Human hepatocyte-like cells and uses thereof

• US 9,623,048 B2
• Filed: 02/08/2014
• Issued: 04/18/2017
• Est. Priority Date: 02/08/2013
• Status: Active Grant

• Current Assignee: Center For Excellence In Molecular Cell Sciences, Chinese Academy Of Sciences
• Original Assignee: Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences
• Inventors: Hui, Lijian, Huang, Pengyu, Zhang, Ludi, Gao, Yimeng
• Primary Examiner(s): Carlson, Karen Cochrane
• Application Number:
  US14/422,307
• Publication Number:
  US 20150190427A1
• Time in Patent Office: 1,165 Days
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61K 35/407 Liver; Hepatocytes
• CPC Class Codes: A61K 38/1709 from mammals
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: C12N 2501/60 Transcription factors
• CPC Class Codes: C12N 2506/1307 from adult fibroblasts
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 5/0602 Vertebrate cells
• CPC Class Codes: C12N 5/067 Hepatocytes
• CPC Class Codes: G01N 33/5014 for testing toxicity
• CPC Class Codes: G01N 33/5017 for testing neoplastic activity

Abstract

The present invention relates to hepatocyte-like cells. Also disclosed are methods of making the cells and using the cells.

Claims

1. A method of generating human hepatocyte-like cells, comprising (i) forcing overexpression in a non-hepatic cell of a FOXA polypeptide and a plurality of HNF polypeptides by nucleic acid transfection, virus infection or protein transduction, and (ii) culturing the non-hepatic human cell in a medium for a period of time to obtain one or more progeny cells thereof, thereby generating human hepatocyte-like cells, wherein the HNF polypeptide comprises HNF4A and HNF1, and the FOXA polypeptide comprises FOXA3, wherein the HNF1 polypeptide comprises HNF1A or HNF1B or both, and wherein the non-hepatic human cell does not express a GATA4 or HHEX polypeptide.

2. The method of claim 1, comprising forcing expression of the FOXA3 polypeptide, the HNF4A polypeptide and the HNF1A polypeptide.

3. The method of claim 1, comprising forcing expression of the FOXA3 polypeptide, the HNF4A polypeptide, and both of the HNF1 A polypeptide and HNF1B polypeptide in the non-hepatic human cell.

4. The method of claim 1, wherein the non-hepatic human cell is a somatic cell.

5. The method of claim 1, wherein the non-hepatic human cell is a fibroblast, an epithelium cell, a blood cell, a neuron, an embryonic cell, or a cell derived from a tissue or organ of a subject.

6. The method of claim 1, wherein the method further comprises increasing a number of the human hepatocyte-like cells generated by increasing cell proliferation or by decreasing apoptosis or cell senescence of the non-hepatic human cell or one or more of the progeny cells.

7. The method of claim 6, comprising increasing cell proliferation by forcing expression of a SV40 large T antigen in the non-hepatic human cell or one or more of the progeny cells.

8. The method of claim 6, comprising reducing expression or activity of an Rb family gene in the non-hepatic human cell or one or more of the progeny cells.

9. The method of claim 1, wherein the period of time is about 1-20 days, 2-14 days or 10-14 days.

10. The method of claim 1, wherein the method further comprises forcing expression of one or more additional polypeptides selected from the group consisting of C/EBPβ

,KLF4, and PROX1 in the non-hepatic human cell.

11. A cultured hepatocyte-like cell obtained using the method of claim 1.

27. A method of evaluating toxicity, carcinogenicity, or biotransformation activity of a test substance, comprising

contacting a test substance with the cell of claim 11, and

examining a level of metabolic activity or viability of the cell, wherein the level indicates the toxicity, carcinogenicity, or biotransformation activity of the test substance.

12. A cultured recombinant cell comprising

(i) a plurality of heterologous HNF polypeptides comprising HNF4A and HNF1, wherein the HNF1 polypeptide comprises HNF1A or HNF1B or both;

(ii) a heterologous FOXA polypeptide, wherein FOXA polypeptide comprises FOXA3, wherein the recombinant cell overexpresses the plurality of heterologous HNF polypeptides and the FOXA3 polypeptide and does not express a GATA4 or HHEX polypeptide.

13. The cell of claim 12, wherein the cell does not express C/EBPα

.

14. The cell of claim 12, wherein the cell further comprises an additional heterologous polypeptide.

15. The cell of claim 14, wherein the additional heterologous polypeptide is selected from the group consisting of C/EBPP, KLF4, and PROX.

16. The cell of claim 12, wherein the cell further comprises a heterologous SV40 large T antigen.

17. The cell of claim 12, wherein the cell displays one or more mature hepatic functions.

18. The cell of claim 17, wherein the cell displays biliary excretion.

19. The cell of claim 17, wherein the cell is capable of metabolizing one or more compounds selected from the group consisting of 3-methylcholanthrene, phenobarbital, rifampicin, phenacetin, coumarin, dextromethorphan, testosterone, and diclofenac.

20. The cell of claim 17, wherein the cell has a normal karyotype or does not form tumor after transplantation in an immuno-deficient mouse.

21. A pharmaceutical composition comprising the cell of claim 12 and a pharmaceutically acceptable carrier.

22. The pharmaceutical composition of claim 21, wherein the cell is encapsulated in a microcapsule.

23. The pharmaceutical composition of claim 22, wherein the cell is encapsulated in a microcapsule comprising alginate-poly-L-lysine-alginate (APA).

24. A bioartificial liver device comprising

a reservoir chamber configured to house hepatocytes or hepatocyte-like cells;

a plurality of the cells of claim 12 housed in the reservoir chamber;

an inlet in fluid communication with said chamber and cells, and

an outlet in fluid communication with said chamber and cells.

25. A method for improving the liver function of a subject in need thereof, comprising (i) administering to the subject a plurality of the cells of claim 12, or (ii) administering to the subject a pharmaceutical composition comprising the cells and a pharmaceutically acceptable carrier or (iii) connecting a bioartificial liver device to the subject comprising

a reservoir chamber configured to house hepatocytes or hepatocyte-like cells;

the plurality of cells or the pharmaceutical composition housed in the reservoir chamber;

an inlet in fluid communication with said chamber and cells, and

an outlet in fluid communication with said chamber and cells.

26. The method of claim 25, wherein the plurality of cells are prepared from a non-hepatic cell of said subject.

28. A composition comprising

(i) a plurality of HNF polypeptides comprising HNF4A and HNF1 polypeptides, wherein the HNF1 polypeptide comprises HNF1A or HNF1B or both;

(ii) a FOXA polypeptide,

wherein the FOXA polypeptide comprises FOXA3, and wherein the composition does not include a GATA4 polypeptide or a HHEX polypeptide.

29. The composition of claim 28, wherein the composition further comprises an additional agent selected from a group consisting of an additional polypeptide, wherein the additional polypeptide is selected from the group consisting of SV40 large T antigen, C/EBPβ

,KLF4, and PROX1.