Browsing by Author "Ahlnäs-Piña, Karen"

A robust energy metabolism in humans relies on functional insulin-producing beta cells for maintenance of normoglycemia. Dysfunctional beta cells or malfunctioning insulin is the underlying cause for all forms of diabetes. With current management of different types of diabetes, patients continue to face life-threatening risk of hypoglycemia and other associated pathologies. Research has been challenging due to difficulties in studying beta cells in vitro, as human donor-derived beta cells do not expand well in laboratory conditions. At the time when this study was performed, only a few protocols had been published for beta-cell differentiation. These protocols were often difficult to reproduce in different cell lines and resulted in a low yield of differentiated cells. Both pancreas and liver develop from the same precursor cells called the definitive endoderm (DE). Factors directing the differentiation of definitive endoderm towards a pancreatic endocrine progenitor fate are not fully known. Previous unpublished data indicated the presence of transforming growth factor beta (TGF-β) superfamily inhibitors in the early stages of a developing murine pancreas. In this study, protocols for producing pancreas-specific endoderm cells from human pluripotent stem cells (hPSC) were tested and developed further. The primary aim of this study was to inhibit TGF-β mediated signaling, follow-up and report the effects in definitive endoderm specification. Human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) were differentiated into definitive endoderm (DE) using directed differentiation, followed by five experimental conditions with TGF-β inhibitors. Quantitative real-time PCR (q-PCR), flow-cytometry (FC), immunocytochemistry (ICC), and –fluorescence (IF) techniques were used to examine the gene- and protein expression of the cells at specific time points of the protocol. The gene expression levels of known hepatic and pancreatic markers were analyzed and compared between the cell lines. The current DE differentiation protocol, consisting of the DE stage, followed by a four-day culture, showed a downregulation of hepatic markers with and without TGF-β inhibitors. The examined protocols resulted in heterogenic cell populations, consequent to previously reported challenges in beta-cell progenitor differentiation. This study provided valuable information and a platform for further research on the differentiation of pancreatic endocrine progenitor cells.