DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELLS

• US 20170009212A1
• Filed: 09/23/2016
• Published: 01/12/2017
• Est. Priority Date: 08/31/2010
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Inventors: Fryer, Benjamin
• Granted Patent: US 9,951,314 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C12N 2500/34 Sugars
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/19 Growth and differentiation factors [GDF]
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• CPC Class Codes: C12N 5/0678 Stem cells; Progenitor cells; Precursor cells

Abstract

The present invention provides methods to promote the differentiation of pluripotent stem cells into insulin producing cells. In particular, the present invention provides a method to produce a population of cells, wherein greater than 80% of the cells in the population express markers characteristic of the definitive endoderm lineage.

Claims

1. A method for generating a population of cells wherein greater than 80% of the cells in the population express markers characteristic of the definitive endoderm lineage, comprising the steps of:

a. Culturing a population of pluripotent stem cells,

b. Differentiating the population of pluripotent stem cells to a population of cells wherein greater than 80% of the cells in the population express markers characteristic of the definitive endoderm lineage in medium supplemented with GDF-8, 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜

2,6˜

.1˜

8,12˜

]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one and glucose at a concentration that does not exceed 10.5 mM.

3. The method of claim 1, wherein the cells express CXCR4 and CD99.

4. The method of claim 1, wherein the cells expressing markers characteristic of the definitive endoderm lineage are definitive endoderm cells.

5. The method of claim 1, wherein the pluripotent stem cells are human pluripotent stem cells.

6. The method of claim 1, wherein the pluripotent stem cells are human embryonic stem cells.

7. The method of claim 1, wherein the medium has a pH of 7.6 or higher.

8. The method of claim 1, wherein the medium comprises about 100 ng/ml of GDF-8.

9. The method of claim 1, wherein the medium is serum free.

10. The method of claim 1, wherein the step of differentiation comprises

culturing the pluripotent stem cells for about one day in serum-free medium supplemented with about 100 ng/ml GDF-8, about 2.5 μ

M 2.5 μ

M of 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜

2,6˜

.1˜

8,12˜

]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one and glucose at a concentration that does not exceed 10.5 mM followed by

culturing the cells for about three days in serum-free medium supplemented with about 100 ng/ml GDF-8 and glucose at a concentration that does not exceed 10.5 mM.

2. The method of claim 2, wherein the concentration of glucose does not exceed 5.5 mM.

11. A method of differentiating pluripotent stem cells into definitive endoderm cells comprising culturing pluripotent stem cells in a medium supplemented with GDF-8, 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜

2,6˜

.1˜

8,12˜

]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one and glucose at a concentration that does not exceed 10.5 mM.

12. The method of claim 11, wherein the method further comprises culturing the pluripotent stem cells prior to differentiating.

13. The method of claim 11, wherein the concentration of glucose does not exceed 5.5 mM.

14. The method of claim 11, wherein the pluripotent stem cells are human pluripotent stem cells.

15. The method of claim 11, wherein the pluripotent stem cells are human embryonic stem cells.

16. The method of claim 11, wherein the medium has a pH of 7.6 or higher.

17. The method of claim 11, wherein the medium comprises about 100 ng/ml of GDF-8.

18. The method of claim 11, wherein the medium is serum free.