Functional and molecular analysis of cardiomyocytes derived from reprogrammed pluripotent cells and embryonic stem cells

Pluripotent stem cells can be obtained from embryonic stage or generated by in vitro reprogramming of terminally differentiated somatic cells. Such reprogrammed cells possess similar developmental potential as embryonic stem (ES) cells and therefore are an indispensible source of diverse cell types, including cardiomyocytes (CMs), for basic research. CMs derived from ES cells and reprogrammed cells can provide possibilities for study of development, differentiation processes, stem cell malignancy and genetic diseases in vitro. Such studies can allow for development of novel compounds for drug discovery and toxicity testing which might help to develop more efficient and safer drugs The present thesis is based on analyzing the impact of switching the cell fate on the differentiated state of reprogrammed cells using cardiomyocytes as a model. Detailed structural molecular and functional characterization of CMs derived from ES cells and reprogrammed cells like fusion hybrid (FH) cells and induced pluripotent stem (iPS) cells was performed. Somatic cells (spleenocytes and bone marrow cells) were fused with ES cells to generate FH cells. The formed hybrid cells were morphologically similar to pluripotent ES cells and retained a tetraploid genome through many passages. Spontaneous differentiation led to formation of embryoid bodies (EBs) with appearance of beating areas representing differentiation to cardiac lineage. The EBs derived from FH cells also retained tetraploid genome and expressed major histocompatibility (MHC) class I molecules of both fusing partners. FH derived CM expressed typical cardiac structural proteins and intact β-adrenergic and muscarinic signaling receptors. Atrial, ventricular and pacemaker cardiac subtypes could be found in FH-CMs. Thus, CMs derived from tetraploid FH cells appear to be structurally and functionally intact. Ongoing research proved iPS cell technology to be more robust reprogramming strategy. In order to obtain homogenous population of CMs for further studies an iPS cell line TiB7.4 was genetically manipulated to allow for antibiotic-based purification of cardiomyocytes after spontaneous differentiation. We generated highly purified CMs from transgenic murine iPS and ES cell lines expressing puromycin N-acetyltransferase and EGFP under the control of α-myosin heavy chain promoter. iPS and ES cells differentiated into CMs at comparable efficiencies yielding highly purified CMs after drug selection. Purified iPS- and ES-CMs exhibited indistinguishable structural properties, similarly responded to pharmacological agents, expressed functional voltage-gated sodium, calcium and potassium channels and possessed comparable current densities. Global transcriptional profile and gene ontology signature of transgenic iPS-CMs were very similar to that of ES-CMs but clearly distinct from fibroblasts that were used to generate iPS cells and differentiated cells in iPS or ES cell-derived embryoid bodies. After puromycin selection, iPS-CMs did not contain any residual pluripotent cells nor formed teratoma in immunodeficient mice. Therefore, cell fate switching brought about by reprogramming does not affect the structural, molecular and functional characteristics of cardiac derivatives of the reprogrammed cells.