Engineering a Human Embryonic Stem Cell Reporter Line for Salivary Lineage Screening
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Abstract of the Thesis Engineering a Human Embryonic Stem Cell Reporter Line for Salivary Lineage Screening By Bahar Houshmand Master of Science in Basic Health Sciences (Oral Biology & Pathology) Stony Brook University 2011 Head and neck cancer radiotherapy and Sjogren Syndrome are two major conditions associated with irreversible damage to salivary glands and, a considerable decrease in saliva secretion. Currently there is no treatment available for these patients other than those relieving the symptoms. Cell-based therapy holds a great promise for treatment of these conditions, however the source of salivary progenitor cells is limiting. Human embryonic stem cell (hESC) can be induced to differentiate in to all cells in the body and may provide a potential source of progenitor cells for regeneration of damaged salivary gland. To direct efficient differentiation of hESC to salivary gland progenitor cells however, well-defined culture conditions must be developed. One way to screen culture conditions that support differentiation of hESC to a specific lineage is to generate lineage-specific reporter lines. This is achieved through genetic modification of hESCs with a tissue-specific promoter driving a reporter gene. In addition, generation of reporter progenitor cells will facilitate the purification of specific cell populations from heterogeneous differentiated hESC progeny. Keratin (K) 19 is a marker of salivary gland progenitor cells. Here I describe the construction of a lentiviral vector in which EGFP expression is controlled by human K19 promoter. When compared to ubiquitously expressed EF1<em>f</em>Ç_-EGFP, the K19-EGFP reporter construct exhibited high levels of activity in prostate epithelial cells (PC3) which express high levels of K19, and low levels of activity in fibroblasts and keratinocytes which normally do not express K19. This demonstrated the fidelity and specificity of transgenic K19 promoter. Transgenesis of a hESC line with lentiviral vector encoding K19-EGFP resulted in minimal or no EGFP expression while high levels of EGFP was detected in those transduced with EF1<em>f</em>Ç_-EGFP. This transgenic line could be useful in high through-put assays to define conditions that reproducibly and efficiently induce differentiation of salivary lineage.