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|Title:||Modeling Disease and Development with Pluripotent Stem Cells|
|Department:||Biochemistry and Biomedical Sciences|
|Abstract:||With recent advances in stem cell research, the use of human pluripotent stem cells (hPSC) provides access to disease-relevant cells that are not easily accessible in patients. The ability of hPSCs to self-renew indefinitely and to differentiate into cell types of all three embryonic germ layers provides a model system to investigate the effects of genetic mutations that are relevant in the context of specific diseases. hPSC carrying disease associated genetic mutations provide access to an unlimited supply of disease-relevant tissue while healthy hPSC can be used to model normal human development. Our lab has access to samples from a family with multiple cases of pulmonary fibrosis (PF). The high prevalence of PF in this family led us to believe that there is a strong genetic component to the pathogenesis of PF in these cases. We previously were able to derive iPSC lines from each of the patients and have identified genetic variants via exome sequencing, including PACS1 and ITGB6, that may predispose carriers to PF. In the present study, we examine the phenotypic changes caused by the PACS1 and ITGB6 gene variants. We hypothesized that the N217S mutation in PACS1 conveys a resistance to apoptosis in fibroblasts which could lead to the accumulation of fibrotic tissue. This hypothesis was tested through the use of apoptosis assays including annexin flow cytometry and immunostaining for caspase 3 cleavage after treatment with TNF-related apoptosis-inducing ligands. We found that in transfected cell lines, there were no significant changes to apoptotic sensitivity. The results are inconclusive in the patient-derived fibroblasts because of high inter-test variability. The second mutation we examined was the R15K mutation in ITGB6, which occurs within the signal peptide. Thus, we hypothesized that this mutation affects the efficiency of localization and subsequently, function. This hypothesis was tested through confocal microscopy of endogenous ITGB6 in small airway epithelial cells (SAEC), as well as HEK 293 FT cells transfected with plasmid constructs containing GFP tagged wt or R15K ITGB6. We then tested for changes in the ability of ITGB6 to affect downstream signaling through plating on fibronectin, pulldown of focal adhesion kinase, and analysis for phosphorylated FAK through western blot. We found that endogenous expression of ITGB6 in SAECs, appears mainly in the membrane with a small percentage within the nuclear membrane. In transfected cell lines, there was a shift in localization from plasma membrane to nuclear membrane. The functionality of ITGB6 did not appear to be affected based on our preliminary data. This thesis also explores the utility of intestinal epithelial organoids derived from hPSCs, termed “human enterospheres” (hEnS), in investigating the process that drives intestinal maturation. The hEnS provide a unique novel model for studying maturation, due to the fetal phenotype of the hEnS. We hypothesized that through changes in culturing medium and addition of bacterial products, it is possible to induce maturation of the hEnS. To test this hypothesis, we cultured the hEnS with heat-killed bacteria that play a role in the natural maturation process and examined changes to markers of maturation. We also performed an in-depth study of key maturation markers at specific gestational stages. By immunostaining the gestational tissue for various markers associated with maturation, we sought to create a scale with which to evaluate the maturation status of hPSC-derived intestinal tissue.|
|Appears in Collections:||Open Access Dissertations and Theses|
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|Amos Lim - Thesis Final with corrections 1.pdf||2.4 MB||Adobe PDF||View/Open|
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