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) originally were identified by the expression of c-kit, the stem cell factor receptor. However, in the adult myocardium c-kit alone distinguish CSCs from other c-kit-expressing cardiac cells because the adult heart contains a heterogeneous mixture of c-kitpos cells, mainly composed of mast and endothelial/progenitor cells. This heterogeneity of cardiac c-kitpos cells has not been considered in recent c-kit-expressing cell fate mapping publications, which have equated the contribution of the whole heterogeneous c-kitpos population to cardiomyocyte generation in adulthood, which is minimal, to that of the CSCs, a result at odds with previous publications. To shed light on this issue, we have assessed the identity, abundancy and myogenic potential of true multipotent CSCs within the total c-kitpos cardiac cell cohort. Blood lineage-committed c-kitpos cells were removed by CD45 negative sorting to obtain a CD45negc-kitpos cell population (<10% of the total c-kitpos cells), which is enriched for cells that express c-kit at low levels and possess all properties of multipotent stem/progenitor cells in vitro. These characteristics are absent from the c-kitneg and the lineage-committed c-kitpos cardiac cells. Single Linnegc-kitpos cell-derived CSC clones, representing 1-2% of total c-kitpos cells, when instructed by TGF-b/Wnt molecules, acquire full transcriptome expression, sarcomere organization, spontaneous contraction and electrophysiological properties of differentiated cardiomyocytes. Significantly, clonogenic CSCs have a potent cardio-regenerative/repair capacity in vivo after acute myocardial infarction. CSC myogenic regenerative capacity is dependent on cardiomyocyte commitment through activation of the SMAD2 pathway. Such regeneration was not apparent when freshly-isolated total c-kitpos cardiac cells were administered. In conclusion, only a very small fraction of cardiac c-kitpos cells (~1-2%) have the characteristics of multipotent CSCs but these exhibit robust myogenic properties. ? Overall design: RNA-seq data from CSCs, iCMs, neonatal (neoCMs) and adult cardiac myocytes (aCMs) (triplicated) have been analysed and compared. Also, mRNA was collected from clonogenic undifferentiated rat CSCs, beating colonies of CSC-derived iCMs, freshly isolated cardiomyocytes obtained from the hearts of 1-2 post-natal day and from 12 week-old Wistar rats. ? Please note that the datasets of CSC1,2 and 3 are from a biological triplicate of the same cardiac stem cell clone. In particular, the dataset of the sample entitled CSC1 has also been used in a different experiment with the title of cCSC4. This dataset was compared with three datasets respectively from three different cardiac stem cell clones (cCSC1, 2, and 3) for clonal identity analysis.