivery method is required to promote clinical application and avoid integration of the delivered gene sequences into host chromosomes. Recently, we developed a polycistronic Sendai virus vector expressing GMT (SeV-GMT) and demonstrated its use for successful in vivo direct cardiac reprogramming.⁴⁶ Genome replication and virion production by SeV nonsegmented, negative-stranded RNA occurs exclusively in the cytoplasm, and therefore does not integrate into the host genome. Notably, compared with conventional retroviral GMT, this new integration-free SeV-GMT promoted cardiac reprogramming in vivo and resulted in improved cardiac function and lower levels of fibrosis following acute MI in mice. Although further investigation is needed, in vivo cardiac reprogramming with SeV could be a potential treatment for heart diseases in the future.

Future Challenges: Direct Reprogramming into Chamber-specific Myocytes and Cardiac-progenitor Cells

Cardiomyocytes comprise several subtypes, including pacemaker cells, atrial myocytes, and ventricular myocytes.⁴⁷ The development of cardiac-subtype specifications to target cardiac reprogramming to desired mature cell types is essential to promote the effective treatment of cardiovascular disease. The identification of specific markers and defined culture conditions will potentially enable the acquisition of the desired cardiac cell types from PSC-directed cardiomyocyte differentiation. Directed cardiac differentiation from PSCs using a series of small molecules and cytokines initially produces mixed cardiovascular populations that contain ventricular-like cells together with small numbers of nodal- and atrial-like cells.⁴⁸ This mixture of cell types makes this method problematic for disease modeling and heart regeneration. To overcome this issue, Lee et al.⁴⁹ demonstrated that atrial and ventricular cardiomyocytes develop from distinct mesoderm populations, revealing that efficient atrial/ventricular myocyte generation is dependent upon specific mesodermal markers. Recently, Zhao et al.⁵⁰ developed a method to engineer heteropolar cardiac tissues containing distinct atrial and ventricular ends, resulting in tissue that exhibited spatially confined drug responses. In contrast to PSC-derived cardiomyocytes, iCMs generated using GMT show mostly atrial-type myocytes based on their in vitro action-potential (AP) characteristics.^29,46 This tendency to form atrial-type myocytes has been observed in human cardiac reprogramming. GMT factors plus Mesp1 and myocardin induced iCMs from human CFs that most frequently demonstrated atrial-like APs.⁵¹

Although a GMT cocktail can generate mostly atrial-type myocytes, Hand2 plays a unique role in modulating cell-fate determination and conversion, as evidenced by the fact that GHMT produces a higher number of ventricular-type myocytes than GMT alone does.^23,32 Additionally, Hand2 promotes the development of cardiomyocytes derived from cardiac-progenitor cells during cardiac development.⁵² These findings suggest that the addition to GHMT of other developmental cardiac-lineage determinants, such as cardiac-subtype-specific factors or molecules, might promote the conversion of fibroblasts directly into the desired cardiac cell types.

We demonstrated that serum-free culture medium containing cardiogenic growth factors, such as fibroblast growth factor (FGF)2, FGF10, and vascular endothelial growth factor (VEGF), greatly enhanced the in vitro generation of beating mature iCMs by activating the p38 mitogen-activated protein kinase pathway and the phosphoinositide 3-kinase/Akt pathway.⁵³ Although electrophysiological analyses were not performed, microarray analyses revealed that the Gene Ontology terms related to ventricular cardiac muscle tissue were highly associated with the upregulated genes. Moreover, identification of optimal combinations of reprogramming factors also affected ventricular-type iCM reprograming. For example, Akt1 activation increased reprogramming efficiency and produced a higher number of mature iCMs via its downstream targets through activation of mammalian target of mTOR and FoxO3a. Furthermore, RNA-Seq analysis revealed that iCMs generated by Akt1 plus GHMT were more similar to adult mouse ventricular cardiomyocytes than those generated using GHMT alone, which agreed with the results reported by our group.^53,54 Although the route of cardiac generation differs between PSC-derived cardiomyocytes and iCMs, comparative analyses revealed that iCMs generated in vitro demonstrated more adult cardiomyocyte features than did iPSC-derived cardiomyocytes.⁵⁵

For regenerative purposes, highly enriched ventricular-type iCMs are the ideal subtype for remuscularization of the ventricular wall in patients following MI. Interestingly, compared with those generated in vitro, iCMs generated in vivo more closely resembled ventricular-type myocytes and displayed changes in morphology, binucleation, and gene expression corresponding to a more mature cardiomyocyte phenotype. Patch-clamp analyses revealed that in vivo iCMs generated in ventricles displayed endogenous ventricular cardiomyocyte-like APs.^22,41,46 These findings suggest that the in vivo cardiac environment promotes the generation of features resembling ventricular-like cells, presumably via unidentified maturation factors. These maturation factors should be the focus of future studies.

