UBC Theses and Dissertations
Analysis of undifferentiated human embryonic stem cell lines using Serial Analysis of Gene Expression Schnerch, Angelique
Since the first reported isolation of immortalized human embryonic stem cell (hESC) lines in 1998 (Thomson et al., 1998), methods for directing their differentiation to various specialized derivatives has been extensively demonstrated and provides hope for future therapeutic applications. Characterization of the key molecular factors governing hESC self-renewal and pluripotency is necessary for ongoing efforts in deriving therapeutically useful cell types and in modelling human embryonic and oncogenic development. To this end the GSC Gene Expression Laboratory has generated 11 global gene expression profiles of 8 undifferentiated hESC lines using long Serial Analysis of Gene Expression (long SAGE) (NIH stem cell registry code BG01, ES03, ES04, WA01, WA07, WA09, WA13, and WA14). I analysed a database of the hESC long SAGE data consisting of 2,613,475 total tags corresponding to 379,465 transcripts. By employing various comprehensive tag-togene mapping resources I have provided a detailed survey of the genes expressed and differentially expressed in multiple hESC lines. A suite of sternness-associated factors was observed in the hESC SAGE data. We also observed molecular components of several pathways involved in embryonic development, the cell cycle, and programmed cell death. Comprehensive interspecies pair-wise comparisons between the hESC libraries and publicly available human SAGE libraries identified up-regulated transcripts in embryonic stem cells. A robust computational approach was designed and identified tags expressed solely in hESCs compared to 247 normal and malignant cells. A key feature of the approach was to isolate tags derived from sequences conserved across the human, mouse and rat genomes; this led to the identification of 301 candidate novel transcripts that may be integral to human pluripotent stem cells. These studies represent an important step in the development of high throughput approaches to an analysis of early human developmental processes and will be a strategic element in more comprehensive interspecies comparisons of E S cells to identify preserved control mechanisms.
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