EphA4-positive human adult pancreatic endocrine progenitor cells

• US 7,807,459 B2
• Filed: 09/27/2005
• Issued: 10/05/2010
• Est. Priority Date: 09/27/2005
• Status: Active Grant

• Current Assignee: ReNeuron Limited (ReNeuron Group Plc)
• Original Assignee: ReNeuron Limited (ReNeuron Group Plc)
• Inventors: Wang, Yanping, Tang, Jinghua, Tsang, Wen-Ghih
• Primary Examiner(s): Kim; Taeyoon
• Application Number:
  US11/237,094
• Publication Number:
  US 20070072292A1
• Time in Patent Office: 1,834 Days
• Field of Search: 435/325, 435/41, 435/70.1, 435/370, 435/374
• US Class Current: 435/325
• 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 2533/54 Collagen; Gelatin
• CPC Class Codes: C12N 5/0676 Pancreatic cells

Abstract

The invention relates to the discovery of a selective cell surface marker that permits the selection of a unique subset of pancreatic stem cells having a high propensity to differentiate into insulin-producing cells or into insulin-producing cell aggregates.

Claims

1. A method of obtaining a culture of propagating pancreatic cells comprising:

(a) isolating pancreatic cells from a pancreas;

(b) contacting the pancreatic cells with an EphA4 binding reagent;

(c) selecting pancreatic cells that specifically bind to the EphA4 binding reagent; and

(d) separating the selected pancreatic cells from pancreatic cells that do not bind the EphA4 binding reagent to obtain a culture of propagating EphA4⁺ pancreatic cells, wherein the EphA4⁺ pancreatic cells have the capacity to differentiate into insulin producing cells.

2. The method of claim 1, wherein the EphA4 binding reagent is labeled.

3. The method of claim 1, wherein the step of selecting is done by fluorescence activated cell sorting.

4. The method of claim 1, wherein the step of selecting is done by panning.

5. The method of claim 1, wherein the EphA4-binding reagent is an antibody that specifically binds to EphA4 protein.

6. The method of claim 1, wherein the pancreas is from a human.

7. The method of claim 1, further comprising propagating the cells of step (d) and differentiating the cells into an aggregate of insulin producing cells.

8. The method of claim 7, wherein the step of differentiating the cells comprises culturing the cells on plates coated with collagen IV.

9. The method of claim 7, wherein the step of differentiating the cells comprises culturing the cells in a medium comprising a differentiation factor.

10. The method of claim 9, wherein the differentiation factor is selected from the group consisting of hepatocyte growth factor, keratinocyte growth factor, and exendin-4.

11. The method of claim 10, wherein the differentiation factor is hepatocyte growth factor.

12. A method of claim 1, further comprising the steps of:

(a) contacting the pancreatic cells with a CD56 binding reagent;

(b) selecting pancreatic cells that specifically bind to the CD56 binding reagent; and

(c) separating the selected pancreatic cells from pancreatic cells that do not bind the CD56 binding reagent to obtain a culture of propagating CD56⁺ pancreatic cells, wherein the steps utilizing the CD56 binding reagent are performed before or after contacting the pancreatic cells with the EphA4 binding reagent.

13. A method of producing an aggregate of insulin-producing pancreatic cells comprising the steps of:

(a) isolating pancreatic cells from a pancreas;

(b) contacting the pancreatic cells with an EphA4 binding reagent;

(c) selecting pancreatic cells that specifically bind to the EphA4 binding reagent;

(d) separating the selected pancreatic cells from pancreatic cells that do not bind the EphA4 binding reagent to obtain a culture of propagating EphA4⁺ pancreatic cells; and

(e) differentiating the propagating EphA4⁺ pancreatic cell culture into an aggregate of insulin producing pancreatic cells.

14. The method of claim 13, wherein the EphA4-binding reagent is labeled.

15. The method of claim 13, wherein the step of selecting is done by fluorescence activated cell sorting.

16. The method of claim 13, wherein the step of selecting is done by panning.

17. The method of claim 13, wherein the EphA4-binding reagent is an antibody that specifically binds to the EphA4 protein.

18. The method of claim 13, wherein the pancreas is from a human.

19. The method of claim 13, wherein the step of differentiating the cells comprise culturing the cells on plates coated with collagen IV.

20. The method of claim 13, wherein the step of differentiating the cells comprises culturing the cells in a medium comprising a differentiation factor.

21. The method of claim 13, wherein the differentiation factor is selected from the group consisting of hepatocyte growth factor, keratinocyte growth factor, and exendin-4.

22. The method of claim 21, wherein the differentiation factor is hepatocyte growth factor.

23. A method of claim 13, further comprising the steps of:

(a) contacting the pancreatic cells with a CD56 binding reagent;

(b) selecting pancreatic cells that specifically bind to the CD56 binding reagent; and

(c) separating the selected pancreatic cells from pancreatic cells that do not bind the CD56 binding reagent to obtain a culture of propagating CD56⁺ pancreatic cells, wherein the steps utilizing the CD56 binding reagent are performed before or after contacting the pancreatic cells with the EphA4 binding reagent.

24. A method of providing insulin to a mammal in need thereof, the method comprising the steps of:

(a) isolating pancreatic cells from a pancreas;

(b) contacting the pancreatic cells with an EphA4 binding reagent;

(c) selecting pancreatic cells that specifically bind to the EphA4 binding reagent;

(d) separating the selected pancreatic cells from pancreatic cells that do not bind the EphA4 binding reagent to obtain a culture of propagating EphA4⁺ pancreatic cells; and

(e) implanting into the mammal the propagating pancreatic cells in an amount sufficient to produce a measurable amount of insulin in the mammal.

25. The method of claim 24, wherein the EphA4 binding reagent is labeled.

26. The method of claim 24, wherein the step of selecting is done by fluorescence activated cell sorting.

27. The method of claim 24, wherein the step of selecting is done by panning.

28. The method of claim 24, wherein the EphA4 binding reagent is an antibody that specifically binds to the EphA4 protein.

29. The method of claim 24, wherein the pancreas is from a human.

30. The method of claim 24, wherein the propagating pancreatic cells differentiate into aggregates of insulin producing pancreatic cells after implantation into the mammal.

31. The method of claim 24, wherein before implantation into the mammal, the propagating pancreatic cell culture is differentiated into an aggregate of insulin producing pancreatic cells.

32. The method of claim 31, wherein the step of differentiating the cells comprises culturing the cells on plates coated with collagen IV.

33. The method of claim 31, wherein the step of differentiating the cells comprises culturing the cells in a medium comprising a differentiation factor.

34. The method of claim 33, wherein the differentiation factor is selected from the group consisting of hepatocyte growth factor, keratinocyte growth factor, and exendin-4.

35. The method of claim 34, wherein the differentiation factor is hepatocyte growth factor.

36. The method of claim 24, wherein the mammal is a human.

37. A method of claim 24, further comprising the steps of:

(a) contacting the pancreatic cells with a CD56 binding reagent;

(b) selecting pancreatic cells that specifically bind to the CD56 binding reagent; and

(c) separating the selected pancreatic cells from pancreatic cells that do not bind the CD56 binding reagent to obtain a culture of propagating CD56³⁰ pancreatic cells, wherein the steps utilizing the CD56 binding reagent are performed before or after contacting the pancreatic cells with the EphA4 binding reagent.