iCMs do not have proliferative potential; consequently, the direct generation of proliferative myogenic cells from fibroblasts would be ideal.⁵⁶ Because of their multipotency and proliferative capacities, cardiac-mesoderm and -progenitor cells are attractive sources for clinical applications. Although some success has been achieved in recent years, direct cardiac-mesoderm/-progenitor cell reprogramming protocols remain undefined. Previous studies have reported the direct reprogramming of fibroblasts into proliferative and multipotent induced cardiac-progenitor cells (iCPCs) that subsequently differentiated into cardiomyocyte-like cells following co-culture with other cardiomyocytes. These iCPCs differentiated into beating cardiomyocytes, which suggested their original status as immature cardiomyocytes.^57,58

We recently demonstrated that Tbx6 is critical for PSC differentiation into mesoderm and cardiovascular lineages.⁵⁹ Because Tbx6 is a marker of paraxial mesoderm and promotes the formation of somite lineages, this finding was somewhat unexpected. To identify novel regulators, we performed direct reprogramming-based unbiased screening. We transduced 58 candidate factors into fibroblasts to analyze the induction of Mesp1, a marker of early/nascent mesoderm. We found that only Tbx6 strongly induced Mesp1 mRNA expression, and that Tbx6-transduced cells expressed nascent mesoderm genes and specific surface markers, whereas these genes in differentiated derivatives, such as cardiomyocytes, smooth muscle cells, and endothelial cells, were not activated. These results suggested that continuous Tbx6 expression induced and maintained a nascent mesoderm program in fibroblasts. Moreover, we found that temporal Tbx6 expression in PSCs induced mesoderm differentiation and further mesodermal-lineage diversification. Interestingly, transient Tbx6 expression induced PSC differentiation into a cardiovascular lineage, whereas continuous Tbx6 expression led to paraxial- and somite-lineage differentiation, suggesting that temporal and spatial expression of Tbx6 is critical for lineage determination. Furthermore, Tbx6-induced mesodermal cells expressed cardiac mesoderm markers but not Gata4, Nkx2.5, or Mef2c, suggesting that they were at an earlier stage of differentiation relative to iCPCs.⁵² Although these findings of cardiac-mesoderm/-progenitor cell generation represent an important step in identifying novel pathways of cardiovascular reprogramming, further studies are needed to allow clinical application of this strategy (Fig. 2).

Fig. 2.

Tbx6 expression induces a cardiac mesoderm program in mouse fibroblasts and PSCs. Schematic of cardiac mesoderm induction and cardiovascular differentiation after Tbx6 overexpression.

Conclusions and Outstanding Questions

Substantial progress has been made in the field of heart regeneration in recent years, and direct reprogramming therapy represents a promising and beneficial approach to heart regeneration (Fig. 3). Despite progress in this field, there remain several challenges that must be addressed prior to clinical application. First, experiments in chronic heart-failure models are required to assess the efficacy and safety of the various approaches. Although in vivo cardiac reprogramming has resulted impressive improvements in cardiac function and fibrosis, all in vivo studies to date have been performed during the acute stage of MI;^{18,22,34,41,46} therefore, it remains unknown whether in vivo reprogramming is applicable to chronic heart-failure models, where regenerative medicine is in high demand. Second, human cardiac reprogramming is more challenging than the process demonstrated in mouse cells. Although critical factors have been identified in mouse models, these factors are insufficient for human cardiac reprogramming because of differences in cellular contexts, including the epigenetic landscape, endogenous signaling, and transgene expression. Despite considerable successes, direct cardiac-reprogramming protocols for humans need to be improved (Table 2).^{29,34,51, 60, 61} There is a high demand for new regenerative therapies, and there remain significant opportunities and potential benefits for optimizing the approaches used for direct cardiac reprogramming.

Fig. 3.

In vitro and in vivo application of iCMs. Reprogramming cocktails can promote the direct conversion of cardiac fibroblasts into iCMs. iCMs generated in vitro might be a suitable source for individualized drug discovery, cardiotoxicity analysis, and disease modeling. Direct cardiac reprogramming in vivo can induce the conversion of resident CFs into functional iCMs in situ and improve cardiac function.

Table 2.Summary of direct cardiac reprogramming of human fibroblasts.

Reprogramming factors	References
Gata4, Hand2, Tbx5, Myocd, miR-1, miR-133	60
Gata4, Mef2c, Tbx5 (GMT) + Mesp1, Myocd	51
GMT + Esrrg, Mesp1, Myocd, ZFPM2	61
GMT + Mesp1, Myocd, miR-133	29
GMT + Myocd, Wnt/TGF-β inhibitor	34

Acknowledgements

M.I. was supported by research grants from the Research Center Network for Realization of Regenerative Medicine and the Practical Research Project for Rare/Intractable Diseases funded by the Japan Agency for Medical Research and Development (AMED), the Japan Society for the Promotion of Science (JSPS; 17H04179 and 17K19678), and the Takeda Science Foundation. T.S. was supported by research grants from JSPS (16K19426 and 18K08114), a Japan Heart Foundation Research Grant, the Nakatomi Foundation, and the Miyata Cardiac Research Promotion Foundation

Conflicts of Interest

M.I. and T.S. are filing a patent application based on the methods for producing cardiac precursor cells (WO2017/159643 A1, WO2017/159644 A1).

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