Differentiation of pluripotent embryonic stem cells

• US 8,012,751 B2
• Filed: 03/31/2006
• Issued: 09/06/2011
• Est. Priority Date: 03/31/2005
• Status: Active Grant

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Zwaka, Thomas P., Thomson, James A.
• Primary Examiner(s): DRISCOLL, LORA E BARNHART
• Application Number:
  US11/395,657
• Publication Number:
  US 20060223179A1
• Time in Patent Office: 1,985 Days
• Field of Search: None
• US Class Current: 435/377
• CPC Class Codes: C12N 2501/48 Regulators of apoptosis
• CPC Class Codes: C12N 2501/70 Enzymes
• 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

Abstract

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.

Claims

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 μ

M to 200 μ

M; 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).

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.

10. The method of claim 1 or 9 wherein the inhibitor is a peptide inhibitor.

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.

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 μ

M to 200 μ

M; and

(c) yielding the population of multipotent cells.

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).