Generation of pancreas-spheres from human embryonic stem cells

X. Xu, V. Browning, J. Odorico
University of Wisconsin - Madison, Surgery, Madison, WI, USA

Objective: Human embryonic stem cells (hESCs) are a potential source of functional beta cells and serve as an in vitro model for studying human pancreas development. We have studied the roles of specific growth factors and 3D culturing in hESC cell fate determination as cells differentiate through a mesendoderm fate to pancreatic progenitors and, ultimately, to cells expressing insulin.
Methods: Undifferentiated hESCs are treated with various combinations of bFGF, Activin A, and BMP4 (FAB) to induce differentiation to endoderm, followed by growth in suspension culture as EBs. EBs are embedded in Matrigel, plated and grown in serum-free media containing insulin, transferrin, selenium, FGF7, nicotinamide, INGAP, and exendin-4 for an additional 3-4 weeks, during which time pancreatic progenitors (“pancreas-spheres”) are formed. Pancreas-spheres are selected and switched to media containing B27 and nicotinamide for 1-2 additional weeks.
Results: Following early growth factor treatment with FAB, cultures robustly express primitive streak and definitive endoderm-associated genes to a greater degree than in FA, FB, AB, F, or B media. Compared to FAB treated cells, Activin alone treatment yielded similar results but cells did not survive attempts to form EBs. In FAB-treated cells at the end of the EB stage, a number of cells express PDX1, FOXA2, and SOX9, suggesting a commitment to early proliferative foregut endoderm and pancreatic lineage. Nearly all cells within the pancreas-spheres co-express PDX1, FOXA2, HNF1ß, HNF6 and SOX9, and many cells also express CPA, NKX6.1 and PTF1a. By the end of the final stage, there is a marked increase in the number of insulin/C-peptide-positive cells.

Conclusions: We have developed a new differentiation protocol for generation of beta-like cells from human ESCs using chemically-defined culture conditions. This four-step protocol follows in vivo differentiation, as cells move through developmentally defined stages on a path toward commitment to endocrine cells.