Abstract P1038: Exosomal RNAs And Proteins From Non-invasively Derived Human Induced Mesenchymal Stem Cells Protect Cardiomyocytes From Death

Abstract

Background: Current stem cells therapy for ischemic cardiomyopathy is effective only about 2-4% in improving cardiac function. In search of novel therapy, we have developed a safe mRNA-based reprogramming of non-invasively derived human urinary epithelial cells into induced pluripotent stem cells (iPSC), and subsequently differentiated them into mesenchymal stem cells (iMSC). We examined therapeutic potential of iMSC in comparison with adult umbilical cord mesenchymal stem cells (MSC). Hypothesis: Autologous iMSC and their exosomes possess an enhanced cardioprotective characteristics compared to MSC. Methods and Results: The generated iMSC and their exosomes (ExoQuick reagent) offered an enhanced protection of human iPSC-derived cardiomyocytes (iCMC) from injuries of (i) angiotensin-II (Ang, 10 μM for 24 h) and (ii) 6 hrs of 1% hypoxia and 24 hrs of reoxygenation, in comparison with their MSC controls. The cardiomyocyte protection was studied through measurement of mitochondrial membrane potential (JC1 dye), intracellular reactive oxygen species (CM-H2DCFDA), in situ cell death by apoptosis, and qRT-PCR expression of survival and proinflammatory genes (BCL2, BAD, TNFA and IL6). Treatment with iMSC-derived exosomes alone enhanced the expression of NRF2, a major regulator of cytoprotective genes in protecting Ang-challenged iCMC. The specific role of iMSC exosomal RNAs and proteins in mediating cardioprotection against Ang-induced injury was identified through in situ cell death assay using the exosomes treated with either RNase A (0.5 μg/μl for 20 min at 37 0 C) or proteinase K (0.05 μg/μl for 10 min at 37 0 C) (Fig.1) . Exosomal noncoding RNA analysis by qRT-PCR revealed the presence of inflammation-associated small nucleolar RNAs such as SNORD32A, SNORD33, SNORD34 and SNORD35A. The transfer of exosomal RNAs into the host cardiac cells was augmented in presence of Ang, identified using 0.5 mM ethynyl uridine-labelled exosomes and detected using Click-iT RNA imaging reagents. All experiments were carried out in triplicates. In vivo studies are in progress. Conclusion: In comparison with adult MSC, non-invasively derived autologous iMSC and their exosomes elicit enhanced cardioprotection and may provide an alternative source of cells or cell-free therapy for treating ischemic cardiomyopathy.