HEPATOCYTE LINEAGE CELLS

• US 20110294211A1
• Filed: 08/02/2011
• Published: 12/01/2011
• Est. Priority Date: 04/28/2006
• Status: Active Grant

• Current Assignee: Emmanuel E. Baetge, Kevin D'Amour
• Original Assignee: Emmanuel E. Baetge, Kevin D'Amour
• Inventors: D'Amour, Kevin, Baetge, Emmanuel E.
• Granted Patent: US 8,574,905 B2
• Time in Patent Office: Days
• US Class Current: 435/370
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/067 Hepatocytes
• CPC Class Codes: C12N 5/0672 Stem cells; Progenitor cells; Precursor cells; Oval cells

Abstract

Disclosed herein are methods for producing liver precursor cells as well as hepatocyte cells form pluripotent and/or multipotent cells. Also disclosed herein are methods of enriching isolating and/or purifying liver precursor cells and/or hepatocyte cells. Further disclosed are compositions comprising cell cultures and cell populations that are enriched for liver precursor cells or hepatocyte cells.

Claims

1. A method of enriching a population of liver precursor cells, said method comprising providing a cell population comprising liver precursor cells with a reagent that binds to a marker selected from the group consisting of DPP4, NOSTRIN, DOK4, SLC35D1, OCDH17, FN1, HLA-C, HLA-B, CYP4X1, CPHA6, PP1057 HPX and DGKB, and separating cells bound to said reagent from cells that are not bound to said reagent, thereby producing an enriched population of liver precursor cells.

2. The method of claim 1, wherein said marker is DPP4.

3. The method of claim 2, further comprising providing said cell population comprising liver precursor cells with a reagent that binds to a marker selected from the group consisting of APOA1, APOA2, HNF4A, PROX1, ALBUMIN, AFP, NOSTRIN, DGKB, DOK4, HPX, LOC440450, SLC35D1, PCDH17, LOC286167, C3orf15, GLUD1, GLUD2, GSN, FN1, LOC286167, EVI1, SP8, ALDH2, HLA-B, HLA-C, Hs.570199, PLA2G12B, SYTL5, FKBP7, CYP4X1, LOC130576, MUSTN1, EPHA6, PP1057, PFTK1 and CUBN and separating cells bound to said reagent from cells that are not bound to said reagent, thereby producing an enriched population of liver precursor cells.

4. The method of claim 1, further comprising providing said cell population comprising liver precursor cells with a reagent that binds to a marker selected from the group consisting of CYP3A7, Transferrin, HBGZ, ASGR1, SLC17A8, PCDHGB7, OCT4, PDX1 and SLC10A1, separating cells bound by said reagent that binds a marker selected from the group consisting of CYP3A7, Transferrin, HBGZ, ASGR1, SLC17A8, PCDHGB7, OCT4, PDX1 and SLC10A1 from cells that are not bound by said reagent that binds a marker selected from the group consisting of CYP3A7, Transferrin, HBGZ, ASGR1, SLC17A8, PCDHGB7, OCT4, PDX1 and SLC10A1, thereby producing an enriched population of liver precursor cells.

5. The method of claim 1, wherein said liver precursor cells can differentiate into mature liver cells.

6. The method of claim 1, wherein said marker is a cell surface marker.

7. The method of claim 1, wherein said reagent comprises an antibody or fragment thereof.

8. The method of claim 1, wherein said reagent comprises a ligand that binds to said marker.

9. The method of claim 1, wherein said liver precursor cells are derived from definitive endoderm cells.

10. The method of claim 9, wherein the expression of a marker selected from the group consisting of DPP4, APOA1, APOA2, HNF4A, PROX1, ALBUMIN, AFP, NOSTRIN, DGKB, DOK4, HPX, LOC440450, SLC35D1, PCDH17, LOC286167, C3orf15, GLUD1, GLUD2, GSN, FN1, LOC286167, EVI1, SP8, ALDH2, HLA-B, HLA-C, Hs.570199, PLA2G12B, SYTL5, FKBP7, CYP4X1, LOC130576, MUSTN1, EPHA6, PP1057, PFTK1, and CUBN is greater in said liver precursor cells than in said definitive endoderm cells.

11. The method of claim 9, wherein a BMP family growth factor and an FGF family growth factor are provided to said definitive endoderm cells.

12. The method of claim 11, wherein said BMP family growth factor is BMP4.

13. The method of claim 11, wherein said FGF family growth factor is FGF10.

14. The method of claim 11, wherein said definitive endoderm cells are provided with said BMP family growth factor and said FGF family growth factor for about 1 day to about 8 days.

15. The method of claim 14, wherein said definitive endoderm cells are provided with said BMP family growth factor and said FGF family growth factor for about 4 days.

16. The method of claim 1, wherein said liver precursor cells are differentiated from pluripotent cells.

17. The method of claim 16, wherein said pluripotent cells are human pluripotent cells.

18. The method of claim 17, wherein separating cells bound to said reagent from cells that are not bound to said reagent is done by Fluorescence Activated Cell Sorting (FACS).

19. A method of enriching a population of liver precursor cells, said method comprising:

providing a cell population comprising pluripotent cells with a growth factor of the TGFβ

superfamily and a growth factor of the fibroblast growth factor family;

allowing sufficient time for at least a portion of said pluripotent cell population to differentiate into liver precursor cells;

contacting said liver precursor cells with a reagent that binds to a marker selected from the group consisting of DPP4, NOSTRIN, DOK4, SLC35D1, OCDH17, FN1, HLA-C, HLA-B, CYP4X1, CPHA6, PP1057 HPX and DGKB; and

separating cells bound to said reagent from cells that are not bound to said reagent, thereby producing an enriched population of liver precursor cells.

20. A cell culture comprising human cells, wherein at least about 10% of said human cells are liver precursor cells that express DPP4 and a marker selected from the group consisting of APOA1, APOA2, HNF4A, PROX1, ALBUMIN, AFP, NOSTRIN, DGKB, DOK4, HPX, LOC440450, SLC35D1, PCDH17, LOC286167, C3 orf15, GLUD1, GLUD2, GSN, FN1, LOC286167, EVI1, SP8, ALDH2, HLA-B, HLA-C, Hs.570199, PLA2G12B, SYTL5, FKBP7, CYP4X1, LOC130576, MUSTN1, EPHA6, PP1057, PFTK1 and CUBN and which do not substantially express a marker selected from the group consisting of CYP3A7, Transferrin, HBGZ, ASGR1, SLC17A8, PCDHGB7, OCT4, PDX1 and SLC10A1, and wherein said liver precursor cells can differentiate into mature liver cells.