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http://hdl.handle.net/11375/20528
Title: | DERIVING MESENCHYMAL STEM CELLS-LIKE CELLS WITH IMPROVED ADIPOGENIC CAPACITY FROM PLURIPOTENT STEM CELL SOURCES |
Authors: | Hatkar, Rupal |
Advisor: | Szabo, Eva |
Department: | Biochemistry and Biomedical Sciences |
Publication Date: | 2016 |
Abstract: | Mesenchymal stem cells (MSCs) have been used for tissue repair, tissue regenerative purposes, to understand cell biology of various cell types, and as an in vitro model of a disease in vitro. Primary MSCs isolated from adult human tissue (i.e. adipose tissue), have been shown to have limited proliferative and differentiation capacity over passages in vitro. Due to the limited availability of tissue donors, it is difficult to obtain sufficient primary MSCs for therapeutic purposes. Therefore, pluripotent stem cells (human embryonic stem cells, hESCs, and induced pluripotent stem cells, iPSCs) have been used an alternative source to obtain the unlimited source of MSC-like cells. These cells are reported to have similar morphological and phenotypical characteristics but have lower differentiation capability compared to primary MSCs. Additionally, the protocols used for derivation of MSC-like cells from PSC sources are lengthy, expensive, and labour intensive. In this study, I established a method for derivation of MSC-like cells from the cells surrounding H9 hESCs and a-iPSCs (iPSC-derived by reprogramming adipose tissue-derived stem cells (ADSCs)) through a simple trypsinization step. These MSC-like cells exhibited typical fibroblastic morphology and MSC surface marker profiles akin to bonafide adult MSCs, such as ADSCs. Compared to other studies, this protocol is more time and labour efficient, and it results in the derivation of significantly more (60%) lipid droplets forming cells. However, further characterization of these cells as adipocytes is necessary, given that their differentiation capacity remains limited when compared to ADSCs. To improve the differentiation capacity of the MSC-like cells derived from H9 hESC or a-iPSCs, I explored: 1. the role of two transcription factors, peroxisome proliferator-activated receptor γ2 (PPARγ2) and Krüppel-like factor 4 (Klf4), both of which have been shown to enhance adipogenesis in the murine system; and 2. the effect of culture media used for adipocyte differentiation. Through these studies, I discovered that neither Klf4 nor PPARγ2 induction improved adipocyte development in my human system. Accordingly, I did not observe an increase in the adipogenic capacity of the MSC-like cells regardless of the method by which Klf4 was induced (ectopic expression or chemical induction using IBMX), or through induction of PPARγ2 using dexamethasone. However, my studies did show a significant improvement in the appearance of lipid droplet containing cells, akin to adipocytes, upon culturing the MSC-like cells with the specific concentration of MEF (Mouse Embryonic Fibroblast)-condition media compared to well-defined media that have been used for prior differentiation protocols. Overall, compared to ADSCs, H9-MSCs and a-iPSC-MSCs displayed a unique expression pattern of MSCs and early epithelial to mesenchymal transition (EMT) genes, suggesting further analysis of H9-MSCs and a-iPSC-MSCs at global gene expression and an epigenetic level is required to improve quality of these cells to match that of adult MSCs, such as ADSCs. Additional optimization of H9-MSCs and a-iPSC-MSCs differentiation protocols is also necessary to ensure that the MSC-like cells are following the appropriate MSC differentiation trajectory, allowing us to obtain a bona fide MSC with mature adipocyte differentiation capacity, similar to that derived from ADSCs. |
URI: | http://hdl.handle.net/11375/20528 |
Appears in Collections: | Open Access Dissertations and Theses |
Files in This Item:
File | Description | Size | Format | |
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Hatkar_Rupal_R_2016 September_M.Sc..pdf | 5.3 MB | Adobe PDF | View/Open |
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