There is a long history of research on amphibian heart regeneration with the most adopted model the zebrafish given its substantial regenerative capacity and amenability to genetic manipulation. The zebrafish heart fully regenerates after the surgical amputation of the cardiac apex: an injury that corresponds to a loss of approximately 20% of the total ventricular mass.1 Initial experiments suggested that undifferentiated progenitor cells were the principal source of regenerating cardiomyocytes in zebrafish; however, two recent gene mapping studies clearly demonstrate that preexisting committed cardiomyocytes are instead the main source.2,3 These two groups independently generated transgenic zebrafish in which the cardiomyocyte-specific cmlc2 (also known as myl7) promoter drives the expression of tamoxifen-inducible Cre recombinase. These animals were crossed with a reporter line in which Cre-mediated excision of a loxP-flanked stop sequence induces constitutive expression of green fluorescent protein (GFP). In the offspring of this cross, all pre-existing cardiomyocytes and their progeny were induced to express GFP by tamoxifen treatment. Therefore, if the regenerated myocardium was derived from undifferentiated progenitor cells, the new ventricular apex should be GFP−. Instead, both groups found that the vast majority of the newly regenerated cardiomyocytes were GFP+, suggesting that the heart regeneration in zebrafish is principally mediated by the proliferation of pre-existing cardiomyocytes. This is contrary to the previously held belief that the generation of new cardiomyocytes from stem cells was the underlying aetiology.

Although they lack the regenerative capacity of the zebrafish heart, postnatal mammalian hearts also undergo a degree of cardiomyocyte renewal during normal ageing and disease. Recently, a study4 showed that the differences between mammalian and fish hearts may not necessarily apply early in development. Using approaches from the zebrafish model, the authors resected the left ventricular (LV) apex of 1-day-old neonatal mice and observed a brisk regenerative response similar to that in the adult zebrafish. By 3 weeks after injury, the defect had been replaced by normal myocardial tissue, which showed normal contractile function by 8 weeks. Genetic fate-mapping studies indicated that this regeneration was mediated by the proliferation of pre-existing cardiomyocytes, again as in the zebrafish. Notably, this regenerative capacity was not observed in 7-day-old mice, suggesting that its loss may coincide with cardiomyocyte binucleation and reduced cell-cycle activity. Nonetheless, this study indicates that zebrafish-like regenerative mechanisms are latent in mammalian hearts. It also provides a genetically tractable model for dissecting the blocks to these mechanisms in the mammalian adult.

It has recently been demonstrated that fibroblasts in infarcts could potentially be reprogrammed directly to cardiomyocytes. Fifteen years ago, researchers showed that fibroblasts could be differentiated into skeletal muscle in vitro or in the injured heart by overexpressing the gene encoding the myogenic transcription factor, MyoD. However, despite extensive work, no comparable master gene for cardiac muscle was found, and interest in reprogramming waned. Spurred by the discovery of induced pluripotent stem cells (iPSCs), scientists have now returned to this field, using combinations of transcription factors to reactivate core transcriptional networks of desired cell types. In the last 2 years, two groups have made progress to this goal. The first group5 screened a total of 14 cardiac transcription factors finding that a specific combination of three transcription factors, Gata4, Mef2c and Tbx5, was sufficient to generate functional beating cardiomyocytes directly from mouse postnatal cardiac or dermal fibroblasts and that the induced cardiomyocytes were globally reprogrammed to adopt a cardiomyocyte-like gene expression profile. These factors activated the transgene in 20% of fibroblasts of which approximately 4% of the cells expressed endogenous sarcomeric proteins such as cardiac troponin T, with ∼1% showing functional properties such as spontaneous beating. Thus, most of the cells were only partially reprogrammed, although their global gene expression patterns had shifted markedly from fibroblast to cardiomyocyte.

The second group6 used a different method of reprogramming mouse embryonic fibroblasts to cardiomyocytes. They used the ‘Yamanaka factors’—OCT4 (also known as POU5F1), SOX2, KLF4 and c-MYC—to initiate reprogramming, but then blocked signalling through the JAK—STAT pathway, which is required for pluripotency in the mouse, and added the cardiogenic factor BMP4. These modifications yielded minimal generation of iPSCs, but instead activated the cardiac progenitor programme and, within 2 weeks, generated substantial numbers of beating colonies. By 18 days after induction, approximately 40% of the cells expressed cardiac troponin T. It should be noted that this study used mouse embryonic fibroblasts, whereas Leda et al5 principally used postnatal mouse cardiac fibroblasts. Reprogramming the scar-forming fibroblast to a cardiomyocyte is appealing, particularly if it can be done directly in the infarct. To succeed clinically, we need to know how normal these reprogrammed cardiomyocytes are, and the process will have to be much more efficient and transgene-free.

