MESODERM AND DEFINITIVE ENDODERM CELL POPULATIONS
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.
1 .- 15 . (canceled)
16 . A method of isolating a cell population enriched for endoderm cells 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 a cell population enriched for endoderm cells comprising culturing embryonic stem cells in the absence of serum and in the presence of at least 30 ng/ml of activin for two to ten days to induce differentiation of embryonic stem cells to mesendoderm cells that express brachyury, isolating said mesendoderm cells that express brachyury, and culturing said isolated cells to obtain endoderm cells that do not express brachyury.
20 .- 23 . (canceled)
24 . A method of identifying an agent that affects the proliferation, differentiation or survival of endoderm cells comprising generating a cell population of enriched for endoderm cells by the method of any one of claims 16 , 19 , 33 and 41 , culturing the cell population 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.
25 .- 28 . (canceled)
29 . A method for generating mammalian cells comprising culturing embryonic stem cells in the absence of serum and in the presence of at least 30 ng/ml of activin for two to ten days to induce differentiation of embryonic stem cells to mesendoderm cells that express brachyury, isolating said mesendoderm cells that express brachyury, culturing said isolated cells to obtain endoderm cells that do not express brachyury, and culturing the endoderm cells under conditions effective for the differentiation of endoderm into liver cells or pancreatic cells.
30 . (canceled)
31 . The method of claim 19 , wherein the embryonic stem cells are cultured in the presence of 100 ng/ml of the activin.
32 . The method of claim 29 , wherein the embryonic stem cells are cultured in the presence of 100 ng/ml of the activin.
33 . A method of generating a cell population enriched for endoderm cells comprising culturing embryonic stem cells in the presence of at least 30 ng/ml of activin for two to ten days to induce differentiation of the embryonic stem cells to endoderm.
34 . The method of claim 33 , wherein the embryonic stem cells are cultured in the presence of 100 ng/ml of the activin.
35 . The method of claim 33 further comprising isolating endoderm cells by isolating cells that express at least one of HNF3β, Mixl-1, Sox17, Hex-1 or pdx-1.
36 . The method of claim 33 , wherein the embryonic stem cells are cultured in the presence of from about 30 ng/ml to about 100 ng/ml of the activin.
37 . The method of claim 33 , wherein the embryonic stem cells are cultured in the presence of about 100 ng/ml of the activin.
38 . The method of claim 33 , wherein the cells are cultured in the absence of serum.
39 . The method of claim 33 , wherein the embryonic stem cells are human embryonic stem cells.
40 . The method of claim 33 , wherein the embryonic stem cells are human embryonic stem cells and the embryonic stem cells are cultured in the presence of about 100 ng/ml of the activin.
41 . A method of generating a cell population enriched for endoderm cells comprising culturing mammalian embryoid bodies in the presence of a concentration of activin of at least 30 ng/ml, whereby said embryoid bodies differentiate to generate a cell population enriched for endoderm.
42 . The method of claim 41 further comprising isolating endoderm cells wherein said endoderm cells express at least one of HNF3β, Mixl-1, Sox17, Hex-1 and pdx-1.
43 . The method of claim 41 , wherein the concentration of activin is from 30 ng/ml to about 100 ng/ml.
44 . The method of claim 41 , wherein the concentration of activin is about 100 ng/ml.
45 . The method of claim 41 further comprising culturing the embryoid bodies in the absence of serum.
46 . The method of claim 41 , wherein the embryoid bodies are human embryoid bodies.
47 . The method of claim 41 , wherein the embryoid bodies are human embryoid bodies and the concentration of activin is about 100 ng/ml.
48 . A method of identifying an agent that affects the proliferation, differentiation or survival of cells derived from endoderm comprising generating a cell population enriched for endoderm cells by the method of any one of claims 16 , 19 , 33 and 41 , culturing the cell population under conditions effective for differentiation to obtain a population of differentiated cells, culturing the population of differentiated cells in the presence of an agent to be tested and comparing the proliferation, differentiation or survival of said differentiated 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 differentiated cells.
49 . The method of claim 48 , wherein said differentiated cells are hepatocytes, pancreatic cells or lung cells.