Differentiation of human embryonic stem cells

• US 9,951,314 B2
• Filed: 09/23/2016
• Issued: 04/24/2018
• 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
• Primary Examiner(s): HILL, KEVIN KAI
• Application Number:
  US15/274,749
• Publication Number:
  US 20170009212A1
• Time in Patent Office: 578 Days
• 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; and

b) differentiating the population of pluripotent stem cells in medium with a pH of 7.6 or higher and supplemented with;

i) GDF-8,

ii) 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

iii) glucose at a concentration that does not exceed 10.5 mM, thereby generating a population of cells wherein greater than 80% of the cells in the population express markers characteristic of the definitive endoderm lineage.

2. The method of claim 1, wherein the concentration of glucose does not exceed 5.5 mM.

3. The method of claim 1, wherein the population of cells differentiated from the population of pluripotent stem 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 comprises about 100 ng/ml of GDF-8.

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

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

a) culturing the pluripotent stem cells for about one day in a serum-free medium supplemented with

i) about 100 ng/ml GDF-8,

ii) 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

iii) glucose at a concentration that does not exceed 10.5 mM, followed by

b) culturing the cells for about three days in a serum-free medium supplemented with

i) about 100 ng/ml GDF-8 and

ii) glucose at a concentration that does not exceed 10.5 mM.

10. A method of differentiating pluripotent stem cells into definitive endoderm cells comprising culturing pluripotent stem cells in a medium with a pH of 7.6 or higher and supplemented with

i) GDF-8,

ii) 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

iii) glucose at a concentration that does not exceed 10.5 mM.

11. The method of claim 10, wherein the method further comprises culturing the pluripotent stem cells prior to differentiating.

12. The method of claim 10, wherein the concentration of glucose does not exceed 5.5 mM.

13. The method of claim 10, wherein the pluripotent stem cells are human pluripotent stem cells.

14. The method of claim 10, wherein the pluripotent stem cells are human embryonic stem cells.

15. The method of claim 10, wherein the medium comprises about 100 ng/ml of GDF-8.

16. The method of claim 10, wherein the medium is serum free.