Influence of ischemic microenvironment on human Wharton's Jelly mesenchymal stromal cells

Abstract

IntroductionWhile in vivo studies suggest poor survival of mesenchymal stromal cells (MSCs) after transplantation in ischemic conditions, in vitro studies report diverse effects on proliferation, apoptosis and differentiation of stem/precursor cells of different tissue-origin. The present focus is to understand the influence of ischemic microenvironment on the survival, proliferation, apoptosis, ROS-generation, antioxidant levels, immunophenotypic-expression and neurotrophic factor secretion of Wharton's Jelly (WJ)-MSCs. MethodWJ-MSCs were cultured in normoxic and hypoxic conditions in presence and absence of serum and the end-point parameters were measured at 4 time-points. Cell survival, proliferation, apoptosis, ROS-generation and immunophenotypic-expression were quantitatively detected either by fluorimetry or flow cytometry techniques. ELISA-based methods were used for detection of antioxidant-substrate glutathione (GSH) and neurotrophic factors [vascular endothelial factor (VEGF), hepatocyte growth factor (HGF) and brain-derived neurotrophic factor (BDNF)]. Expression of the antioxidants glutathione peroxidase (GPx) and superoxide dismutase 1 (SOD1), was measured by real-time RT-PCR. ResultImmunophenotypic analysis showed reduction in mesenchymal-marker (CD73, CD90, and CD105) expression under ischemic conditions influenced mainly by hypoxia, whereas the decrease in cell-survival under ischemic condition was mainly as a result of nutrition depletion. This was associated with increased ROS-generation and apoptosis and reduction in antioxidants (GSH, GPx, SOD1). For neurotrophic factors, ELISA-readings showed that VEGF and HGF secretion (which were higher in hypoxia) peaked at 48 h and decreased from 72 h, though BDNF release did not decrease. DiscussionTherapeutic benefits rendered by WJ-MSCs in in vitro ischemic microenvironment are highest at the 48 h time-point, declining thereafter with time probably due to failure in cellular defense systems and the onset of apoptosis. ConclusionIt is hence clear that the growth factor deficiency is more lethal to the cells than hypoxia in ischemic microenvironment.