(393c) Directing the Differentiation of Human Stem Cells towards Pancreatic Islet Cells in Static and Scalable Cultures

Human embryonic stem cells (hESCs) can serve as a renewable source of transplantable islet cells for diabetes therapies. A robust method was developed for directing the fate of hESCs towards that of pancreatic islet (PI) cells. Human ESCs were exposed to factors, which drive the development of embryonic pancreas, in a stepwise manner. The differentiating cells transitioned to definitive endoderm (DE), pancreatic endocrine precursor (PEP) cells and PIs. Differentiated hESCs underwent morphological and biochemical changes. The hESC-derived PEP cells are positive for markers including HNF6, PDX1, NGN3, NKX-2.2 and -6.1. Marker expression was assessed by quantitative PCR, immunocytochemistry and flow cytometry. An adenoviral construct encoding the red fluorescence protein gene flanked by the insulin gene promoter was developed to aid in the selection of insulin-producing cells from the mixed population in differentiated cells. Subsequently, we investigated the application of our differentiation protocol in conjunction with the large-scale expansion of hESCs. We have reported the propagation of pluripotent hESCs in a microcarrier bioreactor. To that end, hESCs populated the beads and grew 36-fold in 7-8 days while ~85% of the cells were positive for stem cell markers such as OCT3/4A, SSEA4, NANOG and TRA-1-81. These hESCs on the beads were further directed to DE cells co-expressing SOX17 and FOXA2 and to primitive gut tube expressing HNF1B and HNF4A. Current work focusing on the expansion and differentiation of human induced pluripotent stem cells will also be discussed.