(151b) Regulating Endodermal Cell Lineage through Genetic Manipulation of Developmental Regulatory Gene Circuits

Human Pluripotent stem cells (hPSC’s) are a very useful tool for regenerative medicine allowing for the replacement of damaged tissues by implantation of functional tissue or cells. Of specific interest is the ability to properly differentiate liver, pancreas, and lung cells which all derive from endoderm using hPSC’s. One issue that arises is a result from the heterogeneity in the gene expression of hPSC-derived endoderm which can severely compromise the downstream fate of differentiation. Improper differentiation protocols of embryonic stem cells have also been shown to produce unwanted tissue during transplants including skeletal muscle, bone cell subpopulations, gut epithelial-like structures and columnar cells (Kubo et al. Development and Disease 2004). Hepatocyte Nuclear Factor 3B (Foxa2) is known as a Pioneer Transcription Factor (TF) due to its ability to bind albumin enhancers and open normally inaccessible heterochromatin, effectively priming such for a cascade of regulatory elements to further differentiation through necessary gene activation. Foxa2 has been shown to be required for developmental processes such as transitioning to air breathing at birth (Huajing Wan, Jeffery A. Whitsett 2004.), differentiation of pancreatic α-cells (Cathrine Lee, Klause H. Kaestner 2005), and control of differentiation of goblet and enteroendocrine cells in mice (Diana Z, Klause Kaestner 2009). In combination with Foxa2, Hepatocyte Nuclear Factor 3A (Foxa1) has been shown to equally vital. To better understand how Foxa2 and Foxa1 regulates developmental master regulatory gene circuits (DRGC) for induction, maintenance, and differentiation of endoderm towards liver, we provide a model using human hepatocellular carcinoma (HepG2) cells as well as Human Stem Cells (BXS). HepG2 being a well-studied cell line is also known to express alpha-fetoprotein (AFP) and albumin (Alb). RNA interference is used to transiently knockdown both Foxa1/2 with siRNA and a permeant knockdown cell line was created with short hairpin RNA (shRNA). Using qPCR, it was observed that the transient approach resulted in downregulation of AFP, Alb, and Hhex, the double knockdown of foxa1/2 also resulted in upregulation of Sox7, CXCR4, NANOG, and Pax6. shRNA knockdown in HepG2 resulted in Foxa1/2 ^-/- stable cell lines with permanent Foxa1/Foxa2 knockdown, confirmed by qRT-PCR and Western Blot analysis. These cell lines demonstrated downregulation of Alb, Hhex, HNF4A, HNF1B, PDX1, and SOX9. These data suggest that the Foxa1/2 regulation of albumin enhancer and HNF4A present in liver development may also be modeled in HepG2 cells, suggesting the DRGC’s may be similar when compared to hepatic endoderm cells as well as suggesting possible de-differentiation back to a definitive endoderm like state. Futher analysis of lineage and metabolic changes are explored though RNA-seq. Preliminary data of double knockdown of Foxa1/2 in PSC derived gut tube progenitors reduces the development of liver like morphology. Future work will include analysis of combined effects of Foxa1/2 knockdown and Sox17 overexpression as Sox17 has been shown to be vital for endoderm induction as this TF regulates and maintains endoderm while suppressing differentiation into liver (Pfister et al., Int. J. Dev. Biol. 2010). Future work will also include use of dox inducible Foxa1/2 ^-/- iPSC cell lines to study the effects of time dependent knockdown on differentiation of hPSC-derived endoderm.