Analysis of alternative signaling pathways of endoderm induction of human embryonic stem cells identifies context specific differences

hESCs were induced towards endoderm using activin A and 4 different growth factors (FGF2 (F), BMP4 (B), PI3KI (P), and WNT3A (W)) and their combinations thereof, resulting in 15 total experimental conditions. At the end of differentiation each condition was analyzed by qRT-PCR for 12 relevant endoderm related transcription factors (TFs). As a first approach, we used hierarchical clustering to identify which growth factor combinations favor up-regulation of different genes. In the next step we identified sets of co-regulated transcription factors using a biclustering algorithm. The high variability of experimental data was addressed by integrating the biclustering formulation with bootstrap re-sampling to identify robust networks of co-regulated transcription factors. Our results show that the transition from early to late endoderm is favored by FGF2 as well as WNT3A treatments under high activin. However, induction of late endoderm markers is relatively favored by WNT3A under high activin.Use of FGF2, WNT3A or PI3K inhibition with high activin A may serve well in definitive endoderm induction followed by WNT3A specific signaling to direct the definitive endoderm into late endodermal lineages. Other combinations, though still feasible for endoderm induction, appear less promising for pancreatic endoderm specification in our experiments.Embryonic stem cells have been shown to have tremendous impact in the field of regenerative medicine because of its potential to differentiate to multiple cell types of interest. Efficient harvesting of this potential requires careful development of protocols to evolve the cells through specific signaling pathways which will induce desired lineages and properties in the differentiated phenotypes. Our primary interest lies in differentiation of human embryonic stem cells (hESCs) to insulin producing β-cells of the pancreas as a cellular transplantation strategy for diabetes mellitus. The first and perhaps the most important step in diff