Methods and compositions for enhanced differentiation from embryonic stem cells

• US 9,029,147 B2
• Filed: 06/13/2008
• Issued: 05/12/2015
• Est. Priority Date: 06/15/2007
• Status: Active Grant

• Current Assignee: Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: Colton, Clark K., Millman, Jeffrey R., Powers, Daryl E.
• Primary Examiner(s): BERTOGLIO, VALARIE E
• Application Number:
  US12/664,763
• Publication Number:
  US 20100261277A1
• Time in Patent Office: 2,524 Days
• Field of Search: None
• 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 a cell surface oxygen partial pressure that is less than 142 mmHg throughout a time period sufficient to induce mesoderm specific gene expression, wherein the cell surface oxygen partial pressure is about gas phase oxygen partial pressure,

wherein the pluripotent stem cells are mouse or human pluripotent stem cells,

wherein the pluripotent stem cells are cultured on a silicone rubber membrane.

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

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

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

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

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

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

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

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

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

11. The method of claim 10, wherein the second culture step is performed at a second cell surface oxygen partial pressure of 142 mmHg.

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

13. A method for producing or enriching cardiomyocytes comprising

performing a first culture step comprising culturing ES cells at a first cell surface 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 cell surface oxygen partial pressure that is 142 mmHg for a second time period that is about 15 days,

wherein cell surface oxygen partial pressure is about gas phase partial pressure in the first and second culture steps,

wherein the ES cells are mouse or human ES cells.

14. The method of claim 13, wherein the ES cells are cultured on an oxygen permeable membrane.