Influence of ischemic microenvironment on human Wharton's Jelly mesenchymal stromal cells
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.
Section snippets
Isolation and culture of WJ-MSCs
The isolation of WJ-MSCs was carried out as previously described by Datta et al. (2011). Human umbilical cords were aseptically collected from caesarian section or normal deliveries following the informed consent guidelines approved by the Institutional Committee for Stem Cell Research and Therapy (ICSCRT) and Institutional Ethics committee of Manipal Hospital, Bangalore, India. The vessels were manually removed from umbilical cord segments and the tissue was dissected into 2 cm × 2 cm pieces.
Phenotypic characterization of WJMSC
As represented in Fig. 1, WJ-MSCs were positive for the hallmark mesenchymal markers CD73, CD90, CD105 and were negative for the MHC class II cell surface receptor HLA-DR. Similar immunophenotypic expression of markers was observed under serum-deprivation in normoxia, but the expression of CD73, CD90 and CD105 was significantly (#P < 0.05) reduced under ischemic condition (Fig. 1). A significant decrease (#P < 0.05) in mesenchymal marker expression was also observed in hypoxic condition in the
Discussion
We created the conditions of an ischemic microenvironment through hypoxia (2% oxygen) and serum-deprivation. Though HLADR expression remains negative under ischemic condition, a distinct decrease in mesenchymal marker expression of CD73, CD90 and CD105 was observed under hypoxic conditions (with and without serum). This change in mesenchymal markers was not accompanied by morphological differences between ischemic condition and control, but cell numbers declined with time under ischemic
Conclusion
In conclusion, the result obtained in the present study indicates that with time, under the withdrawal of survival growth factors and hypoxia, WJ-MSCs undergo cell-death through apoptosis accompanied with a decline in the counterbalancing antioxidant defense mechanism. The proliferation capacity and the paracrine effect of WJ-MSCs too get impaired under the influence of ischemic microenvironment. While an earlier report [25] suggests that rat BM-MSCs undergo caspase-dependent apoptosis in
Author disclosure statement
The authors have declared that no competing interests exist.
Acknowledgments
We thank Manipal University for providing the necessary infrastructure and funding for this work. We also thank Dr Shobha Rani, Consultant Obstetrician and Gynecologist, Manipal Hospital, Bangalore for providing human umbilical cord samples for the study. We sincerely acknowledge Mr. Anustup Datta for his assistance in statistical analysis. We are also grateful to the Vice-Chancellor and Registrar of MU for their support.
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