Date of Award

2016

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Biology

First Advisor

Paul Gadue

Abstract

Human pluripotent stem cells (PSCs) provide a powerful model system for the study of early human development, disease modeling and physiology. We chose to focus our studies on monogenic diabetes using this model system. Within the pancreas, β cells are one of the most critical endocrine cells as loss of this cell type disrupts blood glucose homeostasis, leading to diabetes. Due to the limited availability of primary human cells it is difficult to study them in vitro, especially in the context of genetic disease where patient material is even more difficult to obtain. Here, we characterize endodermal progenitor (EP) derived β-like cells as a model system for studying β cell development and function in vitro. EP cells are a population of endodermal stem cells, which can self-renew and be differentiated into multiple endodermal cell lineages. EP derived β-like cells are mono-hormonal for insulin and express a number of genes important for insulin processing and secretion. By use of both static stimulations and perifusion assays we show that EP derived β-like cells are responsive to both glucose and a number of other know secretagogues. Next, we demonstrate that genome editing with a zinc finger nuclease at the AAVS1 safe harbor locus can generate stable gene expression in PSCs during differentiation. We then combine these tools and describe the use of PSCs as an in vitro model system to study GATA6. Heterozygous mutations in this gene are the leading cause of pancreas agenesis while studies in mice do not replicate the human phenotype. Induced pluripotent stem cells were created from a pancreas agenesis patient with a heterozygous mutation in GATA6. Using genome editing technology, additional stem cell lines with mutations in both GATA6 alleles were generated and demonstrate a severe block in definitive endoderm induction. Re-expression of GATA6 or other GATA family members can rescue this endoderm phenotype. Partial rescue could also be achieved by treatment with a basic fibroblast growth factor. Using the EP cell culture system to bypass the developmental block at the endoderm stage, cell lines with mutations in one or both GATA6 alleles were differentiated into β-like cells. The mutant cells were shown to be functionally defective by failure to secrete insulin upon glucose stimulation. Decrease in retinoic acid concentrations used during the differentiation lead to decreased β-like cell differentiation efficiency of the heterozygous GATA6 mutants suggesting a possible mechanism for the patient phenotypes. These data show that GATA6 plays a critical role in endoderm specification and β-like cell functionality in humans while in mice it is dispensable, highlighting the importance of studying a human system.

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