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

Abstract

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.

Identity Crisis for Regenerative Cardiac cKit + Cells

“…A new study from Vicinanza et al has reignited the debate over cardiomyogenesis from cKit + CSCs, as they now report that roughly 99% of primary cKit + cells isolated from the heart lack cardiomyogenic potential and have no rejuvenating benefit when injected into the injured hearts of mice or rats 16 . Instead, only a rare sub-fraction of cardiac cKit + cells defined by: 1) expression of the marker cKit, 2) absence of the hematopoietic marker CD45 and 3) having been clonally derived and expanded in vitro, are able to regenerate the heart with new cardiomyocytes.…”

“…Instead, only a rare sub-fraction of cardiac cKit + cells defined by: 1) expression of the marker cKit, 2) absence of the hematopoietic marker CD45 and 3) having been clonally derived and expanded in vitro, are able to regenerate the heart with new cardiomyocytes. It is this fraction, representing only 1% of total cardiac cKit + cells, that Vicinanza et al 16 re-define as the “true cKit + cardiac stem cell.” Such a model, if correct, warrants re-examination of an entire body of past literature, as it calls into question the identity and phenotype of isolated cKit + CSCs that have already been used in numerous animal studies and in ongoing human clinical trials 2, 14 .…”

“…Cardiac cKit + cells without additional isolation criteria were reported to be self-renewing, clonogenic, and multipotent, as well as widely expressing the cardiogenic transcription factor Nkx2.5 1 . Vicinanza et al now contend that CD45 negative selection is required to designate a cardiac cKit + cell as a CSC, and that among all CD45 − cKit + cells, only a small portion (10%) have the potential to generate clones in culture with cardiomyogenic potential 16 . However, the histologic endpoints for cardiac renewal and the physiologic endpoints for functional improvement in Vicinanza et al 16 versus Beltrami et al 1 appear virtually identical, despite the fact that these new data contend that the population used in the original Beltrami et al study was probably not cardiomyogenic.…”

“…Vicinanza et al now contend that CD45 negative selection is required to designate a cardiac cKit + cell as a CSC, and that among all CD45 − cKit + cells, only a small portion (10%) have the potential to generate clones in culture with cardiomyogenic potential 16 . However, the histologic endpoints for cardiac renewal and the physiologic endpoints for functional improvement in Vicinanza et al 16 versus Beltrami et al 1 appear virtually identical, despite the fact that these new data contend that the population used in the original Beltrami et al study was probably not cardiomyogenic. In addition, the new results by Vicinanza et al are also at odds with their Ellison et al study in which they stated that “many of the c-kit pos eCSCs expressed GATA4 and Nkx2.5, two early transcription factors of the cardiac lineage” 3 .…”

“…More perplexing, Vicinanza et al reported that injection of broadly isolated cKit + cells not negatively sorted for CD45 produced essentially no new cardiomyocytes (0.0001%) and no functional benefits in injured rodent hearts 16 . Again, this result stands in contrast to their original observations from 13 years ago 1 , and it calls into question the newly emerging paracrine hypothesis 14 and the potential validity of ongoing clinical trials that also use broadly isolated cardiac cKit + cells (NCT02501811).…”