Mesoderm and definitive endoderm cell populations

• US 20060276420A1
• Filed: 05/19/2003
• Published: 12/07/2006
• Est. Priority Date: 05/17/2002
• Status: Active Grant

• Current Assignee: Icahn School of Medicine at Mount Sinai
• Original Assignee: Icahn School of Medicine at Mount Sinai
• Inventors: Keller, Gordon M, Fehling, Hans Joerg, Kubo, Atsushi, Kouskoff, Valerie
• Granted Patent: US 7,763,466 B2
• Time in Patent Office: Days
• US Class Current: 514/44.R
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 21/04 for myasthenia gravis
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/06 Antianaemics
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/04 Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• 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: G01N 33/5073 Stem cells

Abstract

The present invention provides cell populations that are enriched for mesendoderm and mesoderm, and cell populations that are enriched for endoderm. The cell populations of the invention are useful for generating cells for cell replacement therapy.

Claims

1. A cell population enriched for mesendoderm and mesoderm cells.

2. The cell population of claim 1 comprising at least about 50% mesendoderm and mesoderm cells.

3. The cell population of claim 1 comprising at least about 75% mesendoderm and mesoderm cells.

4. The cell population of claim 1 comprising at least about 90% mesendoderm and mesoderm cells.

5. The cell population of claim 1 obtained by culturing embryonic stem cells in the presence of serum for a time sufficient to obtain cells that express brachyury, and isolating said cells that express brachyury.

6. The cell population of claim 5 wherein said embryonic stem cells are human embryonic stem cells.

7. The cell population of claim 6 wherein said time is about two to about eighteen days.

8. A method of isolating the cell population of claim 1 comprising culturing embryonic stem cells in the presence of serum for a time sufficient to obtain cells that express brachyury, and isolating said cells that express brachyury.

9. The method of claim 8 wherein a selectable marker gene is inserted into the brachury locus of said embryonic stem cells, and cells that express brachury are isolated by selecting for cells that express the selectable marker.

10. The method of claim 8 wherein the selectable marker is enhanced green fluorescence protein.

23. A method of identifying an agent that affects the proliferation, differentiation or survival of mesendoderm and mesoderm cells comprising culturing the cell population of claim 1 in the presence of an agent to be tested and comparing the proliferaton, differentiation or survival of said cells in the presence and absence of said agent, wherein a difference in the presence of said agent is indicative of the identification of an agent that affects the proliferation, differentiation or survival of said cells.

28. A method for generating mammalian cells comprising culturing the cell population of claim 1 under conditions effective for the differentiation of mesoderm into cardiac muscle, vascular smooth muscle, endothelium or hematopoietic cells.

11. A cell population enriched for endoderm cells.

12. The cell population of claim 11 comprising at least about 50% endoderm cells.

13. The cell population of claim 11 comprising at least about 75% endoderm cells.

14. The cell population of claim 11 comprising at least about 90% endoderm cells.

15. The cell population of claim 11 obtained by culturing human embryonic stem cells in the presence of serum for about two to about ten days, isolating cells that express brachyury, and culturing said cells that express brachyury in the absence of serum for about one to about fifteen days.

16. A method of isolating the cell population of claim 11 comprising culturing human embryonic stem cells in the presence of serum for about two to about ten days, isolating cells that express brachyury, and culturing said cells that express brachyury in the absence of serum for about one to about fifteen days.

17. The method of claim 16 wherein a selectable marker gene is inserted into the brachyury locus of said embryonic stem cells, and cells that express brachyury are isolated by selecting for cells that express the selectable marker.

18. The method of claim 17 wherein the selectable marker is enhanced green fluorescence protein.

19. A method of isolating the cell population of claim 11 comprising culturing embryonic stem cells in the absence of serum and in the presence of a sufficient amount of activin to induce differentiation of embryonic stem cells to endoderm, and isolating cells that express brachyury.

21. A method of identifying endodermal cells capable of functioning as mature hepatocytes comprising isolating the cell population of claim 11, transplanting a subpopulation of said population into an animal exhibiting a disorder associated with hepatocyte destruction or dysfunction, and assaying for liver function, wherein an improvement in liver function in said animal is indicative of the identification of endodermal cells capable of functioning as mature hepatocytes.

22. The method of claim 21 wherein said animal is a fumarylacetoacetate deficient mouse.

24. A method of identifying an agent that affects the proliferation, differentiation or survival of enoderm cells comprising culturing the cell population of claim 11 in the presence of an agent to be tested and comparing the proliferation, differentiation or survival of said cells in the presence and absence of said agent, wherein a difference in the presence of said agent is indicative of the identification of an agent that affects the proliferation, differentiation or survival of said cells.

29. A method for generating mammalian cells comprising culturing the cell population of claim 11 under conditions effective for the differentiation of endoderm into liver cells or pancreatic cells.

20. A method of isolating a cell population enriched for mesoderm cells comprising culturing embryonic stem cells in the absence of serum and in the presence of activin in an amount sufficient to induce differentiation of embryonic stem cells to mesoderm but insufficient to induce differentiation of embryonic stems cells to endoderm, and isolating cells that express brachyury.

25. A method of identifying genes involved in cell differentiation comprising incubating embryonic stem cells under conditions to obtain cells that express brachyury, isolating two populations of cells that express brachyury after two different amounts of time in culture, comparing gene expression profiles in said two populations, and identifying genes that are uniquely expressed in a population, wherein identification of a gene that is uniquely expressed in a population is indicative of identification of a gene involved in cell differentiation.

26. A method of making antibodies that recognize cells that express brachyury but not cells that do not express brachyury comprising inserting a selectable marker into the brachyury locus of embryonic stem cells, culturing said cells, isolating cells that express brachyury by selecting for cells that express the selectable marker to provide an isolated population of cells that express brachyury, and raising antibodies against said isolated population.

27. The method of claim 26 wherein the antibodies are monoclonal or polyclonal.

30. A transgenic non-human mammal having a genome comprising a transgene in which DNA encoding a selectable marker is present in the brachyury locus and the selectable marker is expressed in cells that express brachyury.