(190f) Induction of Definitive Endoderm from Human Pluripotent Stem (hPS) Cells

Saber Meamardoost¹, Natesh Parashurama^1,2,3

Saber Meamardoost: sabermea@buffalo.edu

Natesh Parashurama: nateshp@buffalo.edu

¹ Department of Chemical and Biological Engineering, Furnas Hall, University at Buffalo, State University of New York at Buffalo, Buffalo NY 14260

² Department of Biomedical Engineering, Bonner Hall, University at Buffalo, State University of New York at Buffalo, Buffalo NY 14260

³ Department of Medicine, Clinical Translational Research Center (CTRC), 875 Ellicott St, Buffalo, NY 14214

Currently, we are unable to produce functional hepatocytes, and the precursors to functional liver cells remain poorly understood. Endoderm, which gives rise to liver, pancreas, and lung, is the most poorly understood germ layer, and has never been isolated from humans. Also, human pluripotent stem cell (hPSC)-derived endoderm, is heterogeneous, and further investigation into the factors that regulate its differentiation are essential. Genetic in vivo studies demonstrate that developmental master regulatory gene circuits (DRGC) control endoderm formation, and since relatively large changes in gene expression occur during germ layer formation, the DRGC coordinate large changes in gene expression. In order to achieve a high yield of hepatocytes, it is necessary to obtain pure endoderm lineage from embryonic stem (ES) cells. Since DRGC control downstream transcription factors (TFs) and each other levels, it is critical to define and understand DRGC to better understand endoderm formation, and eventually hepatocyte formation; then, the produced pure cells may be used for different research and clinical applications. Hence, it is of highly importance to understand how different soluble factors and signaling pathways activate or inhibit particular transcription factors and how that affects the expression of downstream genes to govern endoderm formation during early stages of development.

The primary objective of our research is to understand how Foxa1 and Foxa2 (Foxa1/2), regulate DRGC composed of master regulatory transcription factors and can eventually be used to control hPSC-derived endoderm induction, maintenance, and differentiation. Foxa1/2 together with other endodermal transcription factors, 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 Foxa1/2 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.

In order to better understand the relationship between major signaling pathways, soluble factors and main transcription factors that govern endoderm formation, we apply multiple differentiation protocols for endoderm induction. We use UCSF4 human embryonic stem cell line, various growth factors and small molecules to activate or inhibit specific signaling pathways such as Nodal, Wit, BMP and FGF during endoderm differentiation. Our optimized protocol requires less cytokines than previously reported methods and we use serum and feeder free system in low oxygen which resembles in vivo endoderm development condition. Interestingly, we demonstrate that how small changes during hPS cells differentiation towards definitive endoderm results in a different configuration of the DRGC and various expression of pluripotency, mesendoderm and endodermal markers in produced endoderm. We show that activating Nodal pathway is essential but not sufficient for proper endoderm induction because this results in formation of a mixture of various cell types, including undifferentiated cells, mesendoderm and definitive endoderm. Activating Wnt signaling pathway facilitates the differentiation of pluripotent population. After mesendoderm formation, however, Wnt signaling favors mesoderm formation and thus its presence reduces the purity of endoderm. To overcome this contradictory roles of Wnt pathway before and after mesendoderm formation, we stop Wnt activation which reduces mesoderm formation significantly. Further, in order to completely inhibit Wnt pathway we use small molecules that target endogenous Wnt to direct differentiation towards definitive endoderm. In addition, BMP pathway plays a similar role as Wnt and inhibition of both Wnt and BMP pathways results in higher expression of endodermal markers and lower expression of mesendoderm and mesodermal markers. Although, it is yet unknown what configuration of the DRGC is ideal for endoderm but further in vivo analysis can reveal the effect of the transcription factors network on the quality of endoderm and how it undergoes patterning in the next developmental stages.

Furthermore, it has been shown that keratinocyte growth factor (KGF or FGF7), is capable of opening chromatin regions at developmental enhancers so that either Foxa1/2 or PDX1 can bind and in the presence of proper developmental cues they can regulate hepatic or pancreatic differentiation, respectively (Wang et al., Cell Stem Cell 2015). Therefore, definitive endoderm requires epigenetic priming of developmental enhancers, pioneer transcription factors and proper developmental signals in order to differentiate further towards its downstream derivatives. However, it is still unclear how endodermal state and fate change in the absence of any of these factors. We seek to differentiate PSCs towards definitive endoderm, and then by manipulating the DRGC we maintain endoderm and lock the cells in that state. This provides a unique system that results in a better understanding of transcriptional network involved in endoderm formation, maintenance and differentiation and also enables us to reduce heterogeneity of endoderm which is vital for clinical and translational applications. Consistent with in vivo process, our data indicates that endoderm is a transient lineage and its state cannot be maintained unless endoderm DRGC is manipulated. We show that, how genetic manipulation of endoderm can enhance its maintenance and differentiation towards endoderm derivatives.