Molecular characterization of human stem cells
Open Access
- Author:
- Jaishankar, Amritha
- Graduate Program:
- Genetics
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- July 09, 2010
- Committee Members:
- Kent Eugene Vrana, Dissertation Advisor/Co-Advisor
Kent Eugene Vrana, Committee Chair/Co-Chair
Sarah Bronson, Committee Member
Jianming Hu, Committee Member
Willard Freeman, Committee Member
Judith Weisz, Committee Member - Keywords:
- mesenchymal stem cells
embryonic stem cells
proteomics - Abstract:
- Human embryonic stem cells (ES) are characterized by their immortality and pluripotency. Human mesenchymal stem cells (MS), on the other hand, have limited self-renewal and restricted differentiation capabilities. The underlying molecular differences that account for this characteristic self-renewal and plasticity are, however, poorly understood. The objective of this research program was to identify differences in the nuclear proteome of undifferentiated human ES and MS cells. Nuclear proteins (such as transcription modulators and proteins involved in chromatin remodeling) are crucial to the regulation and maintenance of self-renewal and plasticity of stem cells and are thereby potentially important stem cell markers and determinants. We used two-dimensional difference gel electrophoresis (2-DIGE) discovery proteomics to identify differentially-expressed nuclear proteins. Proteins of interest were further studied using quantitative western analysis, and quantitative RT-PCR. Our proteomic screen highlighted dramatic differences in the expression of Reptin52, insulin-like growth factor 2 mRNA binding protein 1 (IMP1) and far upstream element-binding protein 1 (FUBP1). Moreover, several of the proteins we have identified interact with each other and more interestingly, their interactions converge on c-myc. To further examine the differences between pluripotent and multipotent stem cells, the gene expression pattern of a panel of genes was examined across different stem cell lines (ES, MS and induced pluripotent stem cells (iPS)) to establish a ‘stemness’ fingerprint. Moreover, promoter-associated CpG island methylation was also examined in ES, MS and iPS cells. In particular, we found that iPS cells and ES cells are virtually indistinguishable in terms of global DNA methylation, selected gene promoter methylation, and mRNA expression levels. This is in contrast to bone marrow-derived MS cells that were very different and more closely resembled fibroblasts (in aspects of mRNA expression). Together, these data help illuminate fundamental differences between human ES and MS cells and similarities between the two pluripotent stem cells (ES and iPS cells), that may be key to understanding and regulating pluripotency and immortality in these cells.