A novel role of vitronectin in promoting survival of mesenchymal stem cells under serum deprivation stress.

BACKGROUND: Due to their immunomodulatory and trophic support functions, mesenchymal stem cells (MSCs) are promising in the field of cell-based regenerative medicine. However, MSC survival post-transplantation is challenged by various microenvironment stress factors. Here, we investigated the role of vitronectin (VTN) in the survival strategy of MSCs under serum deprivation stress condition. METHODS: Proliferation kinetics and cell adhesion of MSCs under serum deprivation were determined from population doublings and cell-matrix de-adhesion studies, respectively. mRNA and protein expression levels of VTN were confirmed by qRT-PCR and Western blotting, respectively. Immunofluorescence technique revealed distribution of VTN under serum deprivation stress. siRNA and inhibitor-based studies were performed to confirm the role and regulation of VTN. Apoptosis and cell cycle status of MSCs were assessed using flow cytometric analysis. RESULTS: Subjecting MSCs to serum deprivation led to significant increase in cell spread area and cell-matrix adhesion. An upregulation of VTN expression was noted with an arrest in G0/G1 phase of cell cycle and no appreciable apoptotic change. Pro-survival PI3kinase pathway inhibition led to further increase in VTN expression with no apoptotic change. siRNA-mediated inhibition of VTN resulted in reversal in G0/G1 cell cycle arrest and a marked increase in apoptosis, suggesting a role of VTN in preventing serum deprivation-induced apoptotic cell death. In addition, p65 knockdown resulted in downregulation of VTN establishing an association between NF-κß pathway and VTN. CONCLUSIONS: VTN was identified as a survival factor in providing protection from serum deprivation-induced apoptosis in MSCs.

Isolation and Molecular Characterization of Progenitor Cells from Human Umbilical Cord.

Mesenchymal stem cells (MSCs) are multipotent precursor cells which have been isolated from different vascularized tissue sources. Due to their paracrine function of secreting trophic and immunomodulatory molecules, MSCs are successfully used in cell-based transplantations and provide an alternative medical paradigm for treating a variety of devastating disorders. Umbilical cord is a medical waste with a large, readily available donor pool. Since umbilical cord is a fetal tissue, MSCs derived from it are considered more primitive with proliferative and differentiation advantages over adult MSCs. We define here a simple, efficient, and reproducible protocol to isolate MSCs from WJ of human umbilical cord using a nonenzymatic procedure. Under the optimized culture conditions, the WJ-MSCs undergo robust proliferation, can be expanded up to 15-20 passages and express the characteristic MSC surface antigens. They can be differentiated into mesodermal lineages in vitro.

p53-NF-κB Crosstalk in Febrile Temperature-Treated Human Umbilical Cord-Derived Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are successful for their therapeutic application in immune and inflammatory contexts due to their anti-inflammatory, trophic, and immunomodulatory roles. However, though MSCs have the potential to provide regenerative treatment toward a wide range of devastating diseases, massive cell death of transplanted MSCs remains an obstacle to overcome. The relation between MSCs and inflammation is multifactorial and challenging to comprehend. Fever is a critical component of the inflamed microenvironment. Also, the choice of MSC source could be critical in determining the fate of transplanted cells under stress conditions. Here we investigated the thermosensitivity of Wharton's jelly MSCs (WJ-MSCs) to elevated temperature in the physiological fever range. We explored the effect of febrile range temperature on morphology, viability, proliferation kinetics, and cell cycle status of WJ-MSCs. WJ-MSCs adopted a flattened morphology at 40°C, and our data from proliferation kinetics study using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and apoptosis assays showed that WJ-MSCs had reduced proliferation and viability at 40°C compared with control cultures. There was also a G0/G1 cell cycle arrest, which was further confirmed by messenger RNA (mRNA) levels of genes specific for different stages of cell cycle. On evaluating p53 status, we observed an increase in p53 protein expression and its nuclear localization in WJ-MSCs exposed to 40°C. Its downstream effector p21 too was upregulated. Moreover, this temperature-induced p53 induction was inhibited on exposure to 40°C in the presence of NF-κB pathway inhibitor, pyrrolidinedithiocarbamate (PDTC) or endonuclease-prepared small interfering RNA (esiRNA) targeting p65. Febrile temperature exposure did not affect the senescence status of WJ-MSCs. The MSC-specific surface antigen profile at 40°C was similar to control WJ-MSCs. Our findings suggest that under febrile temperature stress conditions, WJ-MSCs exhibit G0/G1 cell cycle arrest and reduction in viable cell count, while retaining their basic characteristics, with an underlying interplay of p53 and NF-κB pathway.

Isolation and Establishment of Mesenchymal Stem Cells fromWharton's Jelly of Human Umbilical Cord.

Mesenchymal stem cells (MSCs) are currently considered as 'medicinal signaling cells' and a promising resource in regard to cell-based regenerative therapy. Umbilical cord is a human term perinatal tissue which is easily attainable, and a promising source of stem cells with no associated ethical concerns. MSCs have been isolated from different regions of the umbilical cord and Wharton's jelly (WJ) is the gelatinous matrix that surrounds and provides protection to the umbilical cord blood vessels. Being more primitive, MSCs from human umbilical cord exhibit greater proliferative capacity and immunosuppressive ability as compared to adult stem cells which gives them a therapeutic advantage. To meet the requirements for cell therapy, it is important to generate MSCs at a clinical scale by following steps which are not time consuming or labor intensive. Here we present a simple, efficient protocol for isolation of MSCs from WJ of human umbilical cord by explant culture method which is reproducible and also, cost effective.

Evaluating Wharton's Jelly-Derived Mesenchymal Stem Cell's Survival, Migration, and Expression of Wound Repair Markers under Conditions of Ischemia-Like Stress.

The efficacy of mesenchymal stem cell (MSC) therapy is currently limited by low retention and poor survival of transplanted cells as demonstrated by clinical studies. This is mainly due to the harsh microenvironment created by oxygen and nutrient deprivation and inflammation at the injured sites. The choice of MSC source could be critical in determining fate and cellular function of MSCs under stress. Our objective here was to investigate the influence of ischemia-like stress on Wharton's jelly MSCs (WJ-MSCs) from human umbilical cord to assess their therapeutic relevance in ischemic diseases. We simulated conditions of ischemia in vitro by culturing WJ-MSCs in 2% oxygen in serum deprived and low glucose medium. Under these conditions, WJ-MSCs retained viable population of greater than 80%. They expressed the characteristic MSC surface antigens at levels comparable to the control WJ-MSCs and were negative for the expression of costimulatory molecules. An upregulation of many ECM and adhesion molecules and growth and angiogenic factors contributing to wound healing and regeneration was noted in the ischemic WJ-MSC population by a PCR array. Their migration ability, however, got impaired. Our findings provide evidence that WJ-MSCs might be therapeutically beneficial and potent in healing wounds under ischemic conditions.