Methods for identifying factors for differentiating definitive endoderm

• US 8,216,836 B2
• Filed: 06/02/2009
• Issued: 07/10/2012
• 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., Eliazer, Susan, Baetge, Emmanuel E.
• Primary Examiner(s): SHEN, WU CHENG WINSTON
• Application Number:
  US12/476,570
• Publication Number:
  US 20090311703A1
• Time in Patent Office: 1,134 Days
• Field of Search: 435/366, 435/325
• US Class Current: 435/366
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: C12N 15/1086 Preparation or screening of expression libraries, e.g. reporter assays
• 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 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• 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 2501/385 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 2502/02 embryonic cells
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2503/00 Use of cells in diagnostics
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 5/0018 Culture media for cell or tissue culture media for specific animal cell type C12N5/06
• 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 5/0676 Pancreatic cells
• CPC Class Codes: C12N 5/0679 Cells of the gastro-intestinal tract
• CPC Class Codes: C12Q 1/6881 : for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 2600/158 : Expression markers
• CPC Class Codes: G01N 33/5023 : on expression patterns
• CPC Class Codes: G01N 33/5073 : Stem cells
• CPC Class Codes: G01N 33/56966 : Animal cells

Abstract

Disclosed herein are methods of identifying one or more differentiation factors that are useful for differentiating cells in a cell population comprising definitive endoderm cells into cells which are capable of forming tissues and/or organs that are derived from the gut tube.

Claims

1. An in vitro cell culture comprising a medium, human extraembryonic endoderm cells and human definitive endoderm cells, wherein said definitive endoderm cells are multipotent cells that can differentiate into cells of the gut tube or organs derived therefrom, and wherein said extraembryonic endoderm cells comprise less than 5% of the cells in said cell culture.

2. The cell culture of claim 1, wherein said extraembryonic endoderm cells and said definitive endoderm cells together comprise greater than 10% of the cells in said cell culture.

3. The cell culture of claim 1, wherein said extraembryonic endoderm cells and said definitive endoderm cells together comprise greater than 15% of the cells in said cell culture.

4. The cell culture of claim 1 further comprising a human pluripotent stem cell.

5. The cell culture of claim 4, wherein said human pluripotent stem cell comprises an embryonic stem cell.

6. The cell culture of claim 5, wherein said embryonic stem cell is derived from a tissue selected from the group consisting of the morula and the inner cell mass (ICM) of an embryo.

7. The cell culture of claim 5, wherein said human pluripotent cell is derived from a gondal ridge of an embryo.

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

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

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

superfamily.

11. The cell culture of claim 10, wherein said growth factor of the Nodal/Activin subgroup of the TGFβ

superfamily is selected from the group consisting of Nodal, Activin A, Activin B and combinations thereof.

12. The cell culture of claim 10, wherein said medium comprises a Wnt family member.

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

14. The cell culture of claim 11, wherein said medium comprises Activin B.

15. The cell culture of claim 1, wherein said extraembryonic endoderm cells comprise less than 3% of the cells in said cell culture.

16. The cell culture of claim 1, wherein said extraembryonic endoderm cells comprise visceral endoderm cells.

17. The cell culture of claim 16, wherein said extraembryonic endoderm cells comprise less than 2% visceral endoderm cells.

18. The cell culture of claim 1, wherein said extraembryonic endoderm cells comprise parietal endoderm cells.

19. The cell culture of claim 18, wherein said extraembryonic endoderm cells comprise less than 1% parietal endoderm cells.