Induced hepatocytes and uses thereof

• US 9,808,490 B2
• Filed: 11/25/2015
• Issued: 11/07/2017
• Est. Priority Date: 11/26/2014
• Status: Active Grant

• Current Assignee: Accelerated Biosciences Corp.
• Original Assignee: Accelerated Biosciences Corp.
• Inventors: Lee, Jau-Nan, Lee, Tony Tung-Yin, Lee, Yuta, Tsai, Eing-Mei
• Primary Examiner(s): Fox, Allison
• Application Number:
  US14/952,023
• Publication Number:
  US 20160199416A1
• Time in Patent Office: 713 Days
• CPC Class Codes: A61K 35/407 Liver; Hepatocytes
• CPC Class Codes: A61K 35/545 Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• 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 2501/20 Cytokines; Chemokines
• CPC Class Codes: C12N 2501/237 Oncostatin M [OSM]
• CPC Class Codes: C12N 2501/39 Steroid hormones
• CPC Class Codes: C12N 2501/40 Regulators of development
• CPC Class Codes: C12N 2501/65 MicroRNA
• CPC Class Codes: C12N 2506/025 from extra-embryonic cells, e.g. trophoblast, placenta
• CPC Class Codes: C12N 5/067 Hepatocytes
• CPC Class Codes: C12N 5/0671 Three-dimensional culture, tissue culture or organ culture; Encapsulated cells

Abstract

Disclosed herein are induced hepatocytes from a trophoblast stem cell, methods for inducing the cells, and compositions thereof. Also disclosed herein are methods of treating a disease or disorder (e.g., liver-associated) by utilizing an induced hepatocyte disclosed herein.

Claims

1. A method of inducing a trophoblast stem (TS) cell to differentiate into an induced hepatocyte in vitro, comprising:

contacting the trophoblast stem cell with a fibroblast growth factor (FGF) that binds to FGFR1 to induce formation of a definitive endoderm (DE) cell, and

contacting the DE cell with a medium to induce differentiation into an induced hepatocyte,

wherein the medium comprises a FGF that binds to FGFR1, a glucocorticoid steroid, and an interleukin 6 group cytokine.

2. The method of claim 1, wherein the induced hepatocyte is a hepatic progenitor cell.

3. The method of claim 1, wherein the TS cell is a human TS cell.

4. The method of claim 1, wherein the glucocorticoid steroid is dexamethasone.

5. The method of claim 1, wherein the interleukin 6 group cytokine is oncostatin M.

6. The method of claim 5, wherein the oncostatin M is a human oncostatin M.

7. The method of claim 6, wherein the human oncostatin M is a recombinant human oncostatin M.

8. The method of claim 1, wherein the medium further comprises a bone morphogenetic protein (BMP).

9. The method of claim 8, wherein the BMP is BMP4.

10. The method of claim 1, wherein the medium further comprises a hepatic growth factor (HGF).

11. The method of claim 1, wherein the induced hepatocyte is immune privileged.

12. The method of claim 1, wherein the induced hepatocytes recruit CD4⁺Foxp3⁺Treg cells.

13. The method of claim 1, wherein the induced hepatocytes form tissue of a 3-dimensional structure.

14. The method of claim 1, wherein the induced hepatocytes cluster or aggregate.

15. The method of claim 1, wherein the induced hepatocytes form a crescent cell mass.

16. The method of claim 1, wherein the induced hepatocytes comprise a peripheral compartment and a central compartment.

17. The method of claim 16, wherein the induced hepatocytes distribute irregularly along extracellular matrix (ECM) beyond basement membrane in the peripheral compartment.

18. The method of claim 16, wherein the induced hepatocytes distribute from basal towards central areas in the central compartment.

19. The method of claim 1, wherein the induced hepatocyte expresses one or more markers selected from the group consisting of transforming growth factor beta 1 (TaFβ

1), human leukocyte antigen G (HLA-G), human cytoplasmic marker stem 121 (stem

     121), mast/stem cell growth factor receptor C-kit (C-kit), cytokeratin 19 (CK19), cytokeratin 18(CK18), albumin (ALB), alpha-fetoprotein (AFP), betatrophin, alcohol dehydrogenase-1 (ADH1), apolipoprotein F (APOF), carbamoyl-phosphate synthase 1 (CPS1), GATA transcription factor 4 (GATA4), cytochrome P450 family 1 subfamily A polypeptide 1 (CYP1A1), cytochrome P450 2B6(CYP2B6), asialoglycoprotein receptor 1 (ASGR1), C-X-C chemokine receptor type 4 (CXCR4), bile salt export pump (BSEP), multi-drug resistance protein-2 (MRP2), connexin 32 (Cx32), and any combination thereof.

20. The method of claim 1, wherein the trophoblast stem cell is contacted with the FGF selected from the group consisting of FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF8, FGF10, FGF17, FGF19, FGF20, FGF21, FGF22, and FGF23.

21. The method of claim 20, wherein the FGF is FGF2 (bFGF).

22. The method of claim 20, wherein the FGF is FGF1.

23. The method of claim 1, wherein the glucocorticoid steroid is selected from the group consisting of dexamethasone, betamethasone, budesonide, cortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, and triamcinolone.

24. The method of claim 1, wherein the interleukin 6 group cytokine is selected from the group consisting of interleukin-6, oncostatin M, interleukin-11, leukemia inhibitory factor (LIF), ciliary neurotropic factor (CNTF), cardiotrophin-1(CT-1), and cardiotrophin-like cytokine (CLC).

25. The method of claim 1, wherein the medium comprises:

the FGF selected from the group consisting of;

FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF8, FGF10, FGF17, FGF19, FGF20, FGF21, FGF22, and FGF23;

the glucocorticoid steroid selected from the group consisting of;

dexamethasone, betamethasone, budesonide, cortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, and triamcinolone; and

the interleukin 6group cytokine selected from the group consisting of;

interleukin-6, oncostatin M, interleukin-11, leukemia inhibitory factor (LIF), ciliary neurotropic factor (CNTF), cardiotrophin-1 (CT-1), and cardiotrophin-like cytokine (CLC).

26. The method of claim 25, wherein the medium comprises:

FGF2;

dexamethasone; and

oncostatin M.