Genetic fate mapping and single-cell transcriptomics reveal sca1+ and ckit+ cells as multipotent cardiac progenitors in the adult heart

Introduction Evidence that the mammalian heart contains a pool of cardiac stem cells (CSCs) has been followed by questions about its nature and physiological significance. Previous studies using Cre-lox and other site-specific recombinase cell-fate mapping systems failed to demonstrate a significant contribution of CSCs to cardiomyocyte (CM) formation in adulthood or after injury. A key limitation of these approaches is that they did not efficiently label CSCs nor accurately trace their progeny, leaving their cardiomyogenic potential in vivo unresolved. Methods We generated new Cre-lox mouse systems for constitutive and inducible labeling of c-kit- or sca-1 expressing CSCs to assess their fate from embryonic life to adulthood and after injury. Mice were crossbred to R26mT/mG Cre-reporter mice to label all the c-kit- or Sca-1-expressing cells and map their fate in vivo. Results At birth, hearts of double-mutant c-kit Cre::R26mT/mG (constitutive) show GFP labeling of the vast majority of all myocardial cells, including CMs proving that c-kit labels embryonic cardiac progenitors. On the other hand, at birth the CM population of TgSca-1Cre:R26mT/mG (constitutive) hearts were entirely negative for GFP, showing that the heart and its CM population develop from Sca-1 negative embryonic progenitors. Upon Tamoxifen treatment in vivo, more than half the myocyte-depleted CD45-/CD31-/c-kit+/Sca-1+ cardiac cell fraction from adult double-mutant TgSca-1CreER and c-kit CreER::R26mT/mG inducible mice were GFP+. These labeled cells proved to be multipotent in vitro and single-cell RNA-Seq shows that both the c-kit and Sca-1 Cre/Lox labeling strategies label the same cardiac progenitor pool in vivo. 28 days after permanent LAD coronary ligation, EdU+ newly formed CMs were detected within the infarct remote zone of TAM-treated TgSca-1CreER and c-kit CreER::R26mT/mG mice and all these EdU labeled CMs were GFP positive. Finally, the analysis of heart sections from TgSca-1Cre:R26mT/mG mice from 2 months to 2 years shows that the heart during adult and physiologic aging is progressively replenished of new CMs derived from CSCs whereby GFP+ CMs constitute -60% of all CMs by 2 years of age. Conclusion Our data reinforce the conclusion that CSCs are necessary and sufficient for robust cardiomyogenesis and to support myocardial regeneration/repair replenishing CMs lost by wear and tear and after injury in the adult heart.