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Open Access Research article

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

Shibin Mathew1, Maria Jaramillo2, Xinan Zhang3, Li Ang Zhang1, Alejandro Soto-Gutiérrez4567 and Ipsita Banerjee12*

Author affiliations

1 Department of Chemical and Petroleum Engineering, University of Pittsburgh, 1249 Benedum Hall, 3700 O’Hara Street, Pittsburgh, PA 15261, USA

2 Department of Bioengineering, University of Pittsburgh, 360B CNBIO, 300 Technology Drive, Pittsburgh, PA 15219, USA

3 School of Mathematics and Statistics, Central China Normal University, Wuhan, China

4 Department of Pathology, University of Pittsburgh, Pittsburgh, PA, USA

5 McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, USA

6 Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, USA

7 Center for Innovative Regenerative Therapies, Department of Surgery, Transplantation Section of Children's Hospital of Pittsburgh, Pittsburgh, PA, USA

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Citation and License

BMC Systems Biology 2012, 6:154  doi:10.1186/1752-0509-6-154

Published: 15 December 2012



Lineage specific differentiation of human embryonic stem cells (hESCs) is largely mediated by specific growth factors and extracellular matrix molecules. Growth factors initiate a cascade of signals which control gene transcription and cell fate specification. There is a lot of interest in inducing hESCs to an endoderm fate which serves as a pathway towards more functional cell types like the pancreatic cells. Research over the past decade has established several robust pathways for deriving endoderm from hESCs, with the capability of further maturation. However, in our experience, the functional maturity of these endoderm derivatives, specifically to pancreatic lineage, largely depends on specific pathway of endoderm induction. Hence it will be of interest to understand the underlying mechanism mediating such induction and how it is translated to further maturation. In this work we analyze the regulatory interactions mediating different pathways of endoderm induction by identifying co-regulated transcription factors.


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.

Human embryonic stem cells; Endoderm; Hierarchical clustering; Biclustering; Bootstrap