CULTURE OF STEM CELLS

• US 20080171385A1
• Filed: 01/17/2008
• Published: 07/17/2008
• Est. Priority Date: 01/17/2007
• Status: Active Grant

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Thomson, James A., Bergendahl, Veit
• Granted Patent: US 7,892,830 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• 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

While culture medium and systems have been described that permit the culture and proliferation of human embryonic stem cells in feeder free and animal product free conditions, these conditions will not readily support cloning of an embryonic stem cell culture meaning, at least here, the initiation of a sub-culture using one or a very few originating cells. It has been found here that a class of small molecules that are inhibitors of kinase enzymes will increase the efficiency of cloning of stem cell cultures sufficiently to make such cloning practical in the defined medium and in other media as well.

Claims

1. A method of creating a clonal culture of human pluripotent stem cells comprising the steps of

providing a culture of human pluripotent stem cells in culture in a culture medium in a culture vessel;

selecting a single cell from the stem cells in culture; and

initiating a new culture of the stem cells in a culture medium in a culture vessel with the single cell, the culture medium including an agent selected to increase the cloning efficiency of the stem cell culture, the agent being a small molecule identified by screening for its capacity to increase the cloning efficiency of the stem cell culture.

2. A method as claimed in claim 1, wherein the agent comprises at least a pyridine.

3. A method as claimed in claim 1, wherein the agent is a kinase inhibitor.

4. A method as claimed in claim 1, wherein the agent is a protein kinase A inhibitor, a protein kinase C inhibitor, a protein kinase G inhibitor or a Rho-associated kinase (ROCK) inhibitor.

5. A method as claimed in claim 1, wherein the agent is selected from the group consisting of H-7, iso H-7, H-8, H-9, H-89, HA-100, HA-1004, HA-1077, H-1152 and Y-27632.

6. A method as claimed in claim 1, wherein the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.

7. A method as claimed in claim 1, where the culture medium in the step of providing and in the step of initiating are both a defined medium that supports growth and maintenance of pluripotent, undifferentiated stem cells without feeder cells or serum.

8. A method of creating a clonal culture of human pluripotent stem cells comprising the steps of:

providing a culture of human pluripotent stem cells in culture in a defined medium in a culture vessel;

selecting a single cell from the pluripotent cells in culture; and

initiating a new culture of the pluripotent cells in medium in a culture vessel with the single cell, the culture medium including an agent selected to increase the cloning efficiency of the stem cell culture, the agent being of the formula;

9. A method as claimed in claim 8, wherein the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.

10. A method of creating a clonal culture of human pluripotent stem cells comprising the steps of:

providing a culture of human pluripotent stem cells in culture in a defined medium in a culture vessel;

selecting a single cell from the stem cells in culture; and

initiating a new culture of the human pluripotent stem cells in the defined medium in a culture vessel with the single cell, the culture medium including an agent selected to increase the cloning efficiency of the stem cell culture, the agent being selected from the group consisting of H-7, iso H-7, H-8, H-9, H-89, HA-100, HA-1004, HA-1077, H-1152 and Y-27632.

11. A method as claimed in claim 10, wherein the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.

12. An human pluripotent stem cell culture comprising:

a culture vessel;

a culture medium contained in the vessel;

human pluripotent stem cells growing in the culture medium; and

the culture medium containing an agent selected to increase the cloning efficiency of the stem cell culture, the agent being a kinase inhibitor.

13. A human pluripotent stem cell culture of claim 12, wherein the agent is selected from the group consisting of H-7, iso H-7, H-8, H-9, H-89, HA-100, HA-1004, HA-1077, H-1152 and Y-27632.

14. A human pluripotent stem cell culture of claim 12, wherein the stem cells are embryonic stem cells or induced pluripotent stem cells.

15. An improvement in a medium in which to culture human pluripotent stem cells, the improvement comprising including in the medium an agent selected to increase the cloning efficiency of the stem cell culture, wherein the agent is selected from the group consisting of H-7, iso H-7, H-8, H-9, H-89, HA-100, HA-1004, HA-1077, H-1152 and Y-27632.