Methods for generating hepatocytes and cholangiocytes from pluripotent stem cells

A method of producing hepatocyte and/or cholangiocyte lineage cells from pluripotent embryonic stem cells (PSCs) or induced pluripotent stem cells (iPSCs), the method comprising the following series of steps:

• (a) generating an induced endodermal cell population by either:
• i. culturing the PSCs or iPSCs in a monolayer in a medium comprising a nodal agonist and a Wnt/beta-catenin agonist for 3 days followed by 2 days in a medium comprising a Fibroblast Growth Factor (FGF) agonist and a nodal agonist;
• or
• ii. culturing the PSCs or iPSCs in medium comprising Bone Morphogenetic Protein 4 (BMP4) agonist for 12 to 36 hours to promote formation embryoid bodies (EBs) followed by 5 days in a medium comprising a FGF agonist, a nodal agonist, a Wnt/beta-catenin agonist, and a BMP4 agonist;
• (b) culturing the induced endodermal cell population in a medium comprising a nodal agonist for 1 to 4 days to provide an extended nodal agonist-treated induced endodermal cell population;
• (c) specifying the extended nodal agonist treated induced endodermal cell population to obtain a cell population comprising hepatoblasts by contacting the extended nodal agonist treated induced endodermal cell population with specification media comprising:
• i. a FGF agonist;
• and
• ii. a BMP4 agonist;
• and
• (d) inducing maturation, promoting further lineage specification, and/or inducing expansion of the hepatoblast population, comprising:
• i. dissociating the cell population comprising hepatoblasts;
• ii. generating aggregates of the dissociated cell population comprising hepatoblasts;
• and
• iii. culturing the aggregates in a maturation medium for 1 to 40 days to produce hepatocyte and/or cholangiocyte lineage cells, wherein the maturation medium comprises a CAMP signaling pathway activator and at least one of:
• hepatocyte growth factor (HGF);
• dexamethasone (DEX);
• and Oncostatin M (OSM),
• wherein the nodal agonist is ActA, the Wnt/Beta-catenin agonist is Wnt3a, XAV939, CHIR 99021 or 6-bromo-Indirubin-3′-Oxime, the Fibroblast Growth Factor agonist is FGF-2, FGF-4 or FGF-10, the BMP4 agonist is BMP4, BMP2 or BMP7, and the CAMP signaling pathway activator is 8-bromoadenosine-3′5′-cyclic monophosphate.