US 12,129,490 B2
Methods for generating hepatocytes and cholangiocytes from pluripotent stem cells
Gordon Keller, Toronto (CA); Shinichiro Ogawa, Toronto (CA); Anand Ghanekar, Toronto (CA); Christine Bear, Toronto (CA); Binita M. Kamath, Toronto (CA); Mina Ogawa, Toronto (CA); and James Surapisitchat, Toronto (CA)
Assigned to UNIVERSITY HEALTH NETWORK, Toronto (CA); and THE HOSPITAL FOR SICK CHILDREN, Toronto (CA)
Filed by UNIVERSITY HEALTH NETWORK, Toronto (CA); and THE HOSPITAL FOR SICK CHILDREN, Toronto (CA)
Filed on Aug. 22, 2018, as Appl. No. 16/109,202.
Prior Publication US 2020/0157494 A1, May 21, 2020
Int. Cl. C12N 5/071 (2010.01); A61K 35/407 (2015.01)
CPC C12N 5/067 (2013.01) [C12N 5/0679 (2013.01); A61K 35/407 (2013.01); C12N 2501/01 (2013.01); C12N 2501/115 (2013.01); C12N 2501/119 (2013.01); C12N 2501/12 (2013.01); C12N 2501/155 (2013.01); C12N 2501/16 (2013.01); C12N 2501/237 (2013.01); C12N 2501/415 (2013.01); C12N 2501/42 (2013.01); C12N 2501/724 (2013.01); C12N 2501/727 (2013.01); C12N 2501/999 (2013.01); C12N 2502/00 (2013.01); C12N 2502/14 (2013.01); C12N 2503/02 (2013.01); C12N 2506/02 (2013.01); C12N 2506/45 (2013.01)] 22 Claims
 
1. 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.