Definitive endoderm

• US 8,623,645 B2
• Filed: 03/30/2009
• Issued: 01/07/2014
• Est. Priority Date: 12/23/2003
• Status: Active Grant

• Current Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Original Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Inventors: D'Amour, Kevin Allen, Agulnick, Alan D., Baetge, Emmanuel E.
• Primary Examiner(s): Paras, Jr., Peter
• Assistant Examiner(s): Montanari, David A
• Application Number:
  US12/414,482
• Publication Number:
  US 20090253202A1
• Time in Patent Office: 1,744 Days
• Field of Search: 435/325, 435/366
• 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. An in vitro cell culture comprising a medium human pluripotent cells and human definitive endoderm cells having been differentiated from said human pluripotent cells, wherein at least 1 human definitive endoderm cell is present for every 5 human pluripotent cells present in said cell culture, wherein said medium comprises a Wnt family member.

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

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

4. The cell culture of claim 1, wherein at least 5 human definitive endoderm cells are present for every 1 human pluripotent cells present in said cell culture.

5. The cell culture of claim 1, wherein at least 10 human definitive endoderm cells are present for every 1 human pluripotent cells present in said cell culture.

6. The cell culture of claim 1, wherein said human pluripotent cell comprises an embryonic stem cell.

7. The cell culture of claim 1, wherein said medium lacks B27.

8. The cell culture of claim 1, wherein said medium lacks serum replacement.

9. The cell culture of claim 1, wherein said medium comprises a growth factor of the Nodal/Activin subgroup of the TGFβ

superfamily.

10. The cell culture of claim 9, further comprising a growth factor selected from the group consisting of Nodal, Activin A, Activin B and combinations thereof.

11. The cell culture of claim 1, wherein said medium comprises Nodal.

12. The cell culture of claim 1, wherein said medium comprises Activin B.

13. The cell culture of claim 1, which is substantially free of cells derived from the ectoderm lineage.

14. The cell culture of claim 1, wherein said human cells are nonrecombinant cells.

15. The cell culture of claim 1, wherein said human definitive endoderm cells adhere to a surface of a culture vessel.

16. The cell culture of claim 1, wherein said medium comprises Activin A.

17. The cell culture of claim 1, wherein said Wnt family member comprises Wnt3A.