Rational engineering of self-renewing, endoderm progenitor cell lines

The Parashurama Research Group

The endoderm, labeled above at different stages of development, is the least understood germ layer and gives rise to internal organs like the liver, pancreas, and lung.

Overview

The endoderm represents one of three germ layers present in the early embryo that eventually gives rise to all major tissues in the body. hPSC-derived endoderm is of interest because it gives rise to the epithelial portions of clinically significant tissues/ organs like the liver, pancreas, lung, intestine, thyroid gland, and thymus. The endoderm is the least understood of the three germ layers, and since it has never been isolated from human embryos, and it remains poorly understood. Endoderm was only isolated from mouse ESC in the mid- and late 2000's by others, as well as our lab. These approaches are identical to what is used to isolate endoderm from hPSC, and involves using low serum medium conditions and Activin (Nodal), a TGFb superfamily member. In lower organisms, like flies (Drosophilla) and worms (Xenopus), as well an in mouse, Activin (Nodal) functions by activating developmental, regulatory, master transcription factors (TFs) like Foxa2, Sox17, and GATA4, that interconnect to form a genetic regulatory network (GRN). Unfortunately, each endoderm protocol results in endoderm that is different than endoderm from another protocol, in terms of expression levels of these master TF. Further, mesendoderm TF like Brachyury, Goosecoid, and Eomes are still expressed within endoderm at final stages, even using the same hPSC lines. Not surprisingly, single cell RNA seq studies demonstrate that endoderm is heterogeneous at the single cell level. This may play a role in the fact that fully mature hepatocytes and pancreatic islets have yet to be fully generated.

The goal of this project is to optimize activin concentration, soluble factors that govern lineage formation, like Wnt, BMP, FGF, perturb GRN to develop a homogenous endoderm population. Furthermore, we will investigate the role of endodermal TF in controlling endoderm formation by priming downstream genes within endoderm, and will analyze how epigenetic state controls endoderm formation. Our overall goal is to combine these approaches to develop a reversible endoderm cell line that can be expanded without differentiation, and can be conditionally activate towards differentiation. We feel this will enable production of uniform endoderm with improved differentiation towards liver and pancreas.

Resulting Publications

Parashurama N, Nahmias Y, Cho CH, Berthiaume F, Tilles AW, Yarmush ML. Activin alters kinetics of (hepatic) endoderm induction in collagen gel cultures of embryonic stem cells. Stem Cells. 2008 Feb; 26(2): 474-84. Citations: 25
Cho CH, Parashurama N, Park EY, Suganuma K, Nahmias Y, Park J, Tilles AW, Berthiaume F, Yarmush ML. Homogenous Differentiation of hepatocyte-like cells from embryonic stem cells: Applications for the treatment of liver failure. FASEB. 2008; March; 22(3): 898-909. Citations: 86