Stem cells of the islets of langerhans and their use in treating diabetes mellitus

• US 20030082155A1
• Filed: 04/11/2002
• Published: 05/01/2003
• Est. Priority Date: 12/06/1999
• Status: Abandoned Application

• Current Assignee: Corning Cable Systems LLC (Corning Incorporated), The General Hospital Corporation (Mass General Brigham, Inc.)
• Original Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Inventors: Lechner, Andreas, Leech, Colin A., Nolan, Anna Louise, Thomas, Melissa K., Habener, Joel F., Vallejo, Mario, Abraham, Elizabeth J., Zulewski, Henryk
• Application Number:
  US10/120,687
• Publication Number:
  US 20030082155A1
• Time in Patent Office: Days
• US Class Current: 424/93.21
• CPC Class Codes: A01K 67/0271 Chimeric vertebrates, e.g. comprising exogenous cells
• CPC Class Codes: A61K 2035/122 for inducing tolerance or supression of immune responses
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C12N 2500/34 Sugars
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/335 Glucagon; Glucagon-like peptide [GLP]; Exendin
• CPC Class Codes: C12N 2510/02 Cells for production
• CPC Class Codes: C12N 5/0678 Stem cells; Progenitor cells; Precursor cells

Abstract

Methods and compositions are described for the treatment of type I insulin-dependent diabetes mellitus and other conditions using newly identified stem cells that are capable of differentiation into a variety of pancreatic islet cells, including insulin-producing beta cells, as well as hepatocytes. Nestin and ABCG2 have been identified as molecular markers for pancreatic stem cells, while cytokeratin-19 serves as a marker for a distinct class of islet ductal cells. Methods are described whereby nestin and/or ABCG2-positive stem cells can be isolated from pancreatic islets and cultured to obtain further stem cells or pseudo-islet like structures. Methods for ex vivo differentiation of the pancreatic stem cells are disclosed. Methods are described whereby pancreatic stem cells can be isolated, expanded, and transplanted into a patient in need thereof, either allogeneically, isogeneically or xenogenically, to provide replacement for lost or damaged insulin-secreting cells or other cells.

Claims

1. A method of treating a patient with diabetes mellitus, comprising the steps of:

(a) isolating a nestin-positive pancreatic stem cell from a pancreatic islet of a donor; and

(b) transferring the stem cell into the patient, wherein the stem cell differentiates into an insulin-producing cell.

2. The method of claim 1, wherein said nestin-positive pancreatic stem cell is also ABCG2-positive.

3. The method of claim 1, wherein said nestin-positive pancreatic stem cell is also positive for at least one of the markers selected from the group consisting of Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin, Integrin subunits α

6 and β

1, C-kit, MDR-1, SUR-1, or Kir 6.2.

4. The method of claim 1, wherein said nestin-positive pancreatic stem cell does not express at least one of the markers selected from the group consisting of CD34, CD45, CD133, MHC class I and MHC class II.

6. The method of claim 1 or 5, wherein the patient serves as the donor for said stem cells of step a.

7. The method of claim 1 or 5, wherein, prior to the step of transferring, the stem cell is treated ex vivo with an agent selected from the group consisting of EGF, bFGF-2, high glucose, KGF, HGF/SF, GLP-1, exendin-4, IDX-1, a nucleic acid molecule encoding IDX-1, betacellulin, activin A, TGF-β

, and combinations thereof.

8. The method of claim 1 or 5, wherein the step of transferring is performed via endoscopic retrograde injection.

9. The method of claim 1 or 5, additionally comprising the step of:

(c) treating the patient with an immunosuppressive agent.

10. The method of claim 9, wherein the immunosuppressive agent is selected from the group consisting of FK-506, cyclosporin, and GAD65 antibodies.

5. A method of treating a patient with diabetes mellitus, comprising the steps of:

(a) isolating an ABCG2-positive pancreatic stem cell from a pancreatic islet of a donor; and

(b) transferring the stem cell into the patient, wherein the stem cell differentiates into an insulin-producing cell.

