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. ¿多能成体驻留心脏干细胞（cardiac stem cells, CSCs）最初通过干细胞因子受体c-kit的表达被鉴定。然而，在成体心肌中，仅靠c-kit无法将CSCs与其他表达c-kit的心脏细胞区分开，因为成体心脏内存在异质性的c-kit阳性（c-kitpos）细胞混合群体，其主要组分是肥大细胞与内皮/祖细胞。近期发表的c-kit表达细胞命运图谱研究未考虑心脏c-kit阳性细胞的这种异质性，这些研究将整个异质性c-kit阳性群体在成体心肌细胞生成中的微小贡献，等同于CSCs的贡献，这一结论与既往研究结果相悖。为阐明这一问题，本研究对总c-kit阳性心脏细胞群中真正的多能CSCs的身份、丰度及肌源性潜能进行了评估。通过CD45阴性分选去除血液谱系定向的c-kit阳性细胞，可获得CD45阴性c-kit阳性细胞群（占总c-kit阳性细胞的<10%），该细胞群富集了低水平表达c-kit且在体外具备多能干细胞/祖细胞全部特性的细胞。c-kit阴性（c-kitneg）细胞及谱系定向的c-kit阳性心脏细胞并不具备这些特性。单个谱系阴性（Linneg）c-kit阳性细胞衍生的CSC克隆（占总c-kit阳性细胞的1%~2%），在转化生长因子-β（TGF-β）/Wnt分子的诱导下，可获得分化成熟心肌细胞的完整转录组表达谱、肌节结构、自发性收缩及电生理特性。值得注意的是，克隆形成性CSCs在急性心肌梗死模型体内具有强大的心脏修复/再生能力。CSCs的肌源性再生能力依赖于通过激活SMAD2通路实现的心肌细胞定向分化。当直接输注新鲜分离的总c-kit阳性心脏细胞时，并未观察到此类再生效应。综上，仅极少数心脏c-kit阳性细胞（约1%~2%）具备多能CSCs的特性，且这些细胞展现出强劲的肌源性潜能。