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

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

Mesenchymal stromal cells (MSCs) comprise a heterogeneous population of multipotent stromal cells that can be isolated from different tissue sources and have the potential to self-renew and differentiate to diverse cell types. Being non-tumorogenic, immunomodulatory and avoiding immune-rejection, MSCs represent a promising tool for cell-based therapies [1], [2].

Bone marrow (BM) that was originally used as a source for MSCs involves invasive harvesting procedures and also suffers from age-related decline in the yield and differentiation potential of MSCs. Different perinatal tissues including Wharton's-Jelly (WJ) of umbilical cord are thus being considered as alternative sources [3], [4], [5]. Reports suggest that WJ-MSCs have a higher proliferation rate than, and similar immunomodulatory properties as, BM-MSCs [6], [7].

Evidence suggests that MSC transplantation in neurological disorders has beneficial effects, but the underlying mechanism of the therapeutic effects of MSCs is unclear. It is believed that MSCs can bring about neurogenesis by transdifferentiation and soluble factor release. In our previous in vitro study [8], we report that MSCs demonstrate a certain extent of neuronal plasticity in the presence of embryonic cues but most in vivo neurodegenerative adult animal model studies suggest that there is very low frequency of transdifferentiation to neuronal phenotype [9], [10], [11]. This therefore focuses attention on the alternative mode by which MSCs can bring neural recovery, viz. by secreting neurotrophic factors. Earlier studies on BM-MSCs and adipose derived stem cells (ADSCs) have reported that they can secrete an array of neurotrophic factors such as hepatocyte growth factors (HGF), brain derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF) etc. [12], [13], [14]. This trophic factor release profile again varies among MSCs derived from different tissue sources [12], [13], [14], [15].

At present, the primary limitation of MSCs across tissue sources is the poor survival rate after in vivo transplantation in ischemic conditions [16], [17], [18]. Here we should note that the physiological oxygen tension is nearly 4–7%, while the neurotoxic ischemic microenvironment prevailing in the region of CNS injury primarily features hypoxia (2% oxygen) and deficit of nutrients [19], [20], [21]. Only 1% survival of MSCs has been reported post-transplantation into the left ventricle of an ischemic heart failure adult murine model within 4 days of injection [16], [17], [18]. Therefore, for MSCs to demonstrate their paracrine effect, ensuring their own survival is paramount. The diverse effect of hypoxia on the proliferation, apoptosis and differentiation potential of MSCs from various tissue sources reported by different studies is noteworthy [15], [22], [23], [24], [25], [26], [27]. For instance, Potier et al. (2007) reported apoptosis due to prolonged hypoxia and serum-deprivation in BM-MSCs, whereas Lee et al. (2009) found an increase in proliferation in ADSCs under hypoxia both in presence and absence of serum. While cell death under hypoxic serum-deprived condition has been reported in human BM-MSCs and placenta-derived MSCs [15], [27], the cell defense mechanism of the MSCs and the effect of this condition on their secretory profile are not yet reported, especially for WJ-MSCs, a gap the present study seeks to address. Characterization of the effects of this toxic environment on the anti-oxidative capacity of the graft cells would clearly aid the development of future strategies to enhance cell defense and survival in a pathological condition.