11. A method of treating a patient with diabetes mellitus, comprising the steps of:

(a) isolating a nestin-positive pancreatic stem cell from a pancreatic islet of a donor;

(b) expanding the stem cell ex vivo to produce a progenitor cell; and

(c) transferring the progenitor cell into the patient, wherein the progenitor cell differentiates into an insulin-producing beta cell.

12. The method of claim 11, wherein said nestin-positive pancreatic stem cell is also ABCG2-positive.

13. The method of claim 11, wherein said nestin-positive pancreatic stem cell is also positive for at least one of the markers selected from the group consisting of Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin, Integrin subunits α

6 and β

1, C-kit, MDR-1, SUR-1, or Kir 6.2.

14. The method of claim 11, wherein said nestin-positive pancreatic stem cell does not express at least one of the markers selected from the group consisting of CD34, CD45, CD133, MHC class I and MHC class II.

16. The method of claim 11 or 15, wherein the patient serves as the donor for said stem cells of step a.

17. The method of claim 11 or 15, wherein the step of expanding is performed in the presence of an agent selected from the group consisting of EGF, bFGF-2, high glucose, KGF, HGF/SF, GLP-1, exendin-4, IDX-1, a nucleic acid molecule encoding IDX-1, betacellulin, activin A, TGF-β

, and combinations thereof.

18. The method of claim 11 or 15, wherein the step of transferring is performed via endoscopic retrograde injection.

19. The method of claim 11 or 15 additionally comprising the step of:

(d) treating the patient with an immunosuppressive agent.

20. The method of claim 19, wherein the immunosuppressive agent is selected from the group consisting of FK-506, cyclosporin, and GAD65 antibodies.

15. A method of treating a patient with diabetes mellitus, comprising the steps of:

(a) isolating an ABCG2-positive pancreatic stem cell from a pancreatic islet of a donor;

(b) expanding the stem cell ex vivo to produce a progenitor cell; and

(c) transferring the progenitor cell into the patient, wherein the progenitor cell differentiates into an insulin-producing beta cell.

21. A method of treating a patient with diabetes mellitus, comprising the steps of:

(a) isolating a nestin-positive pancreatic stem cell from a pancreatic islet of a donor;

(b) expanding the stem cell to produce a progenitor cell;

(c) differentiating the progenitor cell in culture to form pseudo-islet like aggregates; and

(d) transferring the pseudo-islet like aggregates into the patient.

22. The method of claim 21, wherein said nestin-positive pancreatic stem cell is also ABCG2-positive.

23. The method of claim 21, wherein said nestin-positive pancreatic stem cell is also positive for at least one of the markers selected from the group consisting of Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin, Integrin subunits α

6 and β

1, C-kit, MDR-1, SUR-1, or Kir 6.2.

24. The method of claim 21, wherein said nestin-positive pancreatic stem cell does not express at least one of the markers selected from the group consisting of CD34, CD45, CD133, MHC class I and MHC class II.

26. The method of claim 21 or 25, wherein the patient serves as the donor for said stem cells of step a.

27. The method of claim 21 or 25, wherein the step of expanding is performed in the presence of an agent selected from the group consisting of EGF, bFGF-2, high glucose, KGF, HGF/SF, GLP-1, exendin-4, IDX-1, a nucleic acid molecule encoding IDX-1, betacellulin, activin A, TGF-β

, and combinations thereof.

28. The method of claim 21 or 25, wherein the step of transferring is performed via endoscopic retrograde injection.

29. The method of claim 21 or 25 additionally comprising the step of:

(e) treating the patient with an immunosuppressive agent.

30. The method of claim 29, wherein the immunosuppressive agent is selected from the group consisting of FK-506, cyclosporin, and GAD65 antibodies.

59. The method of claim 30 further comprising the step of:

contacting the cell with a labeled cytokeratin-19 specific antibody, whereby if the cell does not become labeled with the antibody the cell is identified as a stem cell.

