This editorial refers to ‘Pharmacological response of human cardiomyocytes derived from virus-free induced pluripotent stem cells’ by A. Mehta et al., pp. 577–586, this issue.
The potential of human embryonic stem (hES) cells for therapeutic purposes became immediately clear after their derivation by Thomson's group in 1998.1 It was also clear, though, that their origin represented their main limitation. The ethical and legislative issues challenging hES cell-based research have produced a ‘selective pressure’ in response to which a new type of stem cells has stemmed: human induced pluripotent stem (hiPS) cells.2 The generation of hiPS cells from somatic cells, by virus-mediated ectopic expression of a very limited number of pluripotency genes (Oct4, Nanog, Sox2, Kfl4, and c-myc),3 is undoubtedly one of the most important research advancements of the last decade. hiPS cells have indeed overcome two of the most important drawbacks of hES cells: their ethically questionable origin and their intrinsic immunogenic potential.
The first generations of iPS cells presented nonetheless some important limitations, deriving from the random insertion of the reprogramming genes and of the virus in the genome, a manoeuvre that can repress genes influencing the differentiation potential or activate oncogenes. Recently, virus- and vector-free hiPS cells have been generated using episomal plasmids, small molecules, and microRNA (reviewed in Yoshida and Yamanaka4), which guarantee the integrity of the genome.
hiPS cells hold great promise for cell-based regenerative interventions aimed at repairing the heart, but their use in cell therapy is still far from feasible. At the moment, an attractive and more practical application of these cells in the cardiac field could include the molecular and cellular characterization of human cardiac diseases and the screening for drugs able to correct, in a patient-specific way, the altered phenotype.
In view of the above-mentioned applications, however, it is necessary to verify whether or not hiPS-derived cardiomyocytes represent a suitable cellular substrate. In this regard, Mehta et al. have generated virus-free hiPS cells by employing oriP/EBNA1-based episomal plasmids, developed by Yu et al.5 and containing various pluripotency genes (Oct4, sox2, Nanog, Lin28, klf4, and c-Myc), which, once taken up by the cell, provide the stimuli for reprogramming but do not integrate within the genomic DNA.6 These plasmids are then lost in a few cell cycles due to defects in their synthesis. As expected, no genomic alterations were introduced in these hiPS cells by the reprogramming procedure and, when differentiated, they formed embryoid bodies displaying spontaneously contracting areas. A detailed molecular analysis of several cardiac genes and proteins, including transcription factors, sarcomeric proteins, and ion channels, revealed a cardiogenic pathway compatible with human heart development. Of particular relevance are the functional data which show that field potential recordings (the in vitro equivalent of ECG) displayed predictable alterations when cells were challenged with autonomic agonists and other ion channel modulators. These data demonstrate that this human cellular model presents important signalling pathways that normally regulate cardiomyocyte excitability.6
Thus far, because of the paucity of accessible diseased cardiac tissue and the lack of control (non-diseased) cardiac samples of human origin, most of the data on the molecular, cellular, and functional mechanisms of both inherited and acquired cardiac diseases have been collected from animal models that, however, do not always fully recapitulate human pathophysiology.7 The possibility to generate diseased cardiomyocytes from hiPS cells has made a human cellular model available for in vitro characterization of several cardiac pathologies. Let us consider for example the long QT syndrome: although the mutated proteins, mostly ion channels, causing QT prolongation of the ECG have been extensively characterized in heterologous expression systems, the lack of the cellular milieu specific to cardiomyocytes make the prediction of functional modification difficult.8 The possibility to study the altered proteins directly in a cardiac cell may provide additional important information concerning the effects of the mutation on cell excitability and on the possible pharmacological treatment. Interestingly, hiPS cells have been recently generated from patients with genetic mutations responsible for long-QT (LQT-1 and -2) and for Timothy syndromes.9–11 These studies have demonstrated that cardiomyocytes differentiated from patient-specific, retrovirus-reprogrammed iPS cells show a prolonged repolarization phase, typical of the pathology, when compared with cardiomyocytes derived from healthy controls.
Since it is becoming progressively clearer that the presence of a particular polymorphism may predispose patients carrying a specific mutation to arrhythmias,12 and that an analysis in a heterologous expression system would fail to reveal this aspect, another clear advantage of virus-free human iPS-derived cardiomyocytes is the possibility to evaluate the influence/effect of the patient's genetic background. The proper cellular and genetic context may also be useful to analyse cell responsiveness to a specific pharmacological treatment as indicated by a study by Matsa et al. who reported that cardiomyocytes differentiated from hiPS cells derived from a LQT2 patient respond appropriately to clinically relevant pharmacological agents.13
Although some of the drawbacks of hiPS have already been solved, a few questions remain to be answered before hiPS-derived cardiomyocytes can be definitely used as a model to study human cardiac (patho)physiology: are these cells mature enough to represent fully differentiated cardiomyocytes? And, is it possible to isolate and analyse a specific subtype of cardiomyocyte? Efforts aimed at improving the yield of cardiomyocytes from iPS have been recently made both by strictly controlling and standardizing the differentiation protocol14 and by forcing the differentiation towards specific cardiac subtypes.15
In conclusion, generation of virus-free hiPS cells together with proper differentiation methods will likely allow the de novo generation of mature cardiomyocytes identical to those of a patient and will help in clarifying the molecular and cellular mechanisms of acquired and genetic diseases; it will also permit safe testing of new cardioactive drugs and therapeutic approaches. Furthermore, hiPS cells may also allow the regeneration of the damaged myocardium using a homologous source of cells. All of these future developments, however, will require a strict and detailed characterization of the cell substrate.
Conflict of interest: none declared.