A recent report in this journal drew attention to the great promise of iPSC (reprogrammed somatic cells) as a renewable source of autologous cells.7 These cells were first discovered only 5 years ago by Takahashi and Yamanaka8 following the introduction of genes into adult mouse cells reprogramming them to resemble embryonic stem (ES) cells. Given that the DNA of such cells is identical to that of the patient, it has been assumed that they would not be attacked by the immune system although their immunogenicity has not been vigorously examined. However, a study9 published in Nature in 2011 showed that in a mouse transplantation model, some iPS cells are indeed immunogenic, raising concerns about their therapeutic use. This study examined the immunogenicity of mouse iPS cells, using a teratoma-formation assay. They injected iPS cells into mice that were either immune-compromised or genetically matched with the donor cells. This normally results in the formation of benign tumours called teratomas, which consist of many types of differentiated cells. The approach was validated using a line of genetically matched (autologous) ES cells which gave rise to teratomas, whereas a line of unmatched ES cells was rejected before teratomas were produced. The transplantation of autologous iPS cells derived from fetal fibroblasts into matched mice resulted in the rejection of teratomas, irrespective of the approach used to generate the IPS cells, indicating that, in this assay, matched iPS cells are more immunogenic than matched ES cells.

The study also identified the antigens that may have caused immune rejection of the iPS cells, discovering a group of nine genes that were expressed at abnormally high levels. Inducing the expression of three of these genes (Hormadl, Zgl6 and Cyp3a11) in the non-immunogenic ES cells significantly impaired the cells’ ability to form teratomas on transplantation into genetically matched mice. This study provides more questions than answers with many limitations in relation in clinical studies; however, it highlights that a great deal needs to be understood about the mechanisms underlying cellular reprogramming and the inherent similarities and differences between ES cells and iPS cells.

One of the most exciting developments in regenerative medicine over the past 2 years has been the identification of ‘bona fide source of myocardial progenitors’ (epicardial derived cells)10 which can be induced by thymosin β4 to differentiate into cardiomyocytes. This landmark study by Smart et al11 provides a major step forward in identifying a viable source of stem/progenitor cells that could contribute to new muscle after ischaemic heart disease and acute myocardial infarction (AMI). They demonstrated that in a mouse model the adult heart contains a resident progenitor cell population, which has the potential to become terminally differentiated cardiomyocytes after MI. Progenitor cells were primed with a peptide called thymosin β4 which induced embryonic reprogramming resulting in the mobilisation of this population and subsequent differentiation to give rise to de novo cardiomyocytes. Following experimentally induced MI, these cells were shown to migrate to the site of injury and then differentiate without any evidence of cellular fusion into structurally and functionally active cardiomyocytes. These cardiomyocytes showed evidence of gap junction formation with adjacent cells, synchronous calcium transients and the formation of operational contractile apparatus. Despite a low overall fraction of these cells being present at the site of injury and a relatively poor overall efficiency of differentiation, serial MRI scans revealed significant improvements in ejection fraction, cardiac volumes and scar size in comparison with sham treated animals. The pretreatment with thymosin β4 was crucial to these effects and may suggest a new strategy for promoting myocardial repair in humans.

MicroRNAs (small non-coding RNAs) play a critical role in differentiation and self-renewal of pluripotent stem cells, as well as in the differentiation of cardiovascular lineage cells. As a result, microRNAs have emerged as potential modulators of stem cell differentiation; specifically, miR-1 has been reported to play an integral role in the regulation of cardiac muscle progenitor cell differentiation. A study published in 201112 looked to take this one step further and assessed whether the overexpression of miR-1 in ES cells (miR-1-ES cells) enhances cardiac myocyte differentiation following transplantation into the infarcted myocardium. In this study, mice models of MI had miR-1-ES cells, ES cells or culture medium (control) transplanted into the border zone of the infarcted heart. Overexpression of miR-1 in transplanted ES cells protected host myocardium from MI-induced apoptosis through activation of p-AKT and inhibition of caspase-3, phosphatase and tensin homologue, and superoxide production. A significant reduction in interstitial and vascular fibrosis was quantified in miR-1-ES cells compared with control MI. Finally, mice receiving miR-1-ES cells had significantly improved heart function compared with respective controls. This would suggest that miR-1 drives cardiac myocyte differentiation from transplanted ES cells and inhibits apoptosis post-MI; however, importantly with respect to fibrosis no statistical significance between miR-1-ES cell and ES cell groups was observed suggesting further study in this area is needed. A review13 of the current evidence for the role of microRNAs in stem/progenitor cells and cardiovascular repair has recently been published.