Methods and compositions for expanding and differentiating insulin-producing cells

• US 20040132183A1
• Filed: 05/28/2003
• Published: 07/08/2004
• Est. Priority Date: 05/28/2002
• Status: Abandoned Application

• Current Assignee: Novocell, Inc. (Vertex Pharmaceuticals Incorporated)
• Original Assignee: Novocell, Inc. (Vertex Pharmaceuticals Incorporated)
• Inventors: Coutts, Margaret, McIntyre, Catherine Anne, Latta, Paul Presley, Scharp, David William
• Application Number:
  US10/447,325
• Publication Number:
  US 20040132183A1
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: A61K 2035/126 Immunoprotecting barriers, e.g. jackets, diffusion chambers
• 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 3/08 for glucose homeostasis pancreatic hormones A61P5/48
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2500/46 Amines, e.g. putrescine
• CPC Class Codes: C12N 2501/01 Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/113 Acidic fibroblast growth factor (aFGF, FGF-1)
• 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 2501/12 Hepatocyte growth factor [HGF]
• CPC Class Codes: C12N 2501/135 Platelet-derived growth factor [PDGF]
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/165 Vascular endothelial growth factor [VEGF]
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2501/315 : Prolactin
• CPC Class Codes: C12N 2501/335 : Glucagon; Glucagon-like peptide [GLP]; Exendin
• CPC Class Codes: C12N 2501/34 : Calcitonin; Calcitonin-gene related peptide [CGRO]; Amylin
• CPC Class Codes: C12N 2501/345 : Gastrin; Cholecystokinins [CCK]
• CPC Class Codes: C12N 2501/35 : Vasoactive intestinal peptide [VIP]; Pituitary adenylate cyclase activating polypeptide [PACAP]
• CPC Class Codes: C12N 2501/37 : Parathyroid hormone [PTH]
• CPC Class Codes: C12N 2501/39 : Steroid hormones
• CPC Class Codes: C12N 2501/392 : Sexual steroids
• CPC Class Codes: C12N 2501/41 : Hedgehog proteins; Cyclopamine (inhibitor)
• CPC Class Codes: C12N 2501/83 : Tachykinins, e.g. substance P
• CPC Class Codes: C12N 2501/85 : Hormones derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
• CPC Class Codes: C12N 2501/998 : Proteins not provided for elsewhere
• CPC Class Codes: C12N 2506/22 : from pancreatic cells
• CPC Class Codes: C12N 5/0037 : Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 5/0676 : Pancreatic cells
• CPC Class Codes: Y02A 50/30 : Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

A method of converting differentiated non-hormone producing pancreatic cells into differentiated hormone producing cells is disclosed. The method comprises two steps: first, culturing cells under conditions which convert differentiated non-hormone producing cells into stem cells; and second, culturing stem cells under conditions which provide for differentiating stem cells into hormone-producing cells. The invention provides a new source of large quantities of hormone producing cells such as insulin-producing cells that are not currently available for therapeutic uses such as the treatment of diabetes.

Claims

1. A method of converting differentiated non-hormone producing pancreatic cells into differentiated hormone-producing cells, comprising:

a) culturing said differentiated non-hormone producing pancreatic cells in a first cell culture system with a first cell culture medium, under conditions which provide for converting said differentiated non-hormone producing pancreatic cells into stem cells; and

b) culturing said stem cells in a second cell culture system with a second cell culture medium under conditions which provide for differentiating said stem cells into hormone-producing cells.

2. The method of claim 1, wherein said stem cells proliferate in said first step.

3. The method of claim 1, wherein said stem cells proliferate in said second step.

4. The method of claim 1, wherein said hormone-producing cells produce insulin.

5. The method of claim 1, wherein said hormone-producing cells produce glucagon.

6. The method of claim 1, wherein said differentiated non-hormone producing pancreatic cells are acinar cells.

7. The method of claim 1,

a) wherein said differentiated non-hormone producing pancreatic cells in the first step are cultured with a culture mode selected from the group consisting of:

adherent, suspension and matrix; and

b) wherein said stem cells in said second step are cultured with a culture mode selected from the group consisting of;

adherent, suspension, and matrix.

8. The method of claim 7, wherein said culture mode is an adherent culture mode that comprises culturing cells directly on a surface of a tissue culture container or on a surface of a tissue culture container which is coated with at least one compound selected from the group consisting of collagen, fibronectin, laminin, and hyaluronic acid.

9. The method of claim 1, wherein said differentiated non-hormone producing pancreatic cells are seeded at a density of 10⁵ to 10⁷ cells/cm².

10. The method of claim 7, wherein said culture mode is a suspension culture mode that comprises culturing said differentiated non-hormone producing pancreatic cells in suspension in said culture medium.

11. The method of claim 7, wherein said culture media is a matrix culture mode that comprises culturing said differentiated non-hormone producing pancreatic cells interspersed in a crosslinked polymerizable gel.

