(314c) Analyzing Pathways of Endoderm Induction of hESC by Identification of Co-Regulated Genes

Introduction: Lineage specific differentiation of hESC 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 lot of interest in inducing hESCs to an endoderm fate which serves as a pathway towards more important cell types like the pancreatic and hepatic cells. Research over past decade has established several robust pathways for deriving endoderm from ESCs, with the capability of further maturation into functional cell types. However in our experience endoderm derived through different pathways possess varying maturation capability, specifically to pancreatic lineage. 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.

Methods: hESCs are induced towards endoderm using 4 different growth factors (FGF2 (F), BMP4 (B), PI3KI (P), Wnt3a (W)) and their combinations thereof, resulting in 16 total experimental conditions. At the end of differentiation each condition is analyzed by qRT-PCR for 12 relevant endoderm related transcription factors. The experimental data for the mathematical analysis consists of this 12 x16 data matrix. As a first approach, we used principal component analysis (PCA) to identify which growth factor combinations favor up-regulation of different genes. In the next step we identify sets of co-regulated transcription factors using biclustering algorithm. The biclustering problem is formulated as an optimization problem and solved using evolutionary algorithm. The high variability of experimental data is addressed by integrating the biclustering formulation with bootstrap resampling to identify robust networks of co-regulated transcription factors.

Results and Conclusions: PCA results show that FGF2 and PI3KI favor upregulation of definitive endoderm markers CXCR4 and CER and conditions involving BMP4 and Wnt3a favor the upregulation of other definitive endoderm markers FoxA2, HNF4a and HNF1b. Further, biclustering results show that definitive endoderm induction of hESCs is marked by two robust gene sets, namely PTF1a, GATA4, HNF1b and HNF4a which are co-regulated under conditions of F+P, B+W, F+B+P, F+B+P+W and CER and HNF6 which are co-regulated under conditions of F, W+P, F+W, B+W+P. The current analysis not only captures the regulatory interactions during endoderm differentiation, it also elucidates the similarity between different growth factor combinations. Such information will be instrumental in further optimization of the process of phenotypic maturation.