25. A method of treating a patient with diabetes mellitus, comprising the steps of:

(a) isolating an ABCG2-positive pancreatic stem cell from a pancreatic islet of a donor;

(b) expanding the stem cell to produce a progenitor cell;

(c) differentiating the progenitor cell in culture to form pseudo-islet like aggregates; and

(d) transferring the pseudo-islet like aggregates into the patient.

31. A method of isolating a stem cell from a pancreatic islet of Langerhans, comprising the steps of:

(a) removing a pancreatic islet from a donor;

(b) culturing cells from the pancreatic islet; and

(c) selecting a nestin-positive clone from the culture.

32. The method of claim 31, wherein said nestin-positive clone is also an ABCG2-positive clone.

33. The method of claim 31, wherein said nestin-positive clone is also positive for at least one of the markers selected from the group consisting of Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin, Integrin subunits α

6 and β

1, C-kit, MDR-1, SUR-1, or Kir 6.2.

34. The method of claim 31, wherein said nestin-positive clone does not express at least one of the markers selected from the group consisting of CD34, CD45, CD133, MHC class I and MHC class II.

36. The method of claim 31 or 35, wherein the culturing is first performed in a vessel coated with concanavalin A and then again performed in a vessel not coated with concanavalin A.

37. The method of claim 31 or 35, comprising the additional step of:

(d) expanding the nestin-positive clone by treatment with an agent selected from the group consisting of EGF, bFGF-2, high glucose, KGF, HGF/SF, GLP-1, exendin-4, IDX1, a nucleic acid molecule encoding IDX-1, betacellulin, activin A, TGF-β

, and combinations thereof.

35. A method of isolating a stem cell from a pancreatic islet of Langerhans, comprising the steps of:

(a) removing a pancreatic islet from a donor;

(b) culturing cells from the pancreatic islet; and

(c) selecting an ABCG2-positive clone from the culture.

38. The method of claim 35 comprising the additional step of:

(d) expanding the ABCG2-positive clone by treatment with an agent selected from the group consisting of EGF, bFGF-2, high glucose, KGF, HGF/SF, GLP-1, exendin-4, IDX-1, a nucleic acid molecule encoding IDX-1, betacellulin, activin A, TGF-β

, and combinations thereof.

39. A method of inducing the differentiation of a nestin-positive pancreatic stem cell into a pancreatic progenitor cell, comprising the step of:

treating a nestin-positive pancreatic stem cell with an agent selected from the group consisting of EGF, bFGF-2, high glucose, KGF, HGF/SF, IDX-1, a nucleic acid molecule encoding IDX-1, GLP-1, exendin-4, betacellulin, activin A, TGF-β

, and combinations thereof, whereby the stem cell subsequently differentiates into a pancreatic progenitor cell.

40. The method of claim 39, wherein said nestin-positive pancreatic stem cell is also an ABCG2-positive clone.

41. The method of claim 39, wherein said nestin-positive pancreatic stem cell is also positive for at least one of the markers selected from the group consisting of Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin, Integrin subunits α

6 and β

1, C-kit, MDR-1, SUR-1, or Kir 6.2.

42. The method of claim 39, wherein said nestin-positive pancreatic stem cell does not express at least one of the markers selected from the group consisting of CD34, CD45, CD133, MHC class I and MHC class II.

44. The method of claim 39 or 43, wherein the pancreatic progenitor cell subsequently forms pseudo-islet like aggregates.

43. A method of inducing the differentiation of an ABCG2-positive pancreatic stem cell into a pancreatic progenitor cell, comprising the step of:

treating an ABCG2-positive pancreatic stem cell with an agent selected from the group consisting of EGF, bFGF-2, high glucose, KGF, HGF/SF, IDX-1, a nucleic acid molecule encoding IDX-1, GLP-1, exendin-4, betacellulin, activin A, TGF-β

, and combinations thereof, whereby the stem cell subsequently differentiates into a pancreatic progenitor cell.

