131 Stem cells in the embryonic heart Joo Ferreira-Martins, Barbara Ogrek, Sergio Signore, Francesco Cappetta, Elisabeth

Steadman, Justin Korn, Alex Matsuda, Maria Virginia Caballero, Polina Goichberg, Christian Arranto, Fumihiro Sanada, Hanqiao Zheng, Noriko Ide-Iwata, Toru Hosoda, Annarosa Leri, Piero Anversa, Jan Kajstura, Marcello Rota Brigham and Women's Hospital-Harvard Medical School, Boston, MA, USA The identification of c-kit-positive cardiac stem cells (CSCs) in the adult heart raises the important issue of their origin. Thus, we tested the possibility that the pool of CSCs is established during embryonic development and is maintained throughout life. For this purpose, we utilized transgenic mice in which GFP expression was under the control of the ckit promoter. C-kit positive cells were identified in the cardiac crescent and at later stages of embryonic-fetal life. Time-lapse imaging of ex-vivo preparations by two-photon microscopy, showed that c-kit positive cardiac cells did not originate by translocation from extracardiac regions. Additionally, c-kit positive cells were negative for mesenchymal or hematopoietic lineage markers, excluding their origin from primitive sites of hematopoiesis. In vitro, c-kit positive cells obtained from E12-E19 hearts gave rise to multicellular clones composed of cells expressing the stem cell antigen. When exposed to differentiating medium, c-kit positive cells formed myocytes, smooth muscle cells and endothelial cells. The ability to acquire the myocyte phenotype was also documented in vivo utilizing a fate mapping approach. C-kit positive cells were obtained from embryos of mice in which GFP expression was under the control of the -MHC promoter. Transplantation of c-kit-positive cells in the infarcted LV of wild-type mice resulted in the formation of GFP-positive myocytes. Collectively, these findings indicate that c-kit-positive cells in the embryonic-fetal heart correspond to bona fide CSCs. Fetal CSCs displayed cytosolic Ca2+ elevations originated from Ca2+ mobilization via IP3 receptors. Oscillatory events were maintained in Ca2+ free medium and were not affected by modulation of ryanodine receptors. Since Ca2+ cycling regulates the growth of postnatal CSCs, the impact of Ca2+ oscillations on the proliferative capacity of fetal CSCs was established. Cultured CSCs were exposed to agonist (ATP) or inhibitor (xestospongin-C, U73122) of Ca2+ oscillations and cell proliferation was evaluated. Induction of Ca2+ oscillations with ATP resulted in a 1.9-fold increase in BrdU incorporation in CSCs. Opposite effects were seen with inhibition of IP3R function. In conclusion, c-kit-positive CSCs with properties similar to those identified in the adult heart are present in the embryonic heart. Importantly, the proliferative potential of these cells is modulated by Ca2+ oscillations.