Multipotent adult resident cardiac stem cells (CSCs) were first identified by the expression of c-kit, the stem cell factor receptor. However, in the adult myocardium c-kit alone cannot distinguish CSCs from other c-kit-expressing (c-kitpos) cells. The adult heart indeed contains a heterogeneous mixture of c-kitpos cells, mainly composed of mast and endothelial/progenitor cells. This heterogeneity of cardiac c-kitpos cells has generated confusion and controversy about the existence and role of CSCs in the adult heart. Here, to unravel CSC identity within the heterogeneous c-kit-expressing cardiac cell population, c-kitpos cardiac cells were separated through CD45-positive or -negative sorting followed by c-kitpos sorting. The blood/endothelial lineage-committed (Lineagepos) CD45posc-kitpos cardiac cells were compared to CD45neg(Lineageneg/Linneg) c-kitpos cardiac cells for stemness and myogenic properties in vitro and in vivo. The majority (~90%) of the resident c-kitpos cardiac cells are blood/endothelial lineage-committed CD45posCD31posc-kitpos cells. In contrast, the LinnegCD45negc-kitpos cardiac cell cohort, which represents ⩽10% of the total c-kitpos cells, contain all the cardiac cells with the properties of adult multipotent CSCs. These characteristics are absent from the c-kitneg and the blood/endothelial lineage-committed c-kitpos cardiac cells. Single Linnegc-kitpos cell-derived clones, which represent only 1–2% of total c-kitpos myocardial cells, when stimulated with TGF-β/Wnt molecules, acquire full transcriptome and protein expression, sarcomere organisation, spontaneous contraction and electrophysiological properties of differentiated cardiomyocytes (CMs). Genetically tagged cloned progeny of one Linnegc-kitpos cell when injected into the infarcted myocardium, results in significant regeneration of new CMs, arterioles and capillaries, derived from the injected cells. The CSC’s myogenic regenerative capacity is dependent on commitment to the CM lineage through activation of the SMAD2 pathway. Such regeneration was not apparent when blood/endothelial lineage-committed c-kitpos cardiac cells were injected. Thus, among the cardiac c-kitpos cell cohort only a very small fraction has the phenotype and the differentiation/regenerative potential characteristics of true multipotent CSCs.

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Although reactive oxygen species have been proposed to play a major role in the aging process, the exact molecular mechanisms remain elusive. In this study we investigate the effects of a perturbation in the ratio of Cu/Zn-superoxide dismutase activity (Sod1 dismutases .O2-to H2O2) to glutathione peroxidase activity (Gpx1 catalyses H2O2 conversion to H2O) on cell growth and development. Our data demonstrate that Sod1 transfected cell lines that have an elevation in the ratio of Sod1 activity to Gpx1 activity produce higher levels of H2O2 and exhibit well characterised markers of cellular senescence viz. slower proliferation and altered morphology. On the contrary, Sod1 transfected cell lines that have an unaltered ratio in the activity of these two enzymes, have unaltered levels of H2O2 and fail to show characteristics of senescence. Furthermore, fibroblasts established from individuals with Down syndrome have an increase in the ratio of Sod1 to Gpx1 activity compared with corresponding controls and senesce earlier. Interestingly, cells treated with H2O2 also show features of senescence and/or senesce earlier. We also show that Cip1 mRNA levels are elevated in Down syndrome cells, Sod1 transfectants with an altered Sod1 to Gpx1 activity ratio and those treated with H2O2, thus suggesting that the slow proliferation may be mediated by Cip1. Furthermore, our data demonstrate that Cip1 mRNA levels are induced by exposure of cells to H2O2. These data give valuable insight into possible molecular mechanisms that contribute tribute to cellular senescence and may be useful in the evolution of therapeutic strategies for aging.

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