METHODS AND COMPOSITIONS FOR ENHANCED DIFFERENTIATION FROM EMBRYONIC STEM CELLS

• US 20100261277A1
• Filed: 06/13/2008
• Published: 10/14/2010
• Est. Priority Date: 06/15/2007
• Status: Active Grant

• Current Assignee: Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: Powers, Daryl, Millman, Jeffrey R., Colton, Clark K.
• Granted Patent: US 9,029,147 B2
• Time in Patent Office: Days
• US Class Current: 435/377
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/34 Sugars
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2533/30 Synthetic polymers thermoreactive polymers, e.g. PNIPAm, C12N2539/10
• CPC Class Codes: C12N 5/0657 Cardiomyocytes; Heart cells

Abstract

The invention provides methods for differentiating pluripotent stem cells such as ES cells with improved progenitor and differentiated cell yield using low oxygen conditions and optionally in the absence of exogenously added differentiation factors.

Claims

1. A method for enhancing mesoderm differentiation from pluripotent stem cells comprising

culturing pluripotent stem cells at an oxygen partial pressure that is less than 142 mmHg for a time period sufficient to induce mesoderm specific gene expression.

3. The method of claim 1, wherein the pluripotent stem cells are embryonic stem (ES) cells.

4. The method of claim 1, wherein the pluripotent stem cells are cultured for at least 3 days.

5. The method of claim 1, wherein the pluripotent stem cells are cultured for at least 6 days.

6. The method of claim 1, wherein the pluripotent stem cells are cultures for 3-6 days.

7. The method of claim 1, wherein the pluripotent stem cells are cultured for 5-15 days.

8. The method of claim 1, wherein the oxygen partial pressure is less than 50 mmHg.

9. The method of claim 1, wherein the oxygen partial pressure is less than 40 mmHg.

10. The method of claim 1, wherein the oxygen partial pressure is less than 10 mmHg.

11. The method of claim 1, further comprising a second culture step at an oxygen partial pressure that is greater than the first oxygen partial pressure.

12. The method of claim 11, wherein the second culture step is performed at an oxygen partial pressure of 142 mmHg.

13. The method of claim 11, wherein the second culture step is performed for at least 10 days.

2. A method for enhancing endoderm differentiation from pluripotent stem cells comprising

culturing pluripotent stem cells at an oxygen partial pressure that is less than 142 mmHg for a time period sufficient to induce endoderm specific gene expression.

14. (canceled)

15. A method for producing or enriching cardiomyocytes comprising

performing a first culture step comprising culturing pluripotent stem cells at a first oxygen partial pressure that is less than 142 mmHg for a first time period, and

performing a second culture step comprising culturing the pluripotent stem cells at a second oxygen partial pressure that is greater than the first oxygen partial pressure for a second time period.

16-36. -36. (canceled)

37. A method for producing or enriching cardiomyocytes comprising

performing a first culture step comprising culturing ES cells at a first oxygen partial pressure that is about 36 mmHg for a first time period of 6 days,

performing a second culture step comprising culturing the ES cells at a second oxygen partial pressure that is 142 mmHg for a second time period that is about 15 days.

38-39. -39. (canceled)

40. A method for enhancing cardiomyocyte production comprising

culturing pluripotent stem cells at an oxygen partial pressure that is less than 142 mmHg for a time sufficient to produce cardiomyocytes.

41-55. -55. (canceled)

56. A method for enhancing cardiomyocyte enrichment comprising

culturing pluripotent stem cells at an oxygen partial pressure that is less than 50 mmHg for a time sufficient to produce cardiomyocytes.

57-69. -69. (canceled)

70. A composition comprising

pluripotent stem cells in a culture medium within a culture vessel having an oxygen-permeable membrane.

71-74. -74. (canceled)

75. A method for controlling oxygen partial pressure of pluripotent stem cells in culture comprising

culturing pluripotent stem cells in a liquid medium in contact with a gas phase, wherein

pO_2cell is within 0-15 mmHg of pO_2gas.

76-78. -78. (canceled)

79. A method for enhancing hematopoietic differentiation of pluripotent stem cells comprising

culturing pluripotent stem cells in low oxygen partial pressure for a time period sufficient to generate hematopoietic cells.

80-82. -82. (canceled)