Definitive endoderm

• US 20050158853A1
• Filed: 12/23/2004
• Published: 07/21/2005
• Est. Priority Date: 12/23/2003
• Status: Active Grant

• Current Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Original Assignee: CyThera, Inc. (Vertex Pharmaceuticals Incorporated)
• Inventors: D' Amour, Kevin Allen, Agulnick, Alan D., Baetge, Emmanuel E.
• Granted Patent: US 7,510,876 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• 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 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0603 Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/068 Stem cells; Progenitors

Abstract

Disclosed herein are cell cultures comprising definitive endoderm cells and methods of producing the same. Also disclosed herein are cell populations comprising substantially purified definitive endoderm cells as well as methods for enriching, isolating and purifying definitive endoderm cells from other cell types.

Claims

1. A cell culture comprising human cells wherein at least about 10% of said human cells are definitive endoderm cells, said definitive endoderm cells being multipotent cells that can differentiate into cells of the gut tube or organs derived therefrom.

2. The cell culture of claim 1, wherein at least about 50% of said human cells are definitive endoderm cells.

3. The cell culture of claim 1, wherein at least about 80% of said human cells are definitive endoderm cells.

4. The cell culture of claim 1, wherein said definitive endoderm cells express a marker selected from the group consisting of SOX17 and CXCR4.

5. The cell culture of claim 4, wherein the expression of a marker selected from the group consisting of SOX17 and CXCR4 is greater than the expression of a marker selected from the group consisting of OCT4, alpha-fetoprotein (AFP), Thrombomodulin (TM), SPARC and SOX7 in said definitive endoderm cells.

6. The cell culture of claim 4, wherein said definitive endoderm cells do not express a marker selected from the group consisting of OCT4, AFP, TM, SPARC and SOX7.

7. The cell culture of claim 4, wherein said definitive endoderm cells express a marker selected from the group consisting of MIXL1, GATA4 and HNF3b.

8. The cell culture of claim 4, wherein said definitive endoderm cells express a marker selected from the group consisting of FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1.

9. The cell culture of claim 1, wherein said definitive endoderm cells express SOX17 and CXCR4.

10. The cell culture of claim 9, wherein the expression of SOX17 and CXCR4 is greater than the expression of OCT4, AFP, TM, SPARC and SOX7 in said definitive endoderm cells.

11. The cell culture of claim 9, wherein said definitive endoderm cells do not express OCT4, AFP, TM, SPARC and SOX7.

12. The cell culture of claim 9, wherein said definitive endoderm cells express MIXL1, GATA4 and HNF3b.

13. The cell culture of claim 9, wherein said definitive endoderm cells express a marker selected from the group consisting of FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1.

14. The cell culture of claim 1, wherein at least about 2 definitive endoderm cells are present for about every 1 pluripotent cell in said cell culture.

15. The cell culture of claim 14, wherein said pluripotent cell comprises an embryonic stem cell.

16. The cell culture of claim 15, wherein said embryonic stem cell is derived from a tissue selected from the group consisting of the morula, the inner cell mass (ICM) of an embryo and the gonadal ridges of an embryo.

17. The cell culture of claim 1 further comprising a medium which comprises less than about 10% serum.

18. The cell culture of claim 1 further comprising a growth factor of the Nodal/Activin subgroup of the TGFβ

superfamily.

19. The cell culture of claim 1, further comprising a growth factor selected from the group consisting of Nodal, Activin A, Activin B and combinations thereof.

20. A cell population comprising cells wherein at least about 90% of said cells are human definitive endoderm cells, said human definitive endoderm cells being multipotent cells that can differentiate into cells of the gut tube or organs derived therefrom.

21. The cell population of claim 20, wherein at least about 95% of said cells are human definitive endoderm cells.

22. The cell population of claim 20, wherein at least about 98% of said cells are human definitive endoderm cells.

23. The cell population of claim 20, wherein said human definitive endoderm cells express a marker selected from the group consisting of SOX17 and CXCR4.

24. The cell population of claim 23, wherein the expression of a marker selected from the group consisting of SOX17 and CXCR4 is greater than the expression of a marker selected from the group consisting of OCT4, AFP, TM, SPARC and SOX7 in said human definitive endoderm cells.

25. The cell population of claim 23, wherein said human definitive endoderm cells do not express a marker selected from the group consisting of OCT4, AFP, TM, SPARC and SOX7.

26. The cell population of claim 23, wherein said human definitive endoderm cells express a marker selected from the group consisting of MIXL1, GATA4 and HNF3b.

27. The cell population of claim 23, wherein said definitive endoderm cells express a marker selected from the group consisting of FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1.

28. The cell population of claim 20, wherein said human definitive endoderm cells express SOX17 and CXCR4.

29. The cell population of claim 28, wherein the expression of SOX17 and CXCR4 is greater than the expression of OCT4, AFP, TM, SPARC and SOX7 in said human definitive endoderm cells.

30. The cell population of claim 28, wherein said human definitive endoderm cells do not express OCT4, AFP, TM, SPARC and SOX7.

31. The cell population of claim 28, wherein said human definitive endoderm cells express MIXL1, GATA4 and HNF3b.

32. The cell population of claim 28, wherein said definitive endoderm cells express a marker selected from the group consisting of FGF17, VWF, CALCR, FOXQ1, CMKOR1 and CRIP1.

33. The cell population of claim 20, wherein at least about 2 human definitive endoderm cells are present for about every 1 pluripotent cell in said cell population.

34. The cell population of claim 33, wherein said pluripotent cell comprises an embryonic stem cell.

35. The cell population of claim 34, wherein said embryonic stem cell is derived from a tissue selected from the morula, the ICM of an embryo and the gonadal ridges of an embryo.