Definitive endoderm

• US 7,704,738 B2
• Filed: 12/23/2004
• Issued: 04/27/2010
• 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.
• Primary Examiner(s): SHEN, WU CHENG WINSTON
• Application Number:
  US10/584,338
• Publication Number:
  US 20070154984A1
• Time in Patent Office: 1,951 Days
• Field of Search: 435/377, 435/384
• US Class Current: 435/377
• 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 producing human definitive endoderm cells, said method comprising:

obtaining a cell population comprising pluripotent human stem cells; and

providing said cell population with a TGFβ

superfamily growth factor and a Wnt family member, thereby generating in said cell population definitive endoderm cells expressing at least SOX17 and HNF3β

.

2. The method of claim 1 further comprising removing TGFβ

superfamily growth factor from said cell population.

3. The method of claim 1, wherein at least 50% of said pluripotent cells differentiate into definitive endoderm cells.

4. The method of claim 1, wherein said TGFβ

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

5. The method of claim 1, wherein said TGFβ

superfamily growth factor is activin A.

6. The method of claim 1, wherein said Wnt family member is Wnt3a.

7. The method of claim 1 wherein at least 10 ng/ml activin A is provided.

8. The method of claim 1, wherein at least 100 ng/ml activin A is provided.

9. The method of claim 1 further comprising the step of providing serum to said cell population.

10. The method of claim 1, wherein said pluripotent stem cells comprise embryonic stem cells.

11. The method of claim 10, wherein said embryonic stem cells are derived from tissue selected from the group consisting of the morula and the inner cell mass (ICM) of the embryo.

12. The method of claim 9, wherein providing serum comprises providing increasing concentrations of serum.