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

• US 10,106,777 B2
• Filed: 07/07/2016
• Issued: 10/23/2018
• 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: Brolen, Gabriella, Edsbagge, Josefina
• Primary Examiner(s): Kelly, Robert M
• Application Number:
  US15/204,466
• Publication Number:
  US 20170002327A1
• Time in Patent Office: 838 Days
• Field of Search: None
• 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 composition comprising:

an in vitro derived hepatocyte-like cell in a media comprising one or more GSK3 inhibitors, wherein said in vitro derived hepatocyte-like cell has elevated expression of the genes CYP1A1, CYP1A2, CYP3A4, CYP7A1, and MRP2 compared to a comparable hepatocyte-like cell obtained under conditions without use of any GSK3 inhibitor.

2. The composition of claim 1, wherein said in vitro derived hepatocyte-like cell has enzymatic activities exceeding a fold change of at least 10 in cytochrome P450 activity when compared to saki comparable hepatocyte-like cell obtained under conditions without use of any GSK3 inhibitor.

3. The composition of claim 1, wherein said in vitro derived hepatocyte-like cell has enzymatic activities exceeding a fold change of at least 13 in cytochrome P450 activity when compared to saki comparable hepatocyte-like cell obtained under conditions without use of any GSK3 inhibitor.

4. The composition of claim 1, wherein said one or more GSK3 inhibitors is selected from the group consisting of:

BIO, kenpaullone, SB2167763, Indirubin-3′

-monoxime, and any combination thereof.

5. The composition of claim 1, wherein said one or more GSK3 inhibitors is BIO.

6. The composition of claim 1, wherein said one or more GSK3 inhibitors is kenpaullone.

7. The composition of claim 1, wherein said one or more GSK3 inhibitors is SB2167763.

8. The composition of claim 1, wherein said one or more GSK3 inhibitors is Indirubin-3′

-monoxime.

9. The composition of claim 1, wherein said in vitro derived hepatocyte-like cell further has elevated expression of the gene CYP2C9 compared to the comparable hepatocyte-like cell obtained under conditions without use of any GSK3 inhibitor.

10. The composition of claim 1, wherein said in vitro derived hepatocyte-like cell further has elevated expression of the genes OATP2 and AAT compared to the comparable hepatocyte-like cell obtained under conditions without use of any GSK3 inhibitor.