title: Analysis of alternative signaling pathways of endoderm induction of human embryonic stem cells identifies context specific differences.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Chemical and Petroleum Engineering, University of Pittsburgh, 1249 Benedum Hall, 3700 O'Hara Street, Pittsburgh, PA 15261, USA.
authors.forename: Shibin
authors.initials: S
authors.lastname: Mathew
authors.forename: Maria
authors.initials: M
authors.lastname: Jaramillo
authors.forename: Xinan
authors.initials: X
authors.lastname: Zhang
authors.forename: Li Ang
authors.initials: LA
authors.lastname: Zhang
authors.forename: Alejandro
authors.initials: A
authors.lastname: Soto-Gutiérrez
authors.forename: Ipsita
authors.initials: I
authors.lastname: Banerjee
mesh_terms: D055415:Bone Morphogenetic Protein 4 / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D019070:Cell Lineage / Q000187:drug effects; D016000:Cluster Analysis; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D004707:Endoderm / Q000166:cytology* / Q000187:drug effects; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D006801:Humans; D008954:Models, Biological; D010179:Pancreas / Q000166:cytology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism; D060509:Wnt3A Protein / Q000494:pharmacology
chemical_list: D055415:Bone Morphogenetic Protein 4; D047428:Protein Kinase Inhibitors; D014157:Transcription Factors; D060509:Wnt3A Protein; D016222:Fibroblast Growth Factor 2