Differentiation of Human Embryonic Stem Cells

• US 20100255580A1
• Filed: 07/18/2008
• Published: 10/07/2010
• Est. Priority Date: 07/18/2007
• Status: Abandoned Application

• Current Assignee: Lifescan Incorporated (Duv Holding Corporation)
• Original Assignee: Lifescan Incorporated (Duv Holding Corporation)
• Inventors: Rezania, Alireza
• Application Number:
  US12/669,289
• Publication Number:
  US 20100255580A1
• Time in Patent Office: Days
• US Class Current: 435/377
• CPC Class Codes: A61P 1/18 for pancreatic disorders, e.g. pancreatic enzymes
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 5/50 for increasing or potentiating the activity of insulin
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• 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 2501/23 Interleukins [IL]
• 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 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2533/52 Fibronectin; Laminin
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/067 Hepatocytes
• 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. In particular, the present invention provides an improved method for the formation of pancreatic endoderm, pancreatic hormone expressing cells and pancreatic hormone secreting cells. The present invention also provides methods to promote the differentiation of pluripotent stem cells without the use of a feeder cell layer.

Claims

1. A method for differentiating pluripotent stem cells into cells expressing markers characteristic of the pancreatic endocrine 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, by culturing the pluripotent stem cells in a chemically defined medium comprising medium supplemented with B27 and treating the pluripotent cells with activin A,

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 fibroblast growth factor, or with retinoic acid and at least one fibroblast growth factor, and

d. Differentiating the cells expressing markers characteristic of the pancreatic endoderm lineage into cells expressing markers characteristic of the pancreatic endocrine lineage, by treating the cells expressing markers characteristic of the pancreatic endoderm lineage with a gamma secretase inhibitor.

2. The method of claim 1, wherein the B27 is used at a concentration of about 0.5% to about 1%.

3. The method of claim 1, wherein the B27 is used at a concentration of about 1%.

4. The method of claim 1, wherein the step of differentiating the pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage is achieved by a method comprising the steps of:

a. Plating the pluripotent stem cells on a tissue culture substrate coated with an extracellular matrix, and

b. Culturing the pluripotent stem cells in chemically defined medium comprising medium supplemented with B27.

5. The method of claim 1, wherein the B27 is used at a concentration of about 0.5% to about 1%.

6. The method of claim 1, wherein the B27 is used at a concentration of about 1%.

7. The method of claim 4, wherein the B27 is used at a concentration of about 0.5% to about 1%.

8. The method of claim 4, wherein the B27 is used at a concentration of about 1%.

9. The method of claim 1, wherein the step of differentiating the pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage is achieved by a method comprising the steps of:

a. Plating the pluripotent stem cells on a tissue culture substrate coated with an extracellular matrix,

b. Culturing the pluripotent stem cells with activin A and a Wnt ligand in a first culture medium, and

c. Culturing the pluripotent stem cells with activin A in a second culture medium.

10. The method of claim 1, wherein the step of differentiating the pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage is achieved by a method further comprising plating the pluripotent stem cells on a fibroblast feeder layer.

11. The method of claim 4, wherein the pluripotent stem cells are cultured in chemically defined medium comprising DMEM-F12 supplemented with B27, a Wnt ligand and activin A.

12. The method of claim 4, wherein the pluripotent stem cells are cultured in chemically defined medium comprising DMEM-LG supplemented with B27, a Wnt ligand and activin A.

27. The method of claim 1, wherein the pluripotent stem cells are cultured in chemically defined medium comprising DMEM-F12 supplemented with B27 and activin A.

28. The method of claim 27, wherein the B27 is at a concentration of about 0.5% to about 1%.

29. The method of claim 27, wherein the B27 is at a concentration of about 1%.

30. The method of claim 1, wherein the pluripotent stem cells are cultured in chemically defined medium comprising DMEM-F12 supplemented with B27 and activin A.

31. The method of claim 30, wherein the B27 is at a concentration of about 0.5% to about 1%.

32. The method of claim 30, wherein the B27 is at a concentration of about 1%.

13. A method for differentiating pluripotent stem cells into cells expressing markers characteristic of the pancreatic endoderm lineage, comprising the steps of:

a. Culturing the pluripotent stem cells in chemically defined medium comprising medium supplemented with B27,

b. Differentiating the pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage, by treating the pluripotent cells with activin A, 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 fibroblast growth factor, or with retinoic acid and at least one fibroblast growth factor.

14. The method of claim 13, wherein the B27 is used at a concentration of about 0.5% to about 1%.

15. The method of claim 13, wherein the B27 is used at a concentration of about 1%.

16. The method of claim 13, wherein the step of differentiating the pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage is achieved by a method further comprising plating the pluripotent stem cells on a fibroblast feeder layer.

17. A method for differentiating pluripotent stem cells into cells expressing markers that are characteristic of the definitive endoderm lineage, comprising the steps of:

a. Plating the pluripotent stem cells on a tissue culture substrate coated with an extracellular matrix, and

b. Culturing the pluripotent stem cells in chemically defined medium comprising medium supplemented with B27.

18. The method of claim 17, wherein the B27 is used at a concentration of about 0.5% to about 1%.

19. The method of claim 17, wherein the B27 is used at a concentration of about 1%.

20. The method of claim 17, wherein the step of differentiating the pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage is achieved by a method further comprising plating the pluripotent stem cells on a fibroblast feeder layer.

21. The method of claim 17, wherein the pluripotent stem cells are cultured in chemically defined medium comprising DMEM-F12 supplemented with B27, a Wnt ligand and activin A.

22. The method of claim 21, wherein the B27 is at a concentration of about 0.5% to about 1%.

23. The method of claim 21, wherein the B27 is at a concentration of about 1%.

24. The method of claim 17, wherein the pluripotent stem cells are cultured in chemically defined medium comprising DMEM-LG supplemented with B27, a Wnt ligand and activin A.

25. The method of claim 24, wherein the B27 is at a concentration of about 0.5% to about 1%.

26. The method of claim 24, wherein the B27 is at a concentration of about 1%.