Directed differentiation and maturation of pluripotent cells into hepatocyte like cells by modulation of Wnt-signalling pathway

• US 9,394,522 B2
• Filed: 03/22/2011
• Issued: 07/19/2016
• Est. Priority Date: 03/22/2010
• Status: Active Grant

• Current Assignee: Takara Bio Europe AB (Ningbo Junsheng Investment Group Co., Ltd.)
• Original Assignee: Takara Bio Europe AB (Ningbo Junsheng Investment Group Co., Ltd.)
• Inventors: Broln, Gabriella, Edsbagge, Josefina
• Primary Examiner(s): Kelly, Robert M
• Application Number:
  US13/636,543
• Publication Number:
  US 20130095567A1
• Time in Patent Office: 1,946 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 2501/113 Acidic fibroblast growth factor (aFGF, FGF-1)
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/12 Hepatocyte growth factor [HGF]
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2502/45 Artificially induced pluripotent stem cells
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2506/03 from non-embryonic pluripotent stem cells
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 5/067 Hepatocytes
• CPC Class Codes: C12N 5/0672 Stem cells; Progenitor cells; Precursor cells; Oval cells

Abstract

Provided are improved methods using Glycogen synthase kinase 3 (GSK3) inhibitors by which endodermal cells, notably endodermal cells derived from human pluripotent stem cells (hPS), such as but not limited to hiPS-cells and hES-cells may be differentiated into hepatocyte like cells. The specific modulation of wingless integration gene (WNT)-signalling pathway and use of GSK3 inhibitors achieve direct differentiation and maturation of hepatocytes derived from human pluripotent stem (hPS) cells. GSK-3 inhibitors, when added to the growth medium at certain developmental stages, leads to more mature and functional features for the hepatocyte like cells as well as more pure and homogenous populations of hepatocyte like cells. Provided are also hepatocyte like cells obtained by these methods as well as compositions comprising them.

Claims

1. A method for directing the differentiation of human definitive endoderm cells into human hepatocyte-like cells, wherein said method comprises:

i) differentiating human definitive endoderm cells into human hepatoblasts using a differentiation medium containing 0.05-5% DMSO; and

ii) differentiating said human hepatoblasts obtained in step i) into human hepatocyte-like cells using a differentiation medium containing 0.01-5 μ

M Dexamethasone, 1-50 ng/ml hepatocyte growth factor (HGF), 1-25 ng/ml Oncostatin M (OsM), 0.05-5% DMSO, and 0.1-10 μ

M of

a GSK-3 inhibitor selected from a group consisting of;

BIO (2′

Z,3′

£

)-6-Bromoindirubin-3′

-oxime (GSK3 Inhibitor IX);

BIO-Acetoxime (2′

Z,3′

E)-6-Bromoindirubin-3′

-acetoxime (GSK3 Inhibitor X);

(5-Methyl-1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine (GSK3-Inhibitor XIII);

Pyridocarbazole-cyclopenadienylruthenium complex (GSK3 Inhibitor XV);

TDZD-8 4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (GSK3beta Inhibitor I);

2-Thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole (GSK3beta Inhibitor II);

OTDZT 2,4-Dibenzyl-5-oxothiadiazolidine-3-thione (GSK3beta Inhibitor III);

alpha-4-Dibromoacetophenone (GSK3beta Inhibitor VII);

AR-AO 14418 N-(4-Methoxybenzyl)-N′

-(5-nitro-1,3-thiazol-2-yl)urea (GSK-3beta Inhibitor VIII);

3-(1-(3-Hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione (GSK-3beta Inhibitor XI);

TWSI 19 pyrrolopyrimidine compound (GSK3beta Inhibitor XII);

L803 H-KEAPPAPPQSpP-NH2 (SEQ ID NO;

1) or its Myristoylated form (GSK3beta Inhibitor XIII);

2-Chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone (GSK3beta Inhibitor VI);

Aminopyrimidine CHIR99021;

Kenpaullone (9-Bromo-7,12-dihydro-indolo[3,2-d][1]benzazepin-6(5H)-one;

SB216763 3-(2,4-Dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione; and

Indirubin-3′

-monoxime.

2. The method according to claim 1, wherein the differentiation of human definitive endodermal cells into human hepatocyte-like cells is carried out under xeno-free conditions using xeno-free endodermal cells.

3. The method according to claim 1, wherein the differentiation medium used in step ii) is supplemented with a histone deacetylase (HDAC)-inhibitor in addition to said GSK-3 inhibitor.

4. The method according to claim 3, wherein the HDAC-inhibitor is Sodium Butyrate (NaB).

5. The method according to claim 3, wherein the HDAC-inhibitor is Sodium Butyrate (NaB) and the concentration of NaB in the differentiation medium used in step ii) is from 0.1 mM to 10 mM.

6. The method according to claim 3, wherein the HDAC-inhibitor is Sodium Butyrate (NaB) and the concentration of NaB in the differentiation medium used in step ii) is 0.05 mM to 5 mM.

7. The method according to claim 3, wherein the HDAC-inhibitor is Sodium Butyrate (NaB) and the concentration of NaB in the differentiation medium used in step ii) is 1 mM.

8. The method according to claim 1, wherein the concentration of the GSK-3 inhibitor in the differentiation medium used in step ii) is from 1 μ

M to 5 μ

M.

9. The method according to claim 1, wherein the GSK-3 inhibitor is BIO (2′

Z,3′

£

)-6-Bromoindirubin-3′

-oxime (GSK3 Inhibitor IX).

10. The method according to claim 1, wherein the GSK-3 inhibitor is Kenpaullone (9-Bromo-7,12-dihydro-indolo[3,2-d][1]benzazepin-6(5H)-one.

11. The method according to claim 1, wherein the GSK-3 inhibitor is SB216763 3-(2,4-Dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione.

12. The method according to claim 1, wherein the GSK-3 inhibitor is Indirubin-3′

-monoxime.