(193ao) Controlling Endodermal Cell State by Understanding and Re-engineering Developmental Master Regulatory Gene Circuits

Human pluripotent stem cell-derived (hPSC)-derived endoderm, which gives rise to liver, pancreas, and lung, is heterogeneous, and the further investigation into the factors that regulate its differentiation are warranted. The overall objective of our research is to understand how Foxa2 regulates developmental regulatory gene circuits (DRGC) composed of master regulatory transcription factors (TF) and can eventually be used to control hPSC-derived endoderm induction, maintenance, and differentiation. Foxa1 and Foxa2 (Foxa1/2), together with other endodermal transcription factors (TF), compose the poorly understood DRGC. Foxa2 was identified as the first known pioneer TF, which binds to silent differentiation genes, like Albumin, to prime them for activation. The binding of Foxa2 appears to open normally inaccessible heterochromatin and provides access for other TF of DRGC, like Hnf1, HNF4a, and HNF6 to bind. This enables thousands of genes to be activated, within endoderm derivatives, in a relatively short amount of time during development. Consistent with this, mouse genetic studies of Foxa1/Foxa2 double knockdown (Foxa1/2-/-) within hepatic endoderm, demonstrates a complete absence of the liver bud and a failure to initiate the hepatic differentiation (Lee et al., Nature 2005). Similarly, Foxa1/2-/- phenotype in pancreatic endoderm, severely blocks pancreatic growth and differentiation. The developmental role of Foxa1/2 is in contrast to its role in mature liver tissue, in which Foxa1/2 knockdown in adult hepatocytes has no effect on transcription factors or liver-specific genes. To understand how Foxa1/2 contribute to the endodermal DRGC and orchestrate endoderm/hepatic differentiation, we first analyzed the role of Foxa1/2 in human hepatocellular carcinoma (HepG2) cells. We hypothesized that Foxa1/2 regulates hepatic endoderm transcription factors expressed within the HepG2 cell, thereby serving as a model for endoderm/hepatic endoderm. We used an RNA interference approach for the Foxa1/2 (-/-) phenotype followed by qRT-PCR/western blot to measure mRNA/protein level in components of the DRGC and other master transcription factors involved in differentiation. HepG2 cells were plated in multiple wells of a 24 well plate at 50,000 cells/well density and transfected with 50 nM of siFoxa1 (4 pools), siFoxa2 (4 pools) and both siFoxa1 and siFoxa2 molecules. qRT-PCR was performed after 48 hours of transfection and all the data was normalized to control sample with siPPIB as negative control. Our results indicate that the Foxa1/2(-/-) phenotype, but not the Foxa1(-) or the Foxa2(-) phenotype, is characterized by downregulation of key hepatic endoderm markers like AFP (alphafetoprotein), Alb (albumin) and Hhex (Hematopoietically expressed homeobox) (n=2). We observed heterogeneity of the effects of on Foxa1/2(-/-) phenotype gene expression of DRGC components like Gsc (mesendoderm TF), Hnf4a and Hnf6, as well as the endoderm cell surface marker Cxcr4 (n=2) . Surprisingly, we observed the upregulation of pancreatic markers such as Pdx1 and Pax6 (n=2). We confirmed Foxa1/2(-/-) phenotype by immunofluorescence, which demonstrated a ~80% reduction in Foxa2 levels, and also demonstrated that Foxa1/2(-/-) are intact morphologically and viable. Finally, we performed a western blot of Alb, demonstrating an ~80% knockdown in the Foxa1/2(-/-) phenotype. Overall, our data suggests the Foxa1/2(-/-) phenotype in HepG2 cells result in downregulation of liver differentiation markers, upregulation of pancreatic markers, and heterogeneous changes in DRGC components. Future work includes improving knockdown, testing the Foxa1/2(-/-)
phenotype on hPSC-derived endoderm, and performing analysis of chromatin remodeling to
assess epigenetic effects.