Abstract 1385: Abcg2 Expressing SP Stem Cells Resist Oxidative Stress and Acquire a Cardiac Fate
Stem cell populations occupy a specialized niche and are exposed to hypoxic and oxidative stresses. We have previously established that the multidrug resistance protein, Abcg2, is the molecular determinant of the SP stem cell population. Using Hoechst33342 staining and dual wavelength FACS analysis, we observed that SP cells increase more than eight-fold within three days of injury and co-express cardiac restricted proteins. Transcriptome analysis of SP cells isolated from the injured adult murine heart (0, 3 and 7 days post injury) reveal increased expression of cell cycle regulatory transcripts, cytoprotective transcripts and members of the Notch signaling pathway. These transcriptome results supported the notion that that Abcg2 functions as a cytoprotective factor in stem cell populations and is regulated by HIF-2α, a master regulator of oxidative stress. We identified an evolutionary conserved HIF-2α binding site (HRE) in the 3 kb upstream fragment of the Abcg2 promoter. Using EMSA, mutagenesis and supershift assays we demonstrated that HIF-2α could bind to the HRE in the Abcg2 promoter. We performed transcriptional assays following the cotransfection of the 3kb Abcg2 promoter-luc and HIF-2α plasmids in C2C12 myoblasts and we observed a dose dependent activation of Abcg2 expression by HIF-2α. Furthermore, overexpression of HIF-2α resulted in a 3-fold increase of endogenous Abcg2. These results support the hypothesis that Abcg2 is a direct downstream target of HIF-2α which functions with other factors to initiate a cytoprotective program for the stem cell population. In addition, preclinical studies have been undertaken where Abcg2 expressing human SP cells (labeled with DAPI) were delivered into the border region of the injured heart of the athymic nude rat and DAPI labeled cells isolated 1–3 wks following injury using FACS analysis. These human SP cells expressed cardiac factors using immunohistochemical and RT-PCR analyses. Collectively, these studies support the hypothesis that molecular programs promote cytoprotection in the Abcg2 expressing SP stem cell populations and these cells are capable of acquiring a cardiac fate.