Propensity of Wharton’s Jelly-derived Mesenchymal Stem Cells to Differentiate Towards Chondrogenic Lineage and Pptimization of Serum-free Cryopreservative for Preservation of Umbilical Cord Tissue

Kumari, Poonam (2014) Propensity of Wharton’s Jelly-derived Mesenchymal Stem Cells to Differentiate Towards Chondrogenic Lineage and Pptimization of Serum-free Cryopreservative for Preservation of Umbilical Cord Tissue. Masters thesis, Indian Institute of Science Education and Research Kolkata.

PDF (MS dissertation of Poonam Kumari) Poonam_Kumari.pdf - Submitted Version Restricted to Repository staff only Download (7MB)

Abstract

Mesenchymal Stem Cells (MSCs) show remarkable potential for cell based clinical applications. MSCs isolated from Wharton’s Jelly of umbilical cord (WJMSCs) are promising candidates for cell-based regenerative medicine. They have been shown to have higher proliferative potential than bone marrow derived MSCs. Hence, understanding the nature of MSCs isolated from WJ becomes important for successful therapeutic applications. Large scale expansion of cells in-vitro is necessary for therapeutic applications. The ready availability of MSCs from the same donor at any given time point for MSC based transplantation and the benefit of being able to start from the P0 passage will act as a mile stone for therapeutic application and research purposes. So the focus of the first project was to establish a serum-free cryo protectant solution that can be used to preserve umbilical cord tissue pieces for future use. We have found out that 10%DMSO+0.2M sucrose can be successfully used as the cryo protectant. MSCs isolated from cryopreserved tissue pieces were validated using ISCT guidelines to define MSCs, i.e. 1. Plastic adherence, 2. Surface marker expression, ie, positive for CD73 and CD90 while being negative for hematopoietic marker, i.e. CD34. 3. Ability to differentiate towards adipogenic, osteogenic and chondrogenic lineages. MSCs are found to go towards three basic lineages, i.e. adipogenic, osteogenic and chondrogenic lineage. MSCs derived from bone marrow are considered to be more apt for chondrogenic lineage differentiation due to its source. But we repeatedly observed in the lab that MSCs isolated from WJ of umbilical cord underwent chondrogenic differentiation even without inducing factors as demonstrated by Alcian blue staining. Based on the observation this project is dedicated to understand if WJMSCs have a spontaneous tendency to differentiate towards chondrogenic lineage. Preliminary data showed that there might be some spontaneous chondrogenic differentiation happening in WJMSCs even in the absence of differentiation factors. Alcian blue staining, GAG quantification and gene expression analysis was done to validate the observation which holds true for monolayer as well as pellet culture system. Previously, in our lab it was shown that treatment of WJMSCs with blebbistatin resulted in cell cycle arrest of MSCs at G0/G1 stage. In this part I helped to answer a few questions related to this project. We studied cell cycle analysis after blebbistatin withdrawal, surface marker expression in control and blebbistatin treated cells, differentiation of blebbistatin treated cells towards adipogenic and osteogenic lineages and dose dependent study of blebbistatin effect on WJMSCs. We found out that the cell cycle arrest at G0/G1 was reversed once the blebbistatin treated cells were cultured in a blebbistatin free medium. NMII inhibition using blebbistatin did not affect MSC characteristics since there was no visible difference in immune-phenotypic profile or in vitro differentiation capacity between control and blebbistatin-treated WJ-MSCs.

Actions (login required)

View Item