These conclusions rest mainly on three experimental facts a

These conclusions rest mainly on three experimental facts: a model of myocardial injury with patent coronary circulation to test the spontaneous regenerative capacity of eCSCs; in situ labelling and genetic tracking the fate of c-kit positive cardiac stem mitoxantrone and the replacement of the eCSCs by transplantation of genetically tagged CSCs (Ellison et al., 2013). To that effect, we endeavoured to exclude most of the challenges posed by the potential pitfalls arising from the employed experimental tools. In particular, we demonstrated that newly formed BrdU positive myocytes originate from endogenous and also transplanted CSCs employing multiple and complementary approaches, like genetic tracing, FACS sorting, cell and tissue immunofluorescence, confocal microscopy analysis, and genome pattern of expression. The specificity of the c-kit/cre lentiviral construct to recombine only c-kit expressing cardiac cell lineages in R26/floxed-stop-YFP transgenic mice was demonstrated in vitro and in vivo by cyto/histo-chemistry, FACS and RT-PCR. The effect of 5-fluoro-uracil (5-FU) and Ganciclovir regime for CSC ablation on neighbouring cells and the essential role of resident CSCs in myocardial regeneration were assessed again by multiple assays. Briefly, the replacement of the endogenous CSCs with cloned genetically labelled CSCs, lead to full anatomical and functional recovery of the transplanted animals while the control cohort (untreated or treated with cardiac fibroblasts) developed lethal heart failure. When the transplanted cells were induced to suicide by Ganciclovir treatment the animals were set back in heart failure. The so-called “bystander effect” elicited by Ganciclovir and cell suicide was ruled out with the best obtainable evidence in vitro and in vivo. In addition, these transplanted cells were isolated from the transplanted hosts and proven to maintain their CSC properties in vitro and in a second round of transplantations. We documented that 5-FU administration killed most, if not all, the proliferating myocardial cells after ISO injury. It logically follows that the other cardiac progenitor cell populations were most probably also ablated by the ISO+ 5-FU regimen. However, the clone of a single c-kitpos CSC was able to rescue the failing heart phenotype. Myocardial tissue histology and function became dependent on the transplanted cells, which were selectively killed by Glanciclovir. The latter does not exclude a role and participation of the other described stem/progenitor cells in the recovery process. It remains to be proven whether any of the other stem/progenitor cells described so far are equivalent and indistinguishable from c-kitpos CSCs. We would not be surprised if actually the latter hypothesis turn to be at least partially correct as it would reinforce our view that the majority cardiac stem/progenitor cell are different phenotypes of a unique tissue-specific stem/cell population, at least based on the way they have been characterized so far. Concurrently, a very elegant and well-controlled study by Braun’s group, using a triple transgenic mouse approach, has recently lineage traced cardiomyocyte formation in the adult life to be, at least in part, the progeny of eCSCs, expressing Sca-1 (a fraction of which express, as above described, also c-kit) (Uchida et al., 2013). Thus, these data document that the adult heart has a robust autonomous regenerative capacity, which mainly resides in the eCSCs. This capacity, however, spontaneously is not sufficient to significantly repair segmental tissue loses such as in AMIs produced by occlusion of a main coronary artery. This regeneration deficit also affects the segmental loses of any other tissues, no matter the abundance and potency of their tissue stem cells. The challenge is to manipulate the eCSCs and improve their regenerative potential in order to produce an autologous replacement of the cells lost by the insult.