45. An isolated, nestin-positive human pancreatic or liver stem cell that is not a neural stem cell.

46. The isolated stem cell of claim 45, wherein said cell is also ABCG2-positive.

47. The isolated stem cell of claim 45, wherein said cell is also positive for at least one of the markers selected from the group consisting of Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin, Integrin subunits α

6 and β

1, C-kit, MDR-1, SUR-1, or Kir 6.2.

48. The isolated stem cell of claim 45, wherein said cell does not express at least one of the markers selected from the group consisting of CD34, CD45, CD133, MHC class I and MHC class II.

53. The isolated stem cell of claim 45 or 49, that differentiates to form insulin-producing beta cells.

54. The isolated stem cell of claim 45 or 49, that differentiates to form glucagon-producing alpha cells.

55. The isolated stem cell of claim 45 or 49, that differentiates to form pseudo-islet like aggregates.

56. The isolated stem cell of claim 45 or 49, that differentiates to form hepatocytes.

86. A pharmaceutical composition comprising the isolated stem cell of claim 45 admixed with a physiologically compatible carrier.

87. A pharmaceutical composition comprising the isolated stem cell of claim 46 admixed with a physiologically compatible carrier.

49. An isolated, ABCG2-positive human pancreatic or liver stem cell that is not a neural stem cell.

50. The isolated cell of claim 49, wherein said cell is also positive for at least one of the markers selected from the group consisting of Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin, Integrin subunits α

6 and β

1, C-kit, MDR-1, SUR-1, or Kir 6.2.

51. The isolated cell of claim 49, wherein said cell does not express at least one of the markers selected from the group consisting of CD34, CD45, CD133, MHC class I and MHC class II.

52. The isolated stem cell of claim 49, wherein said cell does not express at least one of the markers selected from the group consisting of CD34, CD45, CD133, MHC class I and MHC class II.

88. A pharmaceutical composition comprising the isolated stem cell of claim 49 admixed with a physiologically compatible carrier.

57. A method of identifying a pancreatic cell as a stem cell, comprising the step of:

contacting a cell with a labeled nestin-specific antibody, whereby if the cell becomes labeled with the antibody the cell is identified as a stem cell.

58. The method of claim 57, further comprising the step of contacting a cell with a labeled antibody that binds to a marker selected from the group consisting of ABCG2, Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin, Integrin subunits α

6 and β

1, C-kit, MDR-1, SUR-1, or Kir 6.2, whereby if the cell becomes labeled with the antibody the cell is identified as a stem cell.

60. The method of claim 57 further comprising the step of:

contacting the cell with a labeled collagen IV specific antibody, whereby if the cell does not become labeled with the antibody the cell is identified as a stem cell.

61. The method of claim 57, further comprising the step of contacting the cell with a labeled antibody that binds to a marker selected from the group consisting of of CD34, CD45, CD133, MHC class I and MHC class II, whereby if the cell does not become labeled with the antibody the cell is identified as a stem cell.

62. A method of inducing a nestin-positive pancreatic stem cell to differentiate into hepatocytes, comprising the step of:

treating the nestin-positive pancreatic stem cell with an effective amount of an agent that induces the stem cell to differentiate into hepatocytes or into progenitor cells that differentiate into hepatocytes.

63. The method of claim 62, wherein said nestin-positive pancreatic stem cell is also ABCG2-positive.

64. The method of claim 62, wherein said nestin-positive pancreatic stem cell is also positive for at least one of the markers selected from the group consisting of Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin, Integrin subunits α

6 and β

1, C-kit, MDR-1, SUR-1, or Kir 6.2.

65. The method of claim 62, wherein said nestin positive pancreatic stem cell does not express at least one of the markers selected from the group consisting of CD34, CD45, CD133, MHC class I and MHC class II.

