The generation of insulin-producing β-cells from human pluripotent stem cells is dependent on efficient endoderm induction and appropriate patterning and specification of this germ layer to a pancreatic fate. In this study, we elucidated the temporal requirements for TGFβ family members and canonical WNT signaling at these developmental stages and show that the duration of nodal/activin A signaling plays a pivotal role in establishing an appropriate definitive endoderm population for specification to the pancreatic lineage. WNT signaling was found to induce a posterior endoderm fate and at optimal concentrations enhanced the development of pancreatic lineage cells. Inhibition of the BMP signaling pathway at specific stages was essential for the generation of insulin-expressing cells and the extent of BMP inhibition required varied widely among the cell lines tested. Optimal stage-specific manipulation of these pathways resulted in a striking 250-fold increase in the levels of insulin expression and yielded populations containing up to 25% C-peptide+ cells.
Copyright: © 2012 M. Cristina Nostro, Farida Sarangi, Shinichiro Ogawa, Audrey Holtzinger, Barbara Corneo, Xueling Li, Suzanne J. Micallef, In-Hyun Park, Christina Basford, Michael B. Wheeler, George Q. Daley, Andrew G. Elefanty, Edouard G. Stanley, and Gordon Keller.