ABSTRACT: The study of how human embryonic stem cells (hESCs) differentiate into insulin-producing beta cells has twofold significance: first, it provides an in vitro model system for the study of human pancreatic development, and second, it serves as a platform for the ultimate production of beta cells for transplantation into patients with diabetes. The delineation of growth factor interactions regulating pancreas specification from hESCs in vitro is critical to achieving these goals. In this study, we describe the roles of growth factors bFGF, BMP4 and Activin A in early hESC fate determination. The entire differentiation process is carried out in serum-free chemically-defined media (CDM) and results in reliable and robust induction of pancreatic endoderm cells, marked by PDX1, and cell clusters co-expressing markers characteristic of beta cells, including PDX1 and insulin/C-peptide. Varying the combinations of growth factors, we found that treatment of hESCs with bFGF, Activin A and BMP4 (FAB) together for 3-4days resulted in strong induction of primitive-streak and definitive endoderm-associated genes, including MIXL1, GSC, SOX17 and FOXA2. Early proliferative foregut endoderm and pancreatic lineage cells marked by PDX1, FOXA2 and SOX9 expression are specified in EBs made from FAB-treated hESCs, but not from Activin A alone treated cells. Our results suggest that important tissue interactions occur in EB-based suspension culture that contribute to the complete induction of definitive endoderm and pancreas progenitors. Further differentiation occurs after EBs are embedded in Matrigel and cultured in serum-free media containing insulin, transferrin, selenium, FGF7, nicotinamide, islet neogenesis associated peptide (INGAP) and exendin-4, a long acting GLP-1 agonist. 21-28days after embedding, PDX1 gene expression levels are comparable to those of human islets used for transplantation, and many PDX1(+) clusters are formed. Almost all cells in PDX1(+) clusters co-express FOXA2, HNF1ß, HNF6 and SOX9 proteins, and many cells also express CPA1, NKX6.1 and PTF1a. If cells are then switched to medium containing B27 and nicotinamide for 7-14days, then the number of insulin(+) cells increases markedly. Our study identifies a new chemically defined culture protocol for inducing endoderm- and pancreas-committed cells from hESCs and reveals an interplay between FGF, Activin A and BMP signaling in early hESC fate determination.
