UBC Theses and Dissertations
Characterization and modulation of adult pancreatic β-cell maturity Szabat, Marta
The functional maturation and dedifferentiation of β-cells is central to diabetes pathogenesis and to beta-cell replacement therapy. Despite its importance, the dynamics of adult β-cell maturation remain poorly understood because it has previously been difficult to study the process directly. A novel dual fluorescent reporter lentiviral vector was developed capable of tracking the differentiation status of single β-cells in culture. Using this labeling tool, an immature β-cell state was identified in adult primary human and mouse islets and beta-cell lines. The immature β-cell state was characterized by Pdx1 promoter activity but undetectable insulin promoter activity. Lineage analysis of labeled single adult human, mouse and MIN6 β-cells revealed that a fraction of the immature β-cells underwent maturation over time in culture by robustly activating the insulin promoter. Immature beta-cells also exhibited a significantly downregulated profile of mature β-cell genes. These cells had increased proliferation and a reduced glucose-stimulated insulin secreting function. In order to manipulate the adult β-cell maturation state, a screen for candidate growth/differentiation factors using image-based approaches was performed. Activin A and its antagonist follistatin were found to modulate adult β-cell maturity. Activin A had a strong negative effect on β-cell maturity, reducing insulin promoter activity, insulin secretion and the expression of mature β-cell genes. Follistatin reversed the effects of endogenous activin A and augmented β-cell maturity. These results uncovered a local autocrine/paracrine regulatory mechanism that controls the maturation state of adult β-cells. In addition, gene expression profiling at the whole genome level was used to analyze purified immature and mature β-cells from humans, mice and the MIN6 β-cell line. These analyses revealed that immature β-cells have increased expression of multiple islet hormones and have enriched expression levels of many genes known to be involved in pancreatic development, stem cell plasticity, proliferation and apoptosis. Conversely, mature β-cells are enriched in genes related to maintaining the mature β-cell phenotype. Collectively, these experiments contribute to the understanding of maturation and plasticity of adult pancreatic β-cells. The results have significant implications for islet regeneration and for in vitro generation of functional β-cells to treat diabetes.
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