Epigenetic Regulation of Pluripotent Stem Cell Differentiation.
Abstract: The isolation and use of pluripotent cells is an exciting and rapidly-evolving field. By controlling the differentiation conditions it is possible to induce embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) to differentiate into any cell type in the human body. Their unlimited expansion potential and ability to generate therapeutically-relevant cell types makes them va... read moreluable candidates for repairing or replacing damaged cells and tissues. Specifically, differentiation into fibroblasts would be essential for therapeutic tissue regeneration, due to their ability to produce extracellular matrix and interact with adjacent cell types. However, before pluripotent cell sources can be implemented in regenerative medicine, rigorous phenotypic analysis and thorough testing of their functional abilities following differentiation is required. In addition, elucidating the underlying epigenetic mechanisms involved in differentiation of pluripotent cells to specific cell types will enhance our understanding of human development and provide insight into the process of lineage specification. I hypothesized that ESC and iPSC could be directed to differentiate towards fibroblast-like cells, and that they would demonstrate functionality within three-dimensional in vitro tissue models. To gain a better understanding of the mechanisms regulating differentiation towards fibroblasts, I investigated changes in DNA methylation involved during specification of this lineage. Using a defined protocol, ESC- and iPSC-derived fibroblast-like cells exhibited many fibroblast characteristics, including morphology and expression of several fibroblast-related markers. These ESC- and iPSC-derived cells also demonstrated the functionality of adult fibroblasts by supporting keratinocyte stratification and formation of a basement membrane in in vitro 3D skin-like tissues. Additionally I also investigated the epigenetic changes occurring in ESCs and iPSCs following differentiation. Interestingly, both cell types demonstrated significant overlap in their DNA methylation profiles. In particular, platelet-derived growth factor-beta was found to be extensively demethylated following differentiation of both ESC and iPSC, which is characteristic of adult fibroblasts. Knockdown of PDGFRβ significantly decreased the migratory ability of these cells as well as their ability to assemble extracellular matrix. The results of this thesis establish the utility of in vitro 3D tissues to provide functional readouts for cells derived from pluripotent sources. This work also demonstrates that ESC- and iPSC-derived cells share similar properties in gene expression, DNA methylation, and function in 3D tissues. As demonstrated by the consistent demethylation of PDGFRβ in ESC- and iPSC-derived fibroblast-like cells, changes in DNA methylation are likely to be important in the functionality of these cells following differentiation.
Thesis (Ph.D.)--Tufts University, 2012.
Submitted to the Dept. of Cell, Molecular & Developmental Biology.
Advisor: Jonathan Garlick.
Committee: Charlotte Kuperwasser, James Schwob, Thomas Linsenmayer, and Peter Jones.
Keyword: Cellular biology.read less