Differentiation Of Functional Islet β Cells From Human Pluripotent Stem Cells

Diabetes mellitus is a kind of chronic metabolic disorders characterized by severe hyperglycemia caused by dysfunction or progressive loss of the insulin-producing isletβ cells,endangering the life and health of hundreds of millions of people worldwide.Currently,the main interventions fail to prevent the progression of diabetes or accurately manage the glycemia,however,pluripotent stem cells with self-renewal and multilineage differentiation capacities provide promise for the cure of diabetes mellitus.Understanding the morphogenesis and expression profiles of key lineage-specific genes during pancreatic development provides a basis for the in vitro islet β cell differentiation from stem cells.Stepwise directed differentiation by sequential supplementation of small molecules and growth factors enables the progression of human pluripotent stem cells from the pluripotent stage toward the pancreatic lineage and finally to insulin-producing islet β cells,thus eventually contributing to the cell replacement therapies and understanding the pathogenesis of diabetes mellitus.Nonetheless,there are two key problems in the directed differentiation of pluripotent stem cells into islet βcells: the differentiation system is not efficient and the function of the islet β cells derived from pluripotent stem cells is not mature enough.In order to generate functional and mature islet β cells more efficiently in vitro,this study first optimized the protocol for differentiating islet β cells from pluripotent stem cells.The results showed that by removing insulin from the culture medium of the specific induction stage of pancreatic progenitor cells,the expression of pancreatic lineage genes was increased,which promoted the differentiation efficiency of PDX1-positive pancreatic progenitor cells from 30% to 80%.On this basis,combination of EGF and Nicotinamide significantly increased the expression of pancreatic lineage genes,and the differentiation efficiency of PDX1 and NKX6-1 double-positive pancreatic progenitor cells was increased from 30% to 75%.Foxy5 could promote the glucose-responsive capacity of islet β cells.At the same time,based on the fact that extended pluripotent stem cells with advanced differentiation potential showed higher maturity in the differentiation of liver and cardiomyocyte lineages,this study aimed to establish an efficient differentiation protocol of extended pluripotent stem cells into isletβ cells as well.Inhibition of PI3 K signal could effectively differentiate extended pluripotent stem cells into definitive endoderm cells,and inhibition of TGF-β signal can significantly promote the generation of pancreatic progenitor cells,resulting in the generation of PDX1 and C-Peptide double-positive islet β cells,and the expression of pancreatic lineage genes is similar to that of conventional pluripotent stem cells.Finally,this study evaluated the in vivo function of islet β cells derived from pluripotent stem cells that could release insulin in response to glucose stimulation in vitro.Results showed that islet β cells derived from pluripotent stem cells improved hyperglycemia symptoms in diabetic mice.In this study,based on the directed differentiation system of human pluripotent stem cells to islet β cells,the differentiation efficiency of pancreatic progenitor cells and islet β cells and the functional maturation of islet β cells were improved by means of signal molecules.Moreover,we established a protocol for the directed differentiation of extended pluripotent stem cells into islet β cells,which expressed the key genes of pancreatic lineage,providing an alternative seed cell for the generation of islet β cells.