Evaluation of Wharton’s Jelly-derived Mesenchymal Stem Cells under Conditions of Ischemia-a Step Towards Cell Therapy

Himal, Iris (2016) Evaluation of Wharton’s Jelly-derived Mesenchymal Stem Cells under Conditions of Ischemia-a Step Towards Cell Therapy. Masters thesis, Indian Institute of Science Education and Research Kolkata.

PDF (MS dissertation of Iris Himal (11MS028)) IRIS_HIMAL-11MS028.pdf - Submitted Version Restricted to Repository staff only Download (4MB)

Abstract

Modern therapeutics focus mainly on stem cell research due to their wide applications in cell transplantation and replacement of tissue lost due to injury. Stem cells can self-renew and differentiate into any specialised cell type. Due to their paracrine and immunomodulatory roles, self-renewal and homing properties and the lack of ethical concerns mesenchymal stem cells (MSCs) are an ideal candidate for cell based therapies. Wharton’s jelly (WJ) is the mucoid connective tissue surrounding the arteries and veins in the umbilical cord and is a rich source of MSCs. Successful MSC based therapy greatly relies on various factors such as survival rate, migration and immunomodulatory property of the cells at the injured or ischemic tissue where the microenvironment is characterized by inadequate amount of oxygen and blood supply. Hence, it is essential to have an in-depth understanding of factors and mechanisms that affect WJ-MSCs in an ischemic environment. We simulated conditions of ischemia in vitro by culturing WJ-MSCs in serum deprived, low glucose media under 2% oxygen for 48 hours. We have evaluated the viability, cell cycle and senescence status of WJ-MSCs under conditions of ischemia. We have tried to assess and compare cell surface marker and costimulatory molecules’ expression between ischemic and control WJ-MSCs. MSCs are known to have a paracrine role in wound healing and tissue regeneration and therefore we evaluated the level of chemokine receptors, growth factors, ECM and adhesion molecule gene expression of ischemic WJ-MSCs by a wound healing PCR array. Next to demonstrate the angiogenic property of ischemic WJ-MSCs, conditioned medium from ischemic WJ-MSC culture was used and angiogenesis assay was performed on HUVECs in comparison against control conditioned medium. The therapeutic property of WJ-MSCs relies also on its homing property therefore we studied the migration of WJ-MSCs under conditions of ischemia. Through scratch wound healing assay and live cell imaging we see that there is decrease in the migration rate of WJ-MSCs under in-vitro ischemic condition. Non Muscle Mysoin is said to play an important role in the migration of WJ-MSCS and therefore we have tried to assess the role of Non muscle myosin II isoforms in the migration of WJ-MSCs under ischemic conditions. Lastly we have tried to test for any change in epigenetic modifications in ischemic WJ-MSCs. Through various experiments which we have carried out we can conclude that WJ-MSCs do not undergo massive cell death under conditions of ischemia and retain a viable population of 70% even under prolonged ischemia. They undergo cell cycle arrest at G0/G1 phase but do not undergo senescence under ischemic conditions. However on prolonged exposure to ischemia they undergo senescence which is seen in control cells also. Characteristic cell surface marker expressions of WJ-MSCs do not change under ischemia or serum deprivation. As seen from the PCR array data ischemic cells show increased expression of certain growth factors, ECM and adhesion molecules responsible for wound repair. From above results we can conclude that WJ-MSCs can survive under conditions of ischemia and retain their characteristic MSC properties and also has the ability to promote wound healing and repair through various factors expressed by them.

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