Abstract

Introduction: Paracrine mechanism of stem cell-based therapy holds importance. However, genes regulating the paracrine factors have not been elucidated in detail. Here in this study, we evaluated the role of HSF1 in exosome biogenesis and their composition in neonatal cardiac mesenchymal stem cells (nMSCs) in concert with ischemic heart generation. Hypothesis: HSF1 in nMSCs promotes production of exosomes, functional exosomal miRs cargo, and exosome acquisition by recipient cells. Methodology: nMSCs and adult MSCs (aMSCs) were respectively isolated from right atrial appendage of neonate (<1month) and adult ( > 40 years) patients. nMSCs were knocked down (KD) for HSF1 gene using CRISPR-CAS9 technique while HSF1 gene was overexpressed (OE) in aMSCs using HSF1 overexpressing lentiviral constructs. Exosome biogenesis was evaluated using nano site technique. We next evaluated effect of HSF1 KD and OE on miRs cargo in nMSCs and aMSCs respectively. Exosome acquisition by target cells was studied by mCherry labeling technique. The potential of nMSCs and aMSCs and their corresponding exosomes to repair rat ischemic heart was evaluated under the effect of HSF1 KD and OE. Results: HSF1-KD in nMSCs resulted in significant reduction in production of exosomes, downregulation of ESCRT complex genes and therapeutic miRs viz, miR-199a, mi590-3P and miR-146. In contrast, HSF-1 over expression in aMSCs statistically increased production of exosomes, ESCRT complex genes and of therapeutic miRs. Moreover, HSF-1 OE in aMSCs resulted in statistical downregulation of miR-122, known to induce apoptosis, and downregulation of p27, a known target of miR-590. We identified that potential of nMSCs to repair ischemic heart decreased statistically after HSF1-KD while that of aMSCs increased significantly after HSF1-OE. Finally, we identified exosomes from these stem cells were preferentially taken up by cells in injured myocardium and not by cells in non-injured one and that HSF1-OE increased the uptake by recipient cells while HSF1-KD had opposite effect. Conclusion: Together, our results demonstrate that HSF-1 plays an important role in exosome biogenesis, miRs cargo enrichment and uptake of exosomes by recipient cells.

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