Adult cardiac stem cells are multipotent and robustly myogenic: c-kit expression is necessary but not sufficient for their identification

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-kit(pos)) cells. The adult heart indeed contains a heterogeneous mixture of c-kit(pos) cells, mainly composed of mast and endothelial/progenitor cells. This heterogeneity of cardiac c-kit(pos) 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-kit(pos) cardiac cells were separated through CD45-positive or -negative sorting followed by c-kit(pos) sorting. The blood/endothelial lineage-committed (Lineage(pos)) CD45(pos)c-kit(pos) cardiac cells were compared to CD45(neg)(Lineage(neg)/Lin(neg)) c-kit(pos) cardiac cells for stemness and myogenic properties in vitro and in vivo. The majority (~90%) of the resident c-kit(pos) cardiac cells are blood/endothelial lineage-committed CD45(pos)CD31(pos)c-kit(pos) cells. In contrast, the Lin(neg)CD45(neg)c-kit(pos) cardiac cell cohort, which represents ⩽10% of the total c-kit(pos) cells, contain all the cardiac cells with the properties of adult multipotent CSCs. These characteristics are absent from the c-kit(neg) and the blood/endothelial lineage-committed c-kit(pos) cardiac cells. Single Lin(neg)c-kit(pos) cell-derived clones, which represent only 1–2% of total c-kit(pos) 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 Lin(neg)c-kit(pos) 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-kit(pos) cardiac cells were injected. Thus, among the cardiac c-kit(pos) cell cohort only a very small fraction has the phenotype and the differentiation/regenerative potential characteristics of true multipotent CSCs.