Overexpression of PAX4 reduces glucagon expression in differentiating hESCs

Abstract

Human embryonic stem cells (hESC s) are pluripotent and capable of generating new β-cells, but current in vitro differentiation protocols generally fail to produce mature, glucose-responsive, unihormonal β-cells. Instead, these methods tend to produce immature polyhormonal endocrine cells which mature in vivo into glucagon-positive α-cells. PAX4 is an established transcription factor in β-cell development and function, and is capable of converting glucagon-positive cells to insulin-positive cells in mice. Work in human and mouse ESC s has shown that constitutive PAX4 expression promotes the development of insulin-positive cells, but whether acute PAX4 expression is sufficient to guide specific endocrine cell fates has not been addressed in hESC s. In this study, we applied recombinant adenovirus to ectopically express human PAX4 in hESC -derived pancreatic progenitors, with the aim of influencing the endocrine developmental cascade away from polyhormonal cells toward unihormonal insulin-positive cells. Gene delivery to pancreatic progenitors was efficient and dose-dependent. By the end of in vitro differentiation, PAX4 reduced AR X expression, but only the high dose tested significantly reduced glucagon release. Single cell analysis revealed that while PAX4 did not alter the proportion of endocrine cells, it did reduce the number of glucagon-positive cells and increased the number of unihormonal insulinpositive cells. These data suggest that acute PAX4 overexpression can reduce expression of AR X and glucagon resulting in improved numbers of unihormonal insulin-positive cells.