EphA4-positive human adult pancreatic endocrine progenitor cells

• US 20070072292A1
• Filed: 09/27/2005
• Published: 03/29/2007
• Est. Priority Date: 09/27/2005
• Status: Active Grant

• Current Assignee: ReNeuron Limited (ReNeuron Group Plc)
• Original Assignee: Amcyte Inc. (ReNeuron Group Plc)
• Inventors: Wang, Yanping, Tsang, Wen-Ghih, Tang, Jinghua
• Granted Patent: US 7,807,459 B2
• Time in Patent Office: Days
• US Class Current: 435/370
• 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 pancreatic 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 the 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 media 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 pancreatic cells.

13. A method of claim 12, wherein steps (a)-(c) are performed before contacting the pancreatic cells with the EphA4 binding reagent.

14. A method of claim 12, wherein steps (a)-(c) are performed after cells binding the EphA4 binding reagent are separated from pancreatic cells that do not bind the EphA4 binding reagent.

15. 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 CD56 binding reagent to obtain a culture of propagating pancreatic cells; and

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

16. The method of claim 15, wherein the EphA4-binding reagent is labeled.

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

18. The method of claim 15, wherein the step of selecting is done by panning.

19. The method of claim 15, wherein the EphA4-binding reagent is an antibody that specifically binds to the EphA4 protein.

20. The method of claim 15, wherein the pancreas is from a human.

21. The method of claim 15, wherein the step of differentiating the cells comprises culturing the cells on plates coated with collagen IV.

22. The method of claim 15, wherein the step of differentiating the cells comprises culturing the cells in a media comprising a differentiation factor.

23. The method of claim 15, wherein the differentiation factor is selected from the group consisting of hepatocyte growth factor, keratinocyte growth factor, and exendin-4.

24. The method of claim 23, wherein the differentiation factor is hepatocyte growth factor.

25. A method of claim 15, 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 pancreatic cells.

26. A method of claim 25, wherein steps (a)-(c) are performed before contacting the pancreatic cells with the EphA4 binding reagent.

27. A method of claim 25, wherein steps (a)-(c) are performed after cells binding the EphA4 binding reagent are separated from pancreatic cells that do not bind the EphA4 binding reagent.

28. A method of providing pancreatic endocrine function to a mammal in need of such function, 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 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.

29. The method of claim 28, wherein the EphA4 binding reagent is labeled.

30. The method of claim 28, wherein the step of selecting is done by fluorescence activated cell sorting.

31. The method of claim 28, wherein the step of selecting is done by panning.

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

33. The method of claim 28, wherein the pancreas is from a human.

34. The method of claim 28, wherein the propagating pancreatic cells differentiate into aggregates of insulin producing pancreatic cells after implantation into the mammal.

35. The method of claim 28, wherein before implantation into the mammal, the propagating pancreatic cell culture is differentiated into an aggregate of insulin producing pancreatic cells.

36. The method of claim 35, wherein the step of differentiating the cells comprises culturing the cells on plates coated with collagen IV.

37. The method of claim 35, wherein the step of differentiating the cells comprises culturing the cells in a media comprising a differentiation factor.

38. The method of claim 37, wherein the differentiation factor is selected from the group consisting of hepatocyte growth factor, keratinocyte growth factor, and exendin-4.

39. The method of claim 38, wherein the differentiation factor is hepatocyte growth factor.

40. The method of claim 28, wherein the mammal is a human.

41. A method of claim 28, 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 pancreatic cells.

42. A method of claim 41, wherein steps (a)-(c) are performed before contacting the pancreatic cells with the EphA4 binding reagent.

43. A method of claim 41, wherein steps (a)-(c) are performed after cells binding the EphA4 binding reagent are separated from pancreatic cells that do not bind the EphA4 binding reagent.

44. A method of monitoring a culture of propagating pancreatic cells by

a) contacting the pancreatic cells with a EphA4 binding reagent;

and

b) determining the quantity of cells that exhibit EphA4 as a cell surface marker.

45. The method of claim 44, wherein the detecting step is done by fluorescence activated cell sorting.

46. The method of claim 44, wherein the EphA4 binding reagent is an antibody that binds specifically to EphA4 protein.

47. The method of claim 44, wherein the pancreas is from a human.

48. A method of claim 44, 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) determining the quantity of cells that exhibit CD56 and EphA4 as cell surface markers.

49. A cell culture produced by 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 pancreatic cells.

50. The cell culture of claim 49, wherein the EphA4 binding reagent is labeled.

51. The cell culture of claim 49, wherein the step of selecting is done by fluorescence activated cell sorting.

52. The cell culture of claim 49, wherein the step of selecting is done by panning.

53. The cell culture of claim 49, wherein the EphA4 binding reagent is an antibody that specifically binds to the EphA4 protein.

54. The cell culture of claim 49, wherein the pancreas is from a human.

55. A cell culture of claim 49, 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.

56. A cell culture of claim 49, wherein steps (a)-(c) are performed before contacting the pancreatic cells with the EphA4 binding reagent.

57. A cell culture of claim 49, wherein steps (a)-(c) are performed after cells binding the EphA4 binding reagent are separated from pancreatic cells that do not bind the EphA4 binding reagent.