D Majumdar1, R Bhonde, I Datta. 1. Manipal Institute of Regenerative Medicine, Manipal University, Bangalore, Karnataka, India.
Abstract
INTRODUCTION: While 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. METHOD: WJ-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. RESULT: Immunophenotypic 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. DISCUSSION: Therapeutic 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. CONCLUSION: It is hence clear that the growth factor deficiency is more lethal to the cells than hypoxia in ischemic microenvironment.
INTRODUCTION: While 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. METHOD: WJ-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. RESULT: Immunophenotypic 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. DISCUSSION: Therapeutic 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. CONCLUSION: It is hence clear that the growth factor deficiency is more lethal to the cells than hypoxia in ischemic microenvironment.