DEFINITIVE ENDODERM

• US 20170159011A1
• Filed: 02/13/2017
• Published: 06/08/2017
• Est. Priority Date: 12/23/2003
• Status: Active Grant

• Current Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Original Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Inventors: Baetge, Emmanuel E., D'Amour, Kevin Allen, Agulnick, Alan D.
• Granted Patent: US 10,421,942 B2
• Time in Patent Office: Days
• 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. An in vitro method of making a cell population comprising definitive endoderm cells, the method comprising:

(a) obtaining a cell population comprising human pluripotent stem cells in medium comprising no serum or serum replacement; and

(b) providing the cell population comprising human pluripotent stem cells with a TGFβ

super family member,

thereby producing a cell population comprising definitive endoderm cells.

2. The method of claim 1, wherein the TGFβ

super family member is activin A.

3. The method of claim 2, comprising providing the cell population comprising human pluripotent stem cells with about 10-100 ng/ml activin A.

4. The method of claim 1, wherein the cell population comprising definitive endoderm further comprises mesendoderm.

5. The method of claim 1, wherein the method further comprises providing a medium comprising less than about 10% volume/volume (v/v) serum about 24 hours after the TGFβ

super family member is provided.

6. The method of claim 1, wherein the method further comprises providing a medium comprising less than about 2% volume/volume (v/v) serum about 24 hours after the TGFβ

super family member is provided.

7. The method of claim 1, wherein the method further comprises providing a medium comprising less than about 2% volume/volume (v/v) serum about 24 hours after the TGFβ

super family member is provided.

8. The method of claim 1, further comprising providing the cell population comprising human pluripotent stem cells with a WNT protein.

9. The method of claim 8, wherein the WNT protein in Wnt3A.

10. The method of claim 1, wherein the cell population comprising definitive endoderm cells is substantially free of cells derived from the ectoderm lineage.

11. An in vitro method of making a cell population comprising definitive endoderm cells comprising:

(a) obtaining a cell population comprising human pluripotent stem cells in a medium comprising less than about 10% serum; and

(b) providing the cell population with a TGFβ

super family member,

thereby producing a cell population comprising definitive endoderm cells.

12. The method of claim 11, wherein the TGFβ

super family member is activin A.

13. The method of claim 12, comprising providing the cell population comprising human pluripotent stem cells with about 10-100 ng/ml activin A.

14. The method of claim 11, wherein the cell population comprising definitive endoderm further comprises mesendoderm.

15. The method of claim 11, wherein the media comprises less than about 2% (v/v) serum.

16. The method of claim 11, wherein the media comprises less than about 0.5% (v/v) serum.

17. The method of claim 11, further comprising providing the cell population comprising human pluripotent stem cells with a WNT family member.

18. The method of claim 11, wherein the WNT family member in Wnt3A.

19. The method of claim 11, wherein the cell population comprising definitive endoderm cells is substantially free of cells derived from the ectoderm lineage.

20. A method of differentiating human pluripotent cells, said method comprising culturing a population of pluripotent human cells in a medium comprising no serum or serum replacement and a TGFβ

superfamily growth factor thereby differentiating the human pluripotent cells.