Differentiation of pluripotent embryonic stem cells
The invention relates to a method to induce primate embryonic stem cells to differentiate into a relatively homogenous population of mesendoderm cells by treatment with caspase-like inhibitors. Also described is a population of mesendoderm cells obtained therefrom. The embryonic stem cell derived mesendoderm cells have the general morphological and cell surface marker characteristics of mesendoderm cells.
1. A method for directing the differentiation of human embryonic stem cells into a population of multipotent cells comprising the steps of
culturing a population of human embryonic stem cells for 2 to 3 days in a culture medium containing at least one caspase inhibitor that inhibits cleavage of poly(ADPribosyl) polymerase (PARP-1), wherein the inhibitor concentration is between 80 μM to 200 μM; and
yielding the population of multipotent cells.
2. The method of claim 1 wherein the medium for culturing human embryonic stem cells into a population of multipotent cells includes mammalian serum.
3. The method of claim 1 wherein the at least one inhibitor is a tetrapeptide inhibitor.
4. The method of claim 3 wherein the tetrapeptide inhibitor is N-benzyloxycarbonyl-Asp-Glu-Val-Asp fluoromethylketone (DEVD.FMK).
5. A multipotent cell population, wherein at least 75% of the cells are Oct4 negative, and exhibit uniform, spindle shaped-morphology with a small nucleus, reduced caspase activity, reduced ability to bind Annexin-V, and increased expression of mesoderm and endoderm genes selected from the group consisting of brachyury variant A, BMP4, GATA-3, WNT5A, WNT3, Nodal, Scl, Flk and DKK4 relative to a population of human embryonic stem cells.
6. The cell culture of claim 5 , wherein over 90% of the cells in the culture test negative for caspase-3 activity.
7. The method of claim 1 wherein the at least one inhibitor inhibits caspase-3 activity.
8. The method of claim 1 wherein at least 75% of the multipotent cells in culture are Oct4 negative, and exhibit uniform, spindle shaped morphology with a small nucleus, reduced caspase activity, reduced ability to bind Annexin-V, and increased expression of mesoderm and endoderm genes, selected from the group consisting of brachyury variant A, BMP4, GATA-3, WNT5A, WNT3, Nodal, Scl, Flk and DKK4 relative to human embryonic stem cells.
9. A method of producing a population of multipotent cells comprising the steps of
a) providing a culture of human embryonic stem cells;
b) culturing the cell population of step (a) for 2 to 3 days in a culture medium containing at least one caspase inhibitor that inhibits cleavage of poly(ADPribosyl) polymerase PARP-1), and inhibits caspase-3 activity, wherein the inhibitor concentration is between 80 μM to 200 μM; and
(c) yielding the population of multipotent cells.
10. The method of claim 1 or 9 wherein the inhibitor is a peptide inhibitor.
11. The method of claim 9 wherein the inhibitor is a tetrapeptide inhibitor.
12. The method of claim 11 wherein the tetrapeptide inhibitor is N-benzyloxycarbonyl-Asp-Glu-Val-Asp fluoromethylketone (DEVD.FMK).