Defining the Role of Vitronectin in Controlling Survival of Umbilical Cord-derived MSCs under Different Conditions of Physiological Stress

Goyal, Umesh (2022) Defining the Role of Vitronectin in Controlling Survival of Umbilical Cord-derived MSCs under Different Conditions of Physiological Stress. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Umesh Goyal (15RS068)) 15RS068_DBS.pdf - Submitted Version Restricted to Repository staff only Download (10MB)

Abstract

The efficacy of Mesenchymal Stem Cell (MSC) therapy is currently limited by poor survival and homing of transplanted cells as demonstrated by clinical studies. This is majorly due to the harsh microenvironment created by oxygen and nutrient deprivation, and inflammatory parameters prevailing at the injured site. The choice of MSC source could be critical in determining the fate and cellular function of MSCs under the different physiological stress conditions. Our study focussed on evaluating the viability of umbilical cord-derived Wharton’s Jelly MSCs (WJ-MSCs) under physiological stress conditions like ischemia, serum deprivation and febrile temperature (40°C). Serum deprivation or 40°C stress exposed WJ-MSCs exhibited reduced proliferation and underwent G0/G1 phase cell cycle arrest with no appreciable apoptotic change. An upregulation in the expression of ECM glycoprotein, vitronectin (VTN), was noted both at mRNA and protein levels with changes in the subcellular distribution pattern as compared to control WJ-MSCs as observed by qRT-PCR, Western blotting and immunofluorescence studies. Investigation into the regulation of VTN expression revealed that inhibition of pro-survival PI3K and ERK signalling pathways, led to further upregulation of VTN expression under both serum deprivation and 40°C conditions, with no significant apoptotic change while WJ-MSCs continued to remain in G0/G1 arrested phase. siRNA mediated knockdown of VTN under both serum deprivation and 40°C stress conditions led to a marked decrease in viability with reversal in G0/G1 cell cycle arrest. Moreover, treatment of VTN knocked down WJ-MSCs with inhibitors of either of the pro-survival ERK or PI3K pathways under serum deprivation or 40°C, resulted in profound increase in cell death. In addition, inhibition of NF-κβ pathway, led to downregulation of VTN, along with a corresponding reduction in the viability and reversal in G0/G1 cell cycle arrest, similar to the observation noted with VTN knockdown. Overall, this study concludes that VTN plays a pro-survival role in maintaining viability of WJ-MSCs under different stress conditions. In addition, VTN enriched conditioned medium derived from WJ-MSCs was able to rescue the massive apoptosis of MCF-7 cells under serum deprivation condition. Overall, the role and importance of VTN as a pro-survival factor in WJ-MSCs under different physiological stress conditions is highlighted.

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