|Authors||Xu X, Kahan B, Forgianni A, Jing P, Jacobson L, Browning V, Treff N, Odorico J|
|Journal||Cloning Stem Cells Volume: 8 Issue: 2 Pages: 96-107|
Human embryonic stem cells (HESCs) are a potential source of insulin-producing tissue for transplantation. Recent studies have begun to define factors that promote definitive endoderm formation from HESCs, but conditions permitting complete islet specification in vitro have not been described. Here, we study spontaneous differentiation of HESCs to definitive endoderm and pancreatic progenitor cells, and begin to determine which aspects of the protocol are required for this cell fate commitment. HESCs were differentiated in culture for up to 10 weeks, including an embryoid body (EB) formation step. Modifications to the protocol included elimination of the EB phase, varying initial cell cluster size when forming EBs, and addition of mesoderm-derived cells to EBs. Differentiated cells were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. HESCs are capable of spontaneous differentiation to cells expressing the definitive endoderm and pancreatic progenitor markers Foxa2, Sox17, and Pdx1, and ultimately, some cells express islet endocrine hormones. This differentiation occurs to a much greater extent when an EB formation step is included. Increased expression of endoderm markers during and after EB formation also correlated strongly with the size of cell clusters used to start EBs, as well as the addition of mesoderm- derived embryonic cells. This study demonstrates that a subset of differentiated HESC progeny adopt an endoderm fate and exhibit the capacity for further pancreatic lineage specification in vitro. Basal conditions were established for examining factors that can commit HESC-derived endoderm cells to specific pancreatic lineages.