CARDIOMYOCYTES DERIVED FROM INDUCED PLURIPOTENT STEM CELL PROLIFERATE AFTER CRYOPRESERVATION AND REPLATING.

NATALIA LUCIA SANTIN VELAZQUE; MARÍA AGUSTINA SCARAFIA; GABRIEL NEIMAN; XIMENA GARATE; LUCÍA NATALIA MORO; ARIEL WAISMAN; ALEJANDRO DAMIÉN LA GRECA; GUSTAVO SEVLEVER; CARLOS DANIEL LUZZANI; SANTIAGO MIRIUKA

Congreso; LXII Reunión de la Sociedad Argentina de Investigación Clínica; 2017

In the human heart, shortly after birth, cardiomyocytes (CM) undergo one last round of division followed by escape from the cell cycle. From then on, the heart develops in absence of CM cytokinesis and generates multinucleated cells. This lack of proliferation is characteristic of CM in the adult mammalian heart accounting for its markedly limited regenerative potential. Production of induced pluripotent stem cell-derived CM (iPSC-CM) is a potentially promising strategy for regenerative therapies, biological studies, disease modeling and drug screening. However, difficulties in generating sufficient quantities of highly purified cardiomyocytes have been a barrier to fully explore their potential.Therefore, it is necessary to develop a cardiac differentiation protocol that allows obtaining large CMs quantities. We then analyzed the ability of the iPSC-CM to re-enter cell cycle after exposure to stress stimuli, such as cryopreservation and replating (shift to new appropriately coated plates). iPSC-CM were obtained by the implementation of a previously reported protocol (Lian et al. 2012). Beating cells were cryopreserved in liquid nitrogen with 20% serum and 10% DMSO at day 20 of the differentiation protocol. Proliferative capacity was assessed at two times after thawing, 24 hours and 7 days, and after replating, by incubation with EdU overnight and subsequent analysis by immunofluorescence and flow cytometry.Our results show that only a small proportion of unstressed CMs were cycling (less than 3%) but this percentage increased when exposed to stressors. In replated CMs we observed that up to 30% of them had re-entered S phase, while cryopreserved CMs showed a 20% of proliferative cells. In all cases, replication activity decreases over time.These findings suggest that iPSC-CMs are able to re-enter cell cycle under appropriate conditions for a limited period of time. Currently our goal is to develop a protocol that allow us to enhance this capacity to generate large numbers of purified CMs.