(138b) Reprogramming of Liver Cells By Understanding and Re-Engineering Developmental Master Regulatory Gene Circuits (DRGC)

Heterogeneity in gene expression of human pluripotent stem cell (hPSC)-derived endoderm severely limits downstream applications of differentiated liver, pancreas, and lung cells which all arise from endoderm. Foxa2, known as a master regulatory or pioneer transcription factor (TF), may be the culprit as the TF binds to albumin enhancer and opens normally inaccessible heterochromatin and allowing for a cascade of regulatory elements to activate genes necessary for further differentiation. Mouse genetic studies with Foxa1/Foxa2 double knockdown (Foxa1/2^-/-) in hepatic endoderm resulted in the lack of liver bud formation and the absence of further hepatic differentiation markers like AFP and albumin (Lee et al., Nature 2005). Similar effects of the knockdown are demonstrated in pancreatic endoderm leading to insufficient pancreatic growth and differentiation. Along with Foxa2, Sox17 is vital for endoderm induction as the TF regulates and maintains endoderm and suppresses differentiation into liver (Pfister et al., Int. J. Dev. Biol. 2010) and pancreas (Spence et al., Dev. Cell. 2009). To understand how Foxa2 and Sox17 regulates developmental regulatory gene circuits (DRGC) for the induction, maintenance, and further differentiation of endoderm towards liver, human hepatocellular carcinoma (HepG2) cells are used as a model for hepatic endoderm. The well-studied cell line is known to express both alphafetoprotein (AFP) and albumin (Alb). RNA interference is used to knockdown Foxa1/2 in a transient approach with siRNA and a permanent knockdown with shRNA. Surprisingly, the transient approach resulted in the downregulation of AFP and Alb, Hhex, and TTR gene expression and an upregulation of Pdx1 and Pax6 (pancreatic markers) and Cdx2 (intestinal marker) gene expression. The shRNA knockdown in HepG2 resulted in Foxa1/2^-/- stable cell lines with permanent Foxa1/2 knockdown, as judged by qRT-PCR and Western Blot. These cell lines demonstrated downregulation of Alb but an upregulation of AFP and Hhex, suggesting compensation for these latter markers, during long term shRNA knockdown. Collectively, the data suggests that mechanisms present during liver development (Foxa2 regulation of the albumin enhancer) may be present in HepG2 cells, suggesting they may share molecular regulation or DRGC similar to hepatic endoderm cells. To understand the role of Sox17 in Foxa1/2^-/- HepG2 cells, Sox17 was overexpressed. The preliminary data indicates in controls Pax6 was repressed, but not Pdx or Sox9, while in Foxa1/2^-/- cells, Pax6, Pdx, and Sox9 were repressed. The data suggests liver and pancreatic genes can be manipulated by the knockdown of Foxa1/2 and the overexpression of Sox17. Future work will include a more detailed characterization of the DRGC during combined effects of Foxa1/2 knockdown and Sox17 overexpression culminating in improved understanding of DRGC, the development of a physical/mathematical model of early endoderm and liver development, and a deeper understanding of hPSC-derived endoderm.