Background. Human induced pluripotent stem cells (iPSCs) are an attractive source of cardiomyocytes for cardiac repair and regeneration. In this study, we aim to determine whether acute electrical stimulation of human iPSCs can promote their differentiation to cardiomyocytes. Methods. Human iPSCs were differentiated to cardiac cells by forming embryoid bodies (EBs) for 5 days. EBs were then subjected to brief electrical stimulation and plated down for 14 days. Results. In iPS(Foreskin)-2 cell line, brief electrical stimulation at 65 mV/mm or 200 mV/mm for 5 min significantly increased the percentage of beating EBs present by day 14 after plating. Acute electrical stimulation also significantly increased the cardiac gene expression of ACTC1, TNNT2, MYH7, and MYL7. However, the cardiogenic effect of electrical stimulation was not reproducible in another iPS cell line, CERA007c6. Beating EBs from control and electrically stimulated groups expressed various cardiac-specific transcription factors and contractile muscle markers. Beating EBs were also shown to cycle calcium and were responsive to the chronotropic agents, isoproterenol and carbamylcholine, in a concentration-dependent manner. Conclusions. Our results demonstrate that brief electrical stimulation can promote cardiac differentiation of human iPS cells. The cardiogenic effect of brief electrical stimulation is dependent on the cell line used.

Human induced pluripotent stem cells (iPSCs) are a valuable tool for studying the cardiac developmental process in vitro, and cardiomyocytes derived from iPSCs are a putative cell source for personalized medicine. Changes in mitochondrial morphology have been shown to occur during cellular reprogramming and pluripotent stem cell differentiation. However, the relationships between mitochondrial dynamics and cardiac mesoderm commitment of iPSCs remain unclear. Here we demonstrate that changes in mitochondrial morphology from a small granular fragmented phenotype in pluripotent stem cells to a filamentous reticular elongated network in differentiated cardiomyocytes are required for cardiac mesodermal differentiation. Genetic and pharmacological inhibition of the mitochondrial fission protein, Drp1, by either small interfering RNA or Mdivi-1, respectively, increased cardiac mesoderm gene expression in iPSCs. Treatment of iPSCs with Mdivi-1 during embryoid body formation significantly increased the percentage of beating embryoid bodies and expression of cardiac-specific genes. Furthermore, Drp1 gene silencing was accompanied by increased mitochondrial respiration and decreased aerobic glycolysis. Our findings demonstrate that shifting the balance of mitochondrial morphology toward fusion by inhibition of Drp1 promoted cardiac differentiation of human iPSCs with a metabolic shift from glycolysis towards oxidative phosphorylation. These findings suggest that Drp1 may represent a new molecular target for future development of strategies to promote the differentiation of human iPSCs into cardiac lineages for patient-specific cardiac regenerative medicine.

Cardiac resident stem cells (CRSCs) hold much promise to treat heart disease but this remains a controversial field. Here, we describe a novel population of CRSCs, which are positive for W8B2 antigen and were obtained from adult human atrial appendages. W8B2 1 CRSCs exhibit a spindle-shaped morphology, are clonogenic and capable of self-renewal. W8B2 1 CRSCs show high expression of mesenchymal but not hematopoietic nor endothelial markers. W8B2 1 CRSCs expressed GATA4, HAND2, and TBX5, but not C-KIT, SCA-1, NKX2.5, PDGFRa, ISL1, or WT1. W8B2 1 CRSCs can differentiate into cardiovascular lineages and secrete a range of cytokines implicated in angiogenesis, chemotaxis, inflammation, extracellular matrix remodeling, cell growth, and survival. In vitro, conditioned medium collected from W8B2 1 CRSCs displayed prosurvival, proangiogenic, and promigratory effects on endothelial cells, superior to that of other adult stem cells tested, and additionally promoted survival and proliferation of neonatal rat cardiomyocytes. Intramyocardial transplantation of human W8B2 1 CRSCs into immunocompromised rats 1 week after myocardial infarction markedly improved cardiac function (~40% improvement in ejection fraction) and reduced fibrotic scar tissue 4 weeks after infarction. Hearts treated with W8B2 1 CRSCs showed less adverse remodeling of the left ventricle, a greater number of proliferating cardiomyocytes (Ki67 1 cTnT 1 cells) in the remote region, higher myocardial vascular density, and greater infiltration of CD163 1 cells (a marker for M2 macrophages) into the border zone and scar regions. In summary, W8B2 1 CRSCs are distinct from currently known CRSCs found in human hearts, and as such may be an ideal cell source to repair myocardial damage after infarction. STEM CELLS 2015;33:3100-3113 SIGNIFICANCE STATEMENTWe have isolated a new type of adult stem cell from human heart tissue, which carries markers different from known cardiac resident stem cells. These cells can be rapidly expanded and have powerful regenerative effects when injected into the hearts of rats after heart attack, such that they greatly improve cardiac performance compared with controls that have also suffered heart attack. We showed these cells protect other cells from dying and promote growth of blood vessels, actions which assist cardiac regeneration. Importantly, their protective actions in this regard are superior to those of other adult stem cells that have shown some benefit in clinical trials after heart attack. These new stem cells have great potential for repairing the heart, and could be derived from the individual patients in need, avoiding any problem of immunorejection.