Chemically defined conditions for long-term maintenance of pancreatic progenitors derived from human induced pluripotent stem cells

Large numbers of hormone-releasing cells, approximately 109 endocrine cells, are required to treat type I diabetes patients by cell transplantation. The SOX9-positive pancreatic epithelium proliferates extensively during the early stages of pancreatic development. SOX9-positive pancreatic epithelium is thought to be an expandable cell source of beta cells for transplantation therapy. In this study, we attempted to expand pancreatic progenitors (PPs: PDX1(+)/SOX9(+)) derived from four human iPSC lines in three-dimensional (3D) culture using a chemically defined medium and examined the potential of the derived PPs to differentiate into beta-like cells. PPs from four human iPSC lines were maintained and effectively proliferated in a chemically defined medium containing epidermal growth factor and R-spondin-1, CHIR99021, fibroblast growth factor-7, and SB431542. PPs derived from one iPSC line can be expanded by more than 10(4)-fold in chemically defined medium containing two of the fives, epidermal growth factor and R-spondin-1. The expanded PPs were also stable following cryopreservation. After freezing and thawing, the PPs proliferated without a decrease in the rate. PPs obtained after 50 days of culture successfully differentiated into insulin-positive beta-like cells, glucagon-positive alpha-like cells, and somatostatin-positive delta-like cells. The differentiation efficiency of expanded PPs was similar to that of PPs without expansion culture.