DEFINITIVE ENDODERM

• US 20140134727A1
• Filed: 11/05/2013
• Published: 05/15/2014
• Est. Priority Date: 12/23/2003
• Status: Abandoned Application

• 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.
• Application Number:
  US14/072,642
• Publication Number:
  US 20140134727A1
• 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. An in vitro cell culture comprising human definitive endoderm cells in a medium comprising serum, wherein less than about 5% (v/v) of the medium is serum.

2. The cell culture of claim 1, wherein the medium further comprises a TGFβ

superfamily growth factor.

3. The cell culture of claim 1, wherein the medium further comprises a TGFβ

superfamily growth factor and a Wnt family member.

4. The cell culture of claim 1, wherein less than about 2% (v/v) of the medium is serum.

5. The cell culture of claim 1, wherein less than about 0.2% (v/v) of the medium is serum.

6. The cell culture of claim 1, wherein the medium does not comprise serum replacement.

7. The cell culture of claim 1, wherein the human definitive endoderm cells are produced by a method comprising:

(a) culturing a population of pluripotent human cells in a growth medium comprising serum and a TGFβ

superfamily growth factor, thereby producing human definitive endoderm cells, wherein less than about 5% (v/v) of the growth medium is serum.

8. The cell culture of claim 7, wherein said growth medium does not comprise serum replacement.

9. The cell culture of claim 7, wherein said TGFβ

superfamily growth factor in said growth medium is selected from the group consisting of Nodal, activin A and activin B.

10. The cell culture of claim 7, wherein said TGFβ

superfamily growth factor in said growth medium is activin A.

11. The cell culture of claim 7, wherein at least 10 ng/ml activin A is present in said growth medium.

12. The cell culture of claim 7, wherein at least 100 ng/ml activin A is present in said growth medium.

13. The cell culture of claim 7, wherein said growth medium further comprises a Wnt family member.

14. The cell culture of claim 7, wherein the Wnt family member is Wnt3a.

15. The cell culture of claim 7, wherein the growth medium comprises less than 2% serum.

16. The cell culture of claim 7, wherein the growth medium comprises less than 0.2% serum.

17. The cell culture of claim 7, wherein the method comprises, prior to step (a), culturing the pluripotent human cells in a first growth medium comprising no serum for about 24 hours.

18. The cell culture of claim 17, wherein the method comprises, prior to step (a) and after said culturing in said first growth medium, culturing the pluripotent human cells in a series of additional growth mediums comprising successively increasing concentrations of serum.

19. An in vitro cell culture comprising human definitive endoderm cells in a medium comprising serum and a TGFβ

superfamily growth factor, wherein less than about 5% (v/v) of the medium is serum.

20. An in vitro cell culture comprising human definitive endoderm cells in a medium comprising serum, a TGFβ

superfamily growth factor and a Wnt family member, wherein less than about 5% (v/v) of the medium is serum.