METHODS AND DEVICES FOR DIFFERENTIATING PLURIPOTENT STEM CELLS INTO CELLS OF THE PANCREATIC LINEAGE

• US 20120264209A1
• Filed: 06/08/2012
• Published: 10/18/2012
• Est. Priority Date: 05/02/2006
• Status: Active Grant

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Odorico, Jon, Xu, Xiaofang
• Granted Patent: US 8,685,730 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• 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 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/335 Glucagon; Glucagon-like peptide [GLP]; Exendin
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0676 Pancreatic cells

Abstract

Methods and devices for culturing human pluripotent stem cells to produce cells of the pancreatic lineage are disclosed. The methods include steps of culturing the stem cells under conditions that induce the expression of mesendoderm/primitive streak and definitive endoderm markers in a chemically defined medium including an effective amount of i) fibroblast growth factor, ii) Activin A, and iii) bone morphogenetic protein. The methods further include the steps of culturing cells under conditions favoring the formation of at least one of intact embryoid bodies and pancreatic progenitor PDX1⁺ Ins⁻ cells.

Claims

1. A method of culturing human pluripotent stem cells to produce cells of the pancreatic lineage, the method comprising the steps of:

(a) culturing the stem cells under conditions that induce formation of mesendoderm/primitive streak and definitive endoderm cells in a chemically defined medium comprising an effective amount of

i) fibroblast growth factor,

ii) Activin A, and

iii) bone morphogenetic protein;

(b) culturing the cells from step (a) in the presence of a chemically defined medium comprising an effective amount of insulin, transferrin, and selenium, wherein the medium further comprises a fibroblast growth factor in an amount that ranges from about 10 ng/ml to about 200 ng/ml; and

(c) culturing the cells under conditions to produce foregut/pancreatic progenitor PDX1⁺ Ins⁻

cells.

2. The method of claim 1, wherein the stem cells are selected from the group consisting of human embryonic stem cells and human induced pluripotent stem cells.

3. The method of claim 1, wherein in step (a) the effective amount of

i) fibroblast growth factor ranges from about 10 ng/ml to about 200 ng/ml,

ii) Activin A ranges from about 10 ng/ml to about 200 ng/ml, and

iii) bone morphogenetic protein ranges from about 10 ng/ml to about 100 ng/ml.

4. The method of claim 3, wherein the fibroblast growth factor comprises basic fibroblast growth factor and the bone morphogenetic protein comprises BMP4.

5. The method of claim 4, wherein in step (a) the effective amount of

i) basic fibroblast growth factor is about 100 ng/ml,

ii) Activin A is about 100 ng/ml, and

iii) BMP4 is about 50 ng/ml.

6. The method of claim 1, wherein step (a) has a duration of about 3-4 days.

7. The method of claim 1, wherein the stem cells of step (b) are cultured under conditions favoring the formation of embryoid bodies.

8. The method of claim 7, wherein step (b) has a duration of about 7 to about 12 days.

9. The method of claim 7, wherein the embryoid bodies of step (b) include definitive endoderm cells with duct-like structures containing FoxA2⁺, Sox17⁺ and PDX1⁺ cells.

10. The method of claim 1, wherein the chemically defined medium is serum free.

11. The method of claim 1, wherein stem cells cultured under the conditions of step (a) co-express FoxA2 and Sox 17.

12. The method of claim 1, wherein the PDX1⁺ Ins⁻

cells may be maintained under the conditions of step (c) for at least 50 days.

13. The method of claim 1, wherein the PDX1⁺ Ins⁻

cells may be further differentiated into PDX1⁺ Ins⁺ cells.

14. The method of claim 1, wherein stem cells are initially seeded in step (a) on a porous substrate.

15. A method of culturing human pluripotent stem cells to produce cells of the pancreatic lineage, the method comprising the steps of:

(a) culturing the stem cells under conditions that induce formation of mesendoderm/primitive streak and definitive endoderm cells in a chemically defined medium comprising an effective amount of

i) fibroblast growth factor,

ii) Activin A, and

iii) bone morphogenetic protein;

(b) culturing the cells from step (a) under conditions favoring the formation of embryoid bodies; and

(c) culturing the embryoid bodies under conditions favoring the formation of pancreas-spheres co-expressing PDX1, HNF1β

, HNF6, and Sox9 proteins.

16. The method of claim 15, wherein the culture conditions in step (c) include culturing the embryoid bodies in a serum-free medium containing insulin, transferrin, selenium, FGF7, nicotinamide, islet neogenesis associated peptide, and exendin-4.

17. The method of claim 15 further comprising culturing the pancreas-spheres under conditions to obtain endocrine specification and differentiation of PDX1⁺ Ins⁺ cells.

18. The method of claim 15, wherein the fibroblast growth factor comprises bFGF and the bone morphogenetic protein comprises BMP4.

19. The method of claim 15, wherein the pancreas-spheres of step (c) are cultured in a suspension culture comprising a serum-free medium containing B27 and nicotinamide.

20. The method of claim 17, wherein the PDX1⁺ Ins⁺ cells express increased levels of insulin compared to the pancreas-spheres.

21. The method of claim 17, wherein the PDX1⁺ Ins⁺ cells secrete C-peptide.

22. A method of culturing pluripotent stem cells in a committed partially differentiated state, comprising the steps of:

(a) culturing the stem cells under conditions that induce formation of embryoid bodies containing multipotent progenitor cells; and

(b) culturing the embryoid bodies on an extracellular matrix in cell culture inserts having a porous floor so as to maintain the multipotent progenitors cells in a non-terminally differentiated state.

23. The method of claim 22, wherein the multipotent progenitors cells are of the pancreatic/foregut lineage.

24. A method of producing progenitor cells of the pancreatic lineage, comprising the steps of:

(a) seeding human pluripotent stem cells in a cell culture vessel comprising an upper chamber and a lower chamber, wherein a bottom surface of the upper chamber comprises a porous substrate;

(b) culturing the cells in a chemically defined medium comprising an effective amount of

i) fibroblast growth factor,

ii) Activin A, and

iii) bone morphogenetic protein; and

(c) obtaining pancreatic progenitor PDX1⁺ Ins⁻

cells.

25. The method of claim 24, wherein the porous substrate comprises pores having a size that restricts migration of cells seeded thereon between the upper and lower chambers and enables the upper and lower chambers to be in fluid communication with one another.

26. The method of claim 25, wherein the pores have a size ranging from 0.01 μ

m to 3 μ

m (±

10%).

27. The method of claim 24, wherein the PDX1⁺ Ins⁻

cells may be maintained under conditions of step (c) for at least 70 days.

28. A pancreatic progenitor cell culture implant platform, comprising:

(a) a bicameral cell culture system with an upper chamber and a lower chamber separated by a porous substrate; and

(b) stem cells cultured in the upper chamber on the porous substrate under conditions that induce commitment to pancreatic progenitor PDX1⁺ Ins⁻

cells,

wherein the stems cells may be treated in a single step to differentiate into insulin producing cells of the pancreatic lineage and either removed from the platform for implantation into a subject or implanted into a subject along with the porous substrate.