Defense Date

2010

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Human Genetics

First Advisor

Shawn Holt

Abstract

A great need exists for an abundant, easily accessible source of patient-specific cells that will function for use in regenerative medicine. One promising source is the adult stem cell derived from adipose tissue (ASCs). Isolated from waste lipoaspiration, these cells could serve as a readily available source for the regeneration of damaged tissues. To further define the biology of ASCs, we have isolated multiple cell strains from different adipose tissue sources, indicating wide-spread distribution in the body. We find that a widely used set of cell surface markers fail to distinguish ASCs from normal fibroblasts. However, our ASC isolations are multipotent while fibroblasts show no differentiation potential. In further contrast to fibroblasts, these cells also show expression of genes associated with pluripotent cells, Oct-4, SOX2, and NANOG. Together, our data suggest that while the cell surface profile of ASCs do not distinguish them from normal fibroblasts and their lack of telomerase shows their limited proliferation capacity, the expression of genes closely linked to pluripotency and their differentiation capacity clearly define ASCs as multipotent stem cells. iPS cells are another promising cell type for tissue regeneration, due to their expression of hTERT and their capacity to differentiate into all three germ layers. Interestingly, telomerase is activated during the induction process, accomplished by the exogenous expression of four genes in normal, non-hTERT-expressing fibroblasts. To elucidate the mechanisms behind this activation, we examined the overexpression of these four factors in BJ fibroblasts and ASCs, which resulted in undetectable hTERT expression. We then demonstrated a lack of an acetylated histone H3K9 with the opposing di-methylation, indicative of a closed chromatin state at the hTERT promoter. Subsequent treatment of cells with TSA alone showed an upregulation of hTERT mRNA without telomerase activity. However, telomerase activity was found when ASCs, but not BJs were treated with TSA and all four factors, indicating differential regulation of hTERT in cells of similar mesenchymal origins. Our data suggest that while hTERT’s expression is universally dependent on the presence of a relaxed chromatin state and sufficient transactivating factors, other cell to cell differences can prevent its expression.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

February 2010

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