Methods for generating hepatocytes and cholangiocytes from pluripotent stem cells

Patent metadata

Field	Value
Publication number	US-12129490-B2
Application number	US-201816109202-A
Country	US
Kind code	B2
Filing date	Aug 22, 2018
Priority date	Feb 18, 2013
Publication date	Oct 29, 2024
Grant date	Oct 29, 2024

Abstract

Methods for producing hepatocyte and/or cholangiocyte lineage cells from pluripotent stem cells, the method comprising (a) specifying the extended nodal agonist treated induced endodermal cell population to obtain a cell population comprising hepatocyte and/or cholangiocyte progenitors by contacting the extended nodal agonist treated induced endodermal cell population with specification media comprising a FGF agonist and a BMP4 agonist and/or active conjugates and/or fragments thereof; (b) inducing maturation, and optionally further lineage specification and/or expansion of the hepatocyte and/or cholangiocyte progenitors of the cell population to obtain a population comprising hepatocyte lineage cells such as hepatoblasts, hepatocytes and/or cholangiocytes, the inducing maturation step comprising generating aggregates of the cell population. Optionally, the method also comprises activating the cAMP pathway within the aggregates and forming co-aggregates.

First claim

The invention claimed is:

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.

2. The method of claim 1 , wherein at least 90% of the induced endodermal cell population is CXCR4 + and cKIT + or CXCR4 + and EPCAM + , and at least 90% of the induced endodermal cell population expresses SOX17 and at least 80% of the induced endodermal cell population expresses FOXA2.

3. The method of claim 1 , wherein the Wnt/Beta-catenin agonist is Wnt3a and/or a GSK-3 selective inhibitor selected from CHIR-99021 or 6-bromo-Indirubin-3′-Oxime.

4. The method of claim 1 , wherein the maturation medium further comprises a Wnt antagonist or a Mek/Erk antagonist, or both, wherein the Wnt antagonist is XAV939 IWP2, DKK1, IWR-1 endo or Wnt Inhibitory Factor-1 and wherein the Mek/Erk antagonist is PD0325901, U0126, PD098059.

5. The method of claim 1 , wherein the maturation medium further comprises a Notch antagonist to promote hepatocyte lineage specification, wherein the Notch antagonist is gamma-secretase inhibitor (GSI) L695.458, DAPT, LY411575 or L-685458.

6. The method of claim 5 , wherein the maturation medium further comprises a Wnt agonist or a TGFβ antagonist, or both, for 6 to 12 days, 8 to 10 days, or 9 days after cell aggregation, wherein the Wnt agonist is GSK3 selective inhibitor CHIR-99021, and wherein the TGFβ antagonist is SB431542, A83-01 or SB525334.

7. The method of claim 1 , wherein the aggregates are cultured in maturation medium comprising EGF, TGFβ1 and HGF for 1 to 10 days after generating cell aggregates, and then Dex for about 1 to 10 days, and wherein the maturation medium further comprises a Notch agonist for 5 days to 90 days, wherein the Notch agonist is added within 1 to 10 days of the generating aggregates step, wherein the Notch agonist is a Notch signaling donor cell selected from OP-9 cell, OP9delta cell, and/or OP9 Jagged-1 cell.

8. The method of claim 5 , wherein the hepatocytes produced are functional hepatocytes.

9. The method of claim 8 , wherein the functional hepatocytes comprise increased expression and/or activity of:
(a) at least one gene or protein selected from the group consisting of ALB, CPS1, G6P, TDO, CYP7A1, CYP3A7, CYP1A2, CYP3A4, CYP2B6, CYP2C9, CYP2D6, NAT2 and UGT1A1;
(b) at least one protein selected from the group consisting of ALB, CPS1, G6P, TDO, CYP7A1, CYP3A7, CYP1A2, CYP3A4, CYP2B6, CYP2C9, CYP2D6, NAT2 and UGT1A1; or
(c) both (a) and (b),

compared to a hepatoblast population.

10. The method of claim 8 , wherein at least 40, 50, 60, 70, 80 or 90% of the functional hepatocytes are ASGPR-1 + .

11. The method of claim 2 , wherein the cholangiocyte fate is specified by including a Notch agonist in the maturation medium, wherein the Notch agonist is a Notch signaling donor cell selected from OP-9 cell, OP9delta cell, and/or OP9 Jagged-1 cell.

12. The method of claim 11 , wherein the hepatoblasts are cultured with the Notch agonist for at least 5, 8, 9, 10, 11, 12, 13, or to about 14 days, to induce further lineage specification, and/or maturation, or both, of the hepatocytes into cholangiocytes or functional cholangiocytes.

13. The method of claim 12 , wherein the functional cholangiocytes comprise increased expression of:
(a) at least 1 gene selected from SOX9, CK19, and CFTR (Cystic fibrosis transmembrane conductance regulator);
(b) at least 1 protein selected from SOX9, CK19, and CFTR; or
(c) both (a) and (b),

compared to a hepatoblast population not treated with a Notch agonist.

14. A method for generating a functional hepatocyte and/or cholangiocyte comprising:
(a) producing a cell population comprising hepatoblasts, according to the method of claim 1 ; and
(b) co-culturing the cell population comprising hepatoblasts for at least 4, 6, 8, 10, 12, 15, 20, 30, 40, 50, 60, or 90 days with:
(i) a culture of aggregates of CD34+ endothelial cells to form a co-culture of chimeric aggregates comprising functional hepatocytes; or
(ii) a culture of Notch signaling donor cells to form a co-culture of chimeric aggregates, wherein the aggregates are cultured with (EGF, TGFβ1, EGF and HGF), or (EGF, TGFβ1 and HGF) to produce a population of cells comprising functional cholangiocytes.

15. The method of claim 1 wherein the aggregates are embedded in a gel/matrix and 3D cultured.

16. The method of claim 8 , further comprising administering the functional hepatocytes to a subject with a chronic liver disease.

17. The method of claim 16 , wherein the chronic liver disease is cystic fibrosis liver syndrome, Alagille Syndrome, primary sclerosing cholangitis, or biliary atresia.

18. The method of claim 13 , further comprising administering the functional hepatocytes to a subject with a chronic liver disease.

19. The method of claim 18 , wherein the chronic liver disease is cystic fibrosis liver syndrome, Alagille Syndrome, primary sclerosing cholangitis, or biliary atresia.

20. The method of claim 6 , wherein the TGFβ antagonist is SB431542.

21. The method of claim 4 , wherein the Wnt antagonist is XAV939, and the Mek/Erk antagonist is PD0325901.

22. The method of claim 7 , wherein the Notch agonist is a Notch signaling donor cell, comprising a Notch ligand bound to a cell surface, a plastic surface, an extracellular matrix (ECM) or the surface of a bead, wherein the Notch ligand is delta, Jagged-1, Jagged1 peptide, o

Assignees

• Univ Health Network
• Hospital For Sick Children

Inventors

• Keller Gordon
• Ogawa Shinichiro
• Ghanekar Anand
• Bear Christine
• Kamath Binita M
• Ogawa Mina
• Surapisitchat James

Classifications

• C12N2503/02

  Drug screening · CPC title

• C12N2502/14

  hepatocytes · CPC title

• C12N2501/999

  Small molecules not provided for elsewhere · CPC title

• C12N2501/727

  Kinases (EC 2.7.) · CPC title

• C12N2501/42

  Notch; Delta; Jagged; Serrate · CPC title