Abstract 12218: In Vitro Production of Niche-like Precursors by Epigenetic Reprogramming of Stroma-Derived Cardiac Cells
INTRODUCTION: We recently isolated a cardiac population of stromal cells (CMSC) that exhibited tissue-specific properties, revealing higher competence for differentiation toward myocardial and vascular lineages than their syngeneic bone marrow counterpart. Despite their plasticity, CMSC did not spontaneously exhibit cardiac stem cell markers, including c-Kit and MDR-1. Since serum and epigenetic drugs, e.g. nitric oxide (NO), retinoic acid (RA), and phenyl butyrate (PB), can modify cell fate and induce functional reprogramming, the aim of the present work was to design an epigenetically-based in vitro strategy to enrich CMSC in a population of putative cardiovascular stem cells.
METHODS AND RESULTS: After a round of expansion in DMEM and 20% fetal bovine serum (FBS), CMSC were exposed, from 3 to 7 days, to low serum (5% FBS) either in the presence or in the absence of a defined “epigenetic cocktail” (EpiC) made of 5 uM ATRA, 5 uM PB and 200 uM DETA/NO. Different parameters were evaluated to assess the biological effects of this treatment. While EpiC significantly inhibited cell proliferation, without increasing apoptosis and senescence, it induced the expression of genes associated to resident cardiac stem cells. Specifically, c-Kit, KDR and MDR-1 proteins were up-regulated, while Nucleostemin (NS, a nucleolar protein associated to proliferating cardiac precursors) was down-regulated compared to cells cultured in complete medium or in low serum alone. In this condition the MDR-1 transporter was functionally active as indicated by the rhodamine extrusion assay. Importantly, the reprogramming was completely reversible as cells transferred to regular medium restarted proliferation and re-expressed NS while silencing c-Kit, KDR and MDR-1.
CONCLUSIONS: Altogether these data suggest that CMSC may be pharmacologically and reversibly reprogrammed in vitro to acquire a cardiovascular quiescent niche-like phenotype (see figure).
- © 2010 by American Heart Association, Inc.