Expansion of definitive endoderm cells

• US 7,625,753 B2
• Filed: 12/22/2005
• Issued: 12/01/2009
• Est. Priority Date: 12/23/2003
• Status: Active Grant

• Current Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Original Assignee: CyThera, Inc. (Vertex Pharmaceuticals Incorporated)
• Inventors: Kelly, Olivia, Baetge, Emmanuel E., Carpenter, Melissa
• Primary Examiner(s): Crouch; Deborah
• Assistant Examiner(s): SHEN, WU CHENG WINSTON
• Application Number:
  US11/317,387
• Publication Number:
  US 20060148081A1
• Time in Patent Office: 1,440 Days
• Field of Search: 435/377, 435/374
• US Class Current: 435/377
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• 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 2501/385 of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
• 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

Abstract

Disclosed herein are cell cultures comprising expanded definitive endoderm cells as well as methods for expanding definitive endoderm cells in culture.

Claims

1. A method of expanding definitive endoderm cells in culture, said method comprising the steps of:

(a) obtaining a cell culture comprising definitive endoderm cells;

(b) isolating at least some of the definitive endoderm cells from at least some of the other cells in the cell culture, thereby producing a cell population enriched in definitive endoderm cells; and

(c) culturing said cell population enriched in definitive endoderm cells under conditions that permit the expansion of said definitive endoderm cells.

2. The method of claim 1, wherein said definitive endoderm cells are multipotent cells that can differentiate into cells of the gut tube or organs derived therefrom.

3. The method of claim 1, wherein said definitive endoderm cells are human definitive endoderm cells.

4. The method of claim 3, wherein said definitive endoderm cells are derived from human embryonic stem cells (hESCs).

5. The method of claim 4, wherein the obtaining step comprises contacting hESCs with at least one growth factor from the TGFβ

superfamily so as to permit differentiation of at least some of said hESCs to definitive endoderm cells.

6. The method of claim 5, wherein said at least one growth factor from the TGFβ

superfamily comprises activin A.

7. The method of claim 1, wherein the step of obtaining said cell culture comprising definitive endoderm further comprises obtaining a portion of an existing definitive endoderm culture.

8. The method of claim 1, wherein said cell population enriched in definitive endoderm cells is substantially free of cells other than definitive endoderm cells.

9. The method of claim 1, wherein said cell population enriched in definitive endoderm cells comprises at least 96% definitive endoderm cells.

10. The method of claim 1, wherein said cell population enriched in definitive endoderm cells comprises at least 98% definitive endoderm cells.

11. The method of claim 1, wherein said cell population enriched in definitive endoderm cells comprises 100% definitive endoderm cells.

12. The method of claim 1, wherein said isolating step comprises providing said cell culture with a reagent which binds to a marker expressed in said definitive endoderm cells but which is not substantially expressed in said other cells present in the cell culture, and separating said definitive endoderm cells bound to said reagent from said other cells present in the cell culture, thereby producing a cell population enriched in definitive endoderm cells.

13. The method of claim 12, wherein said marker is CXCR4.

14. The method of claim 12, wherein said reagent is an antibody.

15. The method of claim 12, wherein said definitive endoderm cells bound to said reagent are separated from said other cells present in the cell culture by fluorescence activated cell sorting (FACS).

16. The method of claim 1, wherein said isolating step comprises separating fluorescently-labeled definitive endoderm cells from unlabeled cells.

17. The method of claim 1, wherein said culturing step comprises plating said population enriched in definitive endoderm cells or a portion thereof.

18. The method of claim 17, wherein said population enriched in definitive endoderm cells or a portion thereof is plated on a surface coated with human fibronectin.

19. The method of claim 18, wherein said surface is coated with poly-ornithine.

20. The method of claim 1, wherein said culturing step comprises incubating said population enriched in definitive endoderm cells or a portion thereof in a medium comprising 2% (v/v) serum.

21. The method of claim 1, wherein said culturing step comprises incubating said population enriched in definitive endoderm cells or a portion thereof in a medium comprising 2% to 20% (v/v) serum.

22. The method of claim 1, wherein said culturing step comprises incubating said population enriched in definitive endoderm cells or a portion thereof in a medium comprising 0% to 2% (v/v) serum.

23. The method of claim 1, wherein said culturing step comprises incubating said population enriched in definitive endoderm cells or a portion thereof in a medium comprising at least one growth factor.

24. The method of claim 23, wherein said at least one growth factor is a growth factor from the TGFβ

superfamily of growth factors.

25. The method of claim 24, wherein said at least one growth factor from the TGFβ

superfamily of growth factors comprises activin A.

26. The method of claim 25, wherein said activin A is present in said medium at a concentration ranging from 1 ng/ml to 5000 ng/ml.

27. The method of claim 23, wherein said at least one growth factor comprises IGF1.

28. The method of claim 27, wherein said IGF1 is present in said medium at a concentration ranging from 1 ng/ml to 5000 ng/ml.

29. The method of claim 23, wherein said at least one growth factor comprises a combination of activin A and IGF1.

30. The method of claim 23, wherein said at least one growth factor comprises bFGF.

31. The method of claim 30, wherein said bFGF is present in said medium at a concentration ranging from 1 ng/ml to 5000 ng/ml.

32. The method of claim 23, wherein said at least one growth factor comprises EGF.

33. The method of claim 32, wherein said EGF is present in said medium at a concentration ranging from 1 ng/ml to 5000 ng/ml.

34. The method of claim 23, wherein said at least one growth factor comprises a combination of activin A, bFGF and EGF.

35. The method of claim 3, wherein the other cells in the cell culture are extraembryonic endoderm cells.

36. The method of claim 3, wherein the other cells in the cell culture are embryonic stem cells.

37. The method of claim 1, wherein the step of culturing said definitive endoderm cells under conditions that permit the expansion of said definitive endoderm cells comprises passaging said definitive endoderm cells, thereby producing a plurality of cell cultures comprising definitive endoderm cells.

38. The method of claim 37, wherein passaging said definitive endoderm cells comprises providing at least one enzyme to said cell culture.

39. The method of claim 38, wherein said at least one enzyme comprises at least one protease.

40. The method of claim 37, wherein passaging said definitive endoderm cells comprises mechanically disrupting contacts between said definitive endoderm cells.