Methods and compositions for increased safety of stem cell-derived populations

• US 9,388,381 B2
• Filed: 07/09/2010
• Issued: 07/12/2016
• Est. Priority Date: 07/09/2009
• Status: Active Grant

• Current Assignee: Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: Millman, Jeffrey R., Colton, Clark K.
• Primary Examiner(s): Claytor, Renee
• Assistant Examiner(s): BERKE-SCHLESSEL, DAVID W
• Application Number:
  US13/382,965
• Publication Number:
  US 20120219532A1
• Time in Patent Office: 2,195 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12N 2500/02 Atmosphere, e.g. low oxygen conditions
• 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
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

Abstract

The invention provides methods and compositions relating to differentiated cell populations that derive from pluripotent stem cells. The methods relate to reducing the number of residual stem cells present in such populations. The compositions include differentiated cell populations that contain reduced number of stem cells or that contain no stem cells. Pluripotent stem cells may be reduced in number and/or function through exposure to low oxygen levels.

Claims

1. A method for reducing the residual pluripotent stem cells in an in vitro differentiated cell population, comprising

culturing an in vitro differentiated cell population at an oxygen partial pressure that is less than or about 50 mm Hg for 10 days or more;

harvesting the differentiated cell population after the culturing step;

wherein the harvested cell population comprises less than 1 pluripotent stem cell per 10³ cells.

2. The method of claim 1, wherein the in vitro differentiated cell population is isolated from a differentiation culture.

3. The method of claim 1, wherein the in vitro differentiated cell population is derived from an embryonic stem cell or an induced pluripotent stem cell.

4. The method of claim 1, wherein the in vitro differentiated cell population comprises cardiomyocyte lineage cells.

5. The method of claim 1, wherein the in vitro differentiated cell population comprises cardiomyocyte precursor cells.

6. The method of claim 1, wherein the oxygen partial pressure is 4-10 mmHg.

7. The method of claim 1, wherein the oxygen partial pressure is 10-50 mmHg.

8. The method of claim 1, wherein the oxygen partial pressure is 36 mmHg.

9. The method of claim 1, wherein the in vitro differentiated cell population is cultured for 20 days.

10. The method of claim 1, wherein the culturing step reduces pluripotent stem cells in the in vitro differentiated cell population by at least 2-fold.

11. The method of claim 1, wherein the in vitro differentiated cell population is cultured in a culture vessel comprising an oxygen permeable membrane.

12. The method of claim 1, wherein the in vitro differentiated cell population is cultured in a culture vessel comprising a silicon rubber membrane.

13. A differentiated cell population obtained according to the method of claim 1.

14. A pharmaceutical preparation comprising the differentiated cell population of claim 13.

15. The method of claim 1, wherein the harvested differentiated cell population comprises less than 1 pluripotent stem cell per 10⁴ cells.

16. The method of claim 1, wherein the harvested differentiated cell population comprises less than 1 pluripotent stem cell per 10⁵ cells.

17. The method of claim 1, further comprising measuring the residual pluripotent stem cells in the cultured differentiated cell population or in the harvested differentiated cell population.