DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELLS

• US 20150252327A1
• Filed: 05/21/2015
• Published: 09/10/2015
• Est. Priority Date: 11/27/2007
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Lifescan Incorporated (Duv Holding Corporation)
• Inventors: Rezania, Alireza
• Granted Patent: US 9,969,982 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61P 1/18 for pancreatic disorders, e.g. pancreatic enzymes
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• CPC Class Codes: C12N 2501/117 Keratinocyte growth factors (KGF-1, i.e. FGF-7; KGF-2, i.e. FGF-12)
• CPC Class Codes: C12N 2501/12 Hepatocyte growth factor [HGF]
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/335 Glucagon; Glucagon-like peptide [GLP]; Exendin
• CPC Class Codes: C12N 2501/385 of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
• CPC Class Codes: C12N 2501/40 Regulators of development
• CPC Class Codes: C12N 2501/41 Hedgehog proteins; Cyclopamine (inhibitor)
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/42 Notch; Delta; Jagged; Serrate
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2501/998 Proteins not provided for elsewhere
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• 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 and the products related to or resulting from such methods. In particular, the present invention provides an improved method for the formation of pancreatic hormone expressing cells and pancreatic hormone secreting cells. In addition, the present invention also provides methods to promote the differentiation of pluripotent stem cells without the use of a feeder cell layer and the products related to or resulting from such methods. The present invention also provides methods to promote glucose-stimulated insulin secretion in insulin-producing cells derived from pluripotent stem cells.

Claims

1. A method for producing cells expressing markers characteristic of the pancreatic endoderm lineage, comprising the steps of:

a. Culturing the pluripotent stem cells,

b. Differentiating the pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage, and

c. Differentiating the cells expressing markers characteristic of the definitive endoderm lineage into cells expressing markers characteristic of the pancreatic endoderm lineage by treating the cells expressing markers characteristic of the definitive endoderm lineage with at least one factor selected from the group consisting of retinoic acid, FGF-2, FGF-4, FGF-7, FGF-10, a sonic hedgehog inhibitor, a factor capable of inhibiting BMP, and a netrin.

2. The method of claim 1, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with at least one factor selected from the group consisting of retinoic acid, FGF-2, FGF-4, FGF-7, FGF-10, a sonic hedgehog inhibitor, a factor capable of inhibiting BMP, and a netrin for about one to about six days.

3. The method of claim 1, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with at least one factor selected from the group consisting of retinoic acid, FGF-2, FGF-4, FGF-7, FGF-10, a sonic hedgehog inhibitor, a factor capable of inhibiting BMP, and a netrin for about six days.

4. The method of claim 1, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with a sonic hedgehog inhibitor, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about one to about three days, followed by treating the cells with a sonic hedgehog inhibitor, retinoic acid, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about one to about four days.

5. The method of claim 4, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with a sonic hedgehog inhibitor, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about three days.

6. The method of claim 4, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with a sonic hedgehog inhibitor, retinoic acid, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about four days.

7. The method of claim 1, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with a sonic hedgehog inhibitor, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about one to about three days, followed by treating the cells with a sonic hedgehog inhibitor, a factor capable of inhibiting BMP, retinoic acid, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about one to about four days.

8. The method of claim 7, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with a sonic hedgehog inhibitor, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about three days.

9. The method of claim 7, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with a sonic hedgehog inhibitor, a factor capable of inhibiting BMP, retinoic acid, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about four days.

10. The method of claim 1, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with a sonic hedgehog inhibitor, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about one to about three days, followed by treating the cells with a sonic hedgehog inhibitor, a factor capable of inhibiting BMP, a netrin, retinoic acid, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about one to about four days.

11. The method of claim 10, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with a sonic hedgehog inhibitor, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about three days.

12. The method of claim 10, wherein the cells expressing markers characteristic of the definitive endoderm lineage are treated with a sonic hedgehog inhibitor, a factor capable of inhibiting BMP, a netrin, retinoic acid, and at least one factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 for about four days.

13. The method of claim 1, wherein the factor selected from the group consisting of FGF-2, FGF-4, FGF-7, and FGF-10 is FGF-7.

14. The method of claim 13, wherein the cells expressing markers characteristic of the definitive endoderm are treated with FGF-7 at a concentration of about 50 pg/ml to about 50 μ

g/ml.

15. The method of claim 13, wherein the cells expressing markers characteristic of the definitive endoderm are treated with FGF-7 at a concentration of 20 ng/ml.

16. The method of claim 1, wherein the cells expressing markers characteristic of the definitive endoderm are treated with retinoic acid at a concentration from about 1 nM to about 1 mM.

17. The method of claim 1, wherein the cells expressing markers characteristic of the definitive endoderm are treated with retinoic acid at a concentration of 1 μ

M.

18. The method of claim 1, wherein the sonic hedgehog inhibitor is cyclopamine.

19. The method of claim 18, wherein cyclopamine is used at a concentration from about 0.1 μ

M to about 10 μ

M.

20. The method of claim 18, wherein cyclopamine is used at a concentration of 0.25 μ

M.

21. The method of claim 1, wherein the factor capable of inhibiting BMP is a BMP4 inhibitor.

22. The method of claim 21, wherein the BMP4 inhibitor is noggin.

23. The method of claim 21, wherein the cells expressing markers characteristic of the definitive endoderm are treated with noggin at a concentration from about 500 ng/ml to about 100 μ

g/ml.

24. The method of claim 21, wherein the cells expressing markers characteristic of the definitive endoderm are treated with noggin at a concentration of 100 ng/ml.

25. The method of claim 1, wherein the cells expressing markers characteristic of the definitive endoderm are treated with the netrin at a concentration from about 500 ng/ml to about 100 μ

g/ml.

26. The method of claim 1, wherein the cells expressing markers characteristic of the definitive endoderm are treated with the netrin at a concentration of 100 ng/ml.

27. The method of claim 1, wherein the netrin is selected from the group consisting of netrin1, netrin 2, and netrin 4.