MESENDODERM AND MESODERM 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 . 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 brachyury locus of said embryonic stem cells, and cells that express brachyury 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.
11 - 22 . (canceled)
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 proliferation, differentiation or survival of said cells or the progeny 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 or the progeny of said cells.
24 - 27 . (canceled)
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.
29 . (canceled)
30 . (canceled)
31 . A cell population enriched for brachyury + mesoderm cells.
32 . The cell population of claim 31 that generates cardiomyocytes under cardiomyocyte differentiation-inducing conditions.
33 . The cell population of claim 31 that generates skeletal muscle cells under skeletal muscle cell differentiation-inducing conditions.
34 . The cell population of claim 31 that generates vascular smooth muscle cells under vascular smooth muscle cell differentiation-inducing conditions.
35 . The cell population of claim 31 that generates endothelial cells under endothelial cell differentiation-inducing conditions.
36 . The cell population of claim 31 that generates hematopoietic cells under hematopoietic cell differentiation-inducing conditions.
37 . A method of generating differentiated cells from embryonic stem cells comprising culturing embryonic stem cells for a time sufficient to obtain cells that express brachyury, isolating said cells that express brachyury, and culturing said isolated cells under conditions sufficient for differentiation to more mature cells.
38 . A cell population of brachyury + /Flk-1 + cells isolated from embryoid bodies.
39 . The cell population of claim 38 wherein the cells express at least one of Scl, Runx1, nodal, BMP2 and BMP4.
40 . A cell population of brachyury + /Flk-1 − cells isolated from embryoid bodies.
41 . The cell population of claim 39 wherein the cells express at least one of Wnt3a, Wnt8a and BMP2.
42 . A cardiomyocyte cell population made by obtaining brachyury + mesoderm cells and culturing said brachyury + mesoderm cells under conditions effective for differentiation of mesoderm to cardiomyocytes.
43 . A vascular smooth muscle cell population made by obtaining brachyury + mesoderm cells and culturing said brachyury + mesoderm cells under conditions effective for differentiation of mesoderm to vascular smooth muscle cells.
44 . A skeletal muscle cell population made by obtaining brachyury + mesoderm cells and culturing said brachyury + mesoderm cells under conditions effective for differentiation of mesoderm to skeletal muscle cells.
45 . An endothelial cell population made by obtaining brachyury + mesoderm cells and culturing said brachyury + mesoderm cells under conditions effective for differentiation of mesoderm to endothelial cells.
46 . A hematopoietic cell population made by obtaining brachyury + mesoderm cells and culturing said brachyury + mesoderm cells under conditions effective for differentiation of mesoderm to hematopoietic cells.
47 . A method of identifying an agent that affects the proliferation, differentiation or survival of cells derived from mesoderm comprising culturing the cell population of claim 1 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.
48 . The method of claim 47 wherein said differentiated cells are selected from the group consisting of cardiac muscle cells, vascular smooth muscle cells, skeletal muscle cells, endothelial cells and hematopoietic cells.
49 . A method of cell replacement comprising transplanting mesoderm-derived cells into a mammal in need of cell replacement wherein said mesoderm-derived cells are obtained by culturing the cell population of claim 1 under conditions effective for the differentiation of mesoderm.
50 . The method of claim 49 wherein the mesoderm-derived cells are selected from the group consisting of cardiac muscle cells, vascular smooth muscle cells, skeletal muscle cells, endothelial cells and hematopoietic cells.