Methods for identifying factors for differentiating definitive endoderm

• US 7,541,185 B2
• Filed: 06/23/2005
• Issued: 06/02/2009
• 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., Eliazer, Susan
• Primary Examiner(s): Ton; Thaian N.
• Assistant Examiner(s): SHEN, WU CHENG WINSTON
• Application Number:
  US11/165,305
• Publication Number:
  US 20060003313A1
• Time in Patent Office: 1,440 Days
• Field of Search: 435/377, 435/4
• US Class Current: 435/377
• 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. A method of identifying a factor that promotes the differentiation of human definitive endoderm cells, said method comprising the steps of:

obtaining a human cell population comprising human definitive endoderm cells, wherein said human definitive endoderm cells comprise at least 10% of the human cells in said cell population;

providing a candidate differentiation factor to said cell population;

determining expression of a marker of a cell differentiated from definitive endoderm in said cell population at a first time point;

determining expression of the same marker in said cell population at a second time point, wherein said second time point is subsequent to said first time point and wherein said second time point is subsequent to providing said cell population with said candidate differentiation factor; and

determining if expression of the marker in said cell population at said second time point is increased as compared to the expression of the marker in said definitive endoderm cell population at said first time point, wherein an increase in expression of said marker in said cell population indicates that said candidate differentiation factor promotes the differentiation of said human definitive endoderm cells.

2. The method of claim 1, wherein human feeder cells are present in said cell population and wherein at least 10% of the human cells other than said feeder cells are definitive endoderm cells.

3. The method of claim 1, wherein said human definitive endoderm cells comprise at least 90% of the human cells in said cell population.

4. The method of claim 1, wherein said human feeder cells are present in said cell population and wherein at least 90% of the human cells other than said feeder cells are definitive endoderm cells.

5. The method of claim 1, wherein said marker is selected from the group consisting of pancreatic-duodenal homeobox factor-1 (PDX1), homeobox A13 (HOXA13) and homeobox C6 (HOXC6) in response to said candidate differentiation factor.

6. The method of claim 1, wherein said marker is selected from the group consisting of albumin, prospero-related homeobox 1 (PROX1) and hepatocyte specific antigen (HSA) in response to said candidate differentiation factor.

7. The method of claim 1, wherein said marker is thyroid transcription factor 1 (TITF1) in response to said candidate differentiation factor.

8. The method of claim 1, wherein said marker is selected from the group consisting of villin and caudal type homeobox transcription factor 2 (CDX2)) in response to said candidate differentiation factor.

9. The method of claim 1, wherein said first time point is prior to providing said candidate differentiation factor to said cell population.

10. The method of claim 1, wherein said first time point is at approximately the same time as providing said candidate differentiation factor to said cell population.

11. The method of claim 1, wherein said first time point is subsequent to providing said candidate differentiation factor to said cell population.

12. The method of claim 1, wherein expression of said marker is increased.

13. The method of claim 1, wherein expression of said marker is determined by quantitative polymerase chain reaction (Q-PCR).

14. The method of claim 1, wherein expression of said marker is determined by immunocytochemistry.

15. The method of claim 1, wherein said candidate differentiation factor comprises a small molecule.

16. The method of claim 1, wherein said candidate differentiation factor comprises a retinoid.

17. The method of claim 1, wherein said candidate differentiation factor comprises retinoic acid.

18. The method of claim 1, wherein said candidate differentiation factor comprises a polypeptide.

19. The method of claim 1, wherein said candidate differentiation factor comprises a growth factor.

20. The method of claim 1, wherein said candidate differentiation factor comprises FGF-10.

21. The method of claim 1, wherein said candidate differentiation factor comprises FGF-2.

22. The method of claim 1, wherein said candidate differentiation factor comprises Wnt3B.

23. The method of claim 1, wherein said candidate differentiation factor is not a foregut differentiation factor.

24. The method of claim 1, wherein said candidate differentiation factor is not a retinoid.

25. The method of claim 1, wherein said candidate differentiation factor is not retinoic acid.

26. The method of claim 1, wherein said candidate differentiation factor is provided to said cell population at a concentration of between 0.1 ng/ml to 10 mg/ml.

27. The method of claim 1, wherein said candidate differentiation factor is provided to said cell population at a concentration of between 1 ng/ml to 1 mg/ml.

28. The method of claim 1, wherein said candidate differentiation factor is provided to said cell population at a concentration of between 10 ng/ml to 100 μ

g/ml.

29. The method of claim 1, wherein said candidate differentiation factor is provided to said cell population at a concentration of between 100 ng/ml to 10 μ

g/ml.

30. The method of claim 1, wherein said candidate differentiation factor is provided to said cell population at a concentration of 1 μ

g/ml.

31. The method of claim 1, wherein said candidate differentiation factor is provided to said cell population at a concentration of 100 ng/ml.