Differentiation of human embryonic stem cells

• US 9,528,090 B2
• Filed: 08/17/2011
• Issued: 12/27/2016
• Est. Priority Date: 08/31/2010
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Inventors: Rezania, Alireza
• Primary Examiner(s): HILL, KEVIN KAI
• Application Number:
  US13/211,959
• Publication Number:
  US 20120052575A1
• Time in Patent Office: 1,959 Days
• Field of Search: 435/366, 435/377
• US Class Current: 1/1
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/19 Growth and differentiation factors [GDF]
• CPC Class Codes: C12N 2501/33 Insulin together with transferrin C12N2500/25; Insulin-like growth factors C12N2501/105
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0608 Germ cells production of embryos, nuclear transfer A01K67/027
• CPC Class Codes: C12N 5/0676 Pancreatic cells
• 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 85% of the cells in the population express markers characteristic of the definitive endoderm lineage.

Claims

1. An in vitro cell culture comprising an isolated population of cells and a culture medium suitable for differentiating pluripotent stem cells,

wherein greater than 85% of the cells are definitive endoderm cells,

wherein said population of cells is obtained by differentiating in vitro pluripotent stem cells,

wherein the culture medium lacks serum and is supplemented with BSA, GDF-8, 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜

2,6˜

0.1˜

8,12˜

]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one and a factor selected from the group consisting of insulin and from about 1 ng/ml to about 50 ng/ml of IGF-1, and

wherein said definitive endoderm cells express CXCR4 and do not express CD9.

2. The in vitro culture of claim 1, wherein the isolated population of cells is obtained without further purifying the cells after differentiation.

3. The in vitro culture of claim 1, wherein the medium is further supplemented with activin A and Wnt-3.

4. The in vitro culture of claim 1, wherein the medium is chemically-defined.

5. A method for generating a population of cells wherein greater than 85% of the cells in the population are definitive endoderm cells, comprising the steps of:

a. culturing a population of pluripotent stem cells; and

b. differentiating the population of pluripotent stem cells to a population of cells wherein greater than 85% of the cells in the population are definitive endoderm cells in medium lacking serum and supplemented with BSA, GDF-8, 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜

2,6˜

0.1˜

8,12˜

]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one and a factor selected from the group consisting of insulin and from about 1 ng/ml to about 50 ng/ml of IGF-1.

6. The method of claim 5, wherein the population of pluripotent stem cells is differentiated in the medium lacking serum and supplemented with BSA and a factor selected from the group consisting of insulin and from about 1 ng/ml to about 50 ng/ml of IGF-1 for a period of at least 6 days.

7. The method of claim 5, wherein the population of pluripotent stem cells is differentiated in the medium lacking serum and supplemented with BSA and a factor selected from the group consisting of insulin and from about 1 ng/ml to about 50 ng/ml of IGF-1 for a period of at least 7 days.

8. The method of claim 5, wherein the step of differentiating comprises treating the pluripotent stem cells with a medium lacking serum and supplemented with BSA, GDF-8, 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜

2,6˜

0.1˜

8,12˜

]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one and from about 1 ng/ml to about 50 ng/ml of IGF-1.

9. The method claim 5, wherein the population of cells is obtained without further purifying the cells after differentiation.

10. The method of claim 5, wherein the step of differentiating comprises treating the pluripotent stem cells with a medium lacking serum and supplemented with BSA, GDF-8, 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜

2,6˜

0.1˜

8,12˜

]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one and insulin.

11. The method of claim 5, wherein the step of differentiating comprises treating the pluripotent stem cells with a medium lacking serum and supplemented with BSA, from about 5 ng/ml to about 500 ng/ml of GDF-8, 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜

2,6˜

0.1˜

8,12˜

]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one and a factor selected from the group consisting of insulin and from about 1 ng/ml to about 50 ng/ml of IGF-1.

12. The method of claim 5, wherein the step of differentiating comprises treating the pluripotent stem cells with a medium lacking serum and supplemented with from about 0.5 to about 2% BSA, GDF-8, 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜

2,6˜

0.1˜

8,12˜

]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one and a factor selected from the group consisting of insulin and from about 1 ng/ml to about 50 ng/ml of IGF-1.

13. The method of claim 5, wherein the step of differentiating comprises treating the pluripotent stem cells with a medium lacking serum and supplemented with BSA, GDF-8, 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜

2,6˜

0.1˜

8,12˜

]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one and from about 1 ng/ml to about 100 ng/ml of insulin.

14. The method of claim 5, wherein the step of differentiating comprises treating the pluripotent stem cells with a medium lacking serum and supplemented with about 2% BSA, GDF-8, 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜

2,6˜

0.1˜

8,12˜

]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one and from about 1 ng/ml to about 50 ng/ml of IGF-1.

15. The method of claim 5, wherein the medium is chemically-defined.

16. An in vitro culture comprising an isolated population of cells in which greater than 85% of the cells are definitive endoderm cells and a cell culture medium,

wherein said population of cells is obtained by differentiating pluripotent stem cells into definitive endoderm cells, and

wherein the cell culture medium lacks serum and is supplemented with BSA, GDF-8, 14- and Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜

2,6˜

0.1-8,12˜

]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one and either insulin or from about 1 ng/ml to about 50 ng/ml of IGF-1.

17. The in vitro culture of claim 16, wherein the population is obtained by differentiating pluripotent stem cells into definitive endoderm cells by treating the pluripotent stem cells with the medium lacking serum and supplemented with BSA, GDF-8, 14- and Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜

2,6˜

0.1˜

8,12˜

]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one and either insulin or from about 1 ng/ml to about 50 ng/ml of IGF-1.