67. The method of claim 62 or 66, wherein the agent is cyclopamine.

66. A method of inducing an ABCG2-positive pancreatic stem cell to differentiate into hepatocytes, comprising the step of:

treating the ABCG2-positive pancreatic stem cell with an effective amount of an agent that induces the stem cell to differentiate into hepatocytes or into progenitor cells that differentiate into hepatocytes.

68. A method of treating a patient with liver disease, comprising the steps of:

(a) isolating a nestin-positive pancreatic stem cell from a pancreatic islet of a donor; and

(b) transferring the stem cell into the patient, wherein the stem cell differentiates into a hepatocyte.

69. The method of claim 68, wherein said nestin-positive pancreatic stem cell is also ABCG2-positive.

70. The method of claim 68, wherein said nestin-positive pancreatic stem cell is also positive for at least one of the markers selected from the group consisting of Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin, Integrin subunits α

6 and β

1, C-kit, MDR-1, SUR-1, or Kir 6.2.

71. The method of claim 68, wherein said nestin positive pancreatic stem cell does not express at least one of the markers selected from the group consisting of CD34, CD45, CD133, MHC class I and MHC class II.

73. The method of claim 68 or 72, wherein the patient serves as the donor for said stem cells of step a.

72. A method of treating a patient with liver disease, comprising the steps of:

(a) isolating an ABCG2-positive pancreatic stem cell from a pancreatic islet of a donor; and

(b) transferring the stem cell into the patient, wherein the stem cell differentiates into a hepatocyte.

74. A method of treating a patient with liver disease, comprising the steps of:

(a) isolating a nestin-positive pancreatic stem cell from a pancreatic islet of a donor;

(b) expanding the stem cell ex vivo to produce a progenitor cell; and

(c) transferring the progenitor cell into the patient, wherein the progenitor cell differentiates into a hepatocyte.

75. The method of claim 74, wherein said nestin-positive pancreatic stem cell is also ABCG2-positive.

76. The method of claim 74, wherein said nestin-positive pancreatic stem cell is also positive for at least one of the markers selected from the group consisting of Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin, Integrin subunits α

6 and β

1, C-kit, MDR-1, SUR-1, or Kir 6.2.

77. The method of claim 74, wherein said nestin positive pancreatic stem cell does not express at least one of the markers selected from the group consisting of CD34, CD45, CD133, MHC class I and MHC class II.

79. The method of claim 74 or 78, wherein the patient serves as the donor for said stem cells of step a.

78. A method of treating a patient with liver disease, comprising the steps of:

(a) isolating an ABCG2-positive pancreatic stem cell from a pancreatic islet of a donor;

(b) expanding the stem cell ex vivo to produce a progenitor cell; and

(c) transferring the progenitor cell into the patient, wherein the progenitor cell differentiates into a hepatocyte.

80. A method of treating a patient with liver disease, comprising the steps of:

(a) isolating a nestin-positive pancreatic stem cell from a pancreatic islet of a donor;

(b) differentiating the stem cell ex vivo to produce a hepatocyte; and

(c) transferring the hepatocyte into the patient.

81. The method of claim 80, wherein said nestin-positive pancreatic stem cell is also ABCG2-positive.

82. The method of claim 80, wherein said nestin-positive pancreatic stem cell is also positive for at least one of the markers selected from the group consisting of Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin, Integrin subunits α

6 and β

1, C-kit, MDR-1, SUR-1, or Kir 6.2.

83. The method of claim 80, wherein said nestin positive pancreatic stem cell does not express at least one of the markers selected from the group consisting of CD34, CD45, CD133, MHC class I and MHC class II.

85. The method of claim 80 or 84, wherein the patient serves as the donor for said stem cells of step a.

84. A method of treating a patient with liver disease, comprising the steps of:

(a) isolating an ABCG2-positive pancreatic stem cell from a pancreatic islet of a donor;

(b) differentiating the stem cell ex vivo to produce a hepatocyte; and

(c) transferring the hepatocyte into the patient.