12. The method of claim 1, wherein said differentiated non-hormone producing pancreatic cells are seeded at a density of 10⁴ to 10⁸ cells/ml in a hydrogel.

13. The method of claim 12, wherein said hydrogel is alginate.

14. The method of claim 1, wherein said culture medium in the first step comprises serum and a basal medium selected from the group consisting of Dulbecco'"'"'s Modified Eagle'"'"'s medium (DMEM), Medium 199 (M199), Ham'"'"'s F12 Nutrient Mixture (Ham'"'"'s F12), RPMI 1640 Medium, CMRL medium, and mixtures thereof.

15. The method of claim 14, wherein said culture medium in the first step further comprises at least three compounds selected from the group consisting of insulin, transferrin, selenium, zinc sulphate, glutathione, ethanolamine, cyclodextrin, biotin, alpha Tocopherol, calcium pantothenate, myoinositol, nicotinamide, IGF1, Prolactin, exendin-4, EGF, VEGF, KGF, and HGF.

16. The method of claim 1, wherein said culture medium in the first step comprises a basal medium without serum selected from the group consisting of Dulbecco'"'"'s Modified Eagle'"'"'s medium (DMEM), Medium 199 (M 199), Ham'"'"'s F12 Nutrient Mixture (Ham'"'"'s F12), RPMI 1640 Medium, CMRL medium, and mixtures thereof.

17. The method of claim 16, wherein said culture medium in the first step further comprises at least three compounds selected from the group consisting of insulin, transferrin, selenium, zinc sulphate, glutathione, ethanolamine, cyclodextrin, biotin, alpha Tocopherol, calcium pantothenate, myoinositol, nicotinamide, IGF1, Prolactin, exendin, EGF, VEGF, KGF, and HGF.

18. The method of claim 1, wherein said culture medium in the second step comprises a basal medium without serum selected from the group consisting of Dulbecco'"'"'s Modified Eagle'"'"'s medium (DMEM), Medium 199 (M199), Ham'"'"'s F12 Nutrient Mixture (Ham'"'"'s F1 2), RPMI 1640 Medium, CMRL medium, neurobasal medium, Johe'"'"'s N2 medium, and mixtures thereof.

19. The method of claim 18, wherein said culture medium in the second step further comprises insulin, transferrin, and selenium.

20. The method of claim 14, wherein said culture medium in the second step further comprises at least two compounds selected from the group consisting of glutathione, ethanolamine, biotin, alpha Tocopherol (Vitamin E), and albumin (human or bovine).

21. The method of claim 19, wherein said culture medium in the second step further comprises at least two compounds selected from the group consisting of glutathione, ethanolamine, biotin, alpha Tocopherol (Vitamin E), and albumin (human or bovine).

22. The method of claim 18, wherein said culture medium in the second step further comprises at least two compounds selected from the group consisting of L-carnitine, corticosterone, D(+) galactose, linoleic acid, linolenic acid, progesterone, putrescine, retinly acetate, triodo-1-thyronin (T3), DL-α

-tocopherol acetate, catalase, superoxide dismutase, apotransferrin and bFGF.

23. The method of claim 19, wherein said culture medium in the second step further comprises at least two compounds selected from the group consisting of L-carnitine, corticosterone, D(+) galactose, linoleic acid, linolenic acid, progesterone, putrescine, retinly acetate, triodo-1-thyronin (T3), DL-α

-tocopherol acetate, catalase, superoxide dismutase, apotransferrin and bFGF.

24. The method of claim 21, wherein said culture medium in the second step further comprises at least two compounds selected from the group consisting of L-carnitine, corticosterone, D(+) galactose, linoleic acid, linolenic acid, progesterone, putrescine, retinly acetate, triodo-1-thyronin (T3), DL-α

-tocopherol acetate, catalase, superoxide dismutase, apotransferrin and bFGF.

52. The method of claim 51, wherein said culture medium further comprises insulin, transferrin, and selenium.

54. The method of claim 52, wherein said culture medium comprises at least two compounds selected from the group consisting of glutathione, ethanolamine, biotin, alpha Tocopherol (Vitamin E), and albumin (human or bovine).

56. The method of claim 52, wherein said culture medium further comprises at least two compounds selected from the group consisting of L-carnitine, corticosterone, D(+) galactose, linoleic acid, linolenic acid, progesterone, putrescine, retinly acetate, triodo-1-thyronin (T3), α

-tocopherol, catalase, superoxide dismutase, apotransferrin and bFGF.

57. The method of claim 54, wherein said culture medium further comprises at least two compounds selected from the group consisting of L-carnitine, corticosterone, D(+) galactose, linoleic acid, linolenic acid, progesterone, putrescine, retinly acetate, triodo-1-thyronin (T3), α

-tocopherol, catalase, superoxide dismutase, apotransferrin and bFGF.

25. A method of converting differentiated non-hormone producing pancreatic cells into stem cells comprising culturing said differentiated non-hormone producing pancreatic cells in a cell culture system with a cell culture medium, under conditions which provide for converting said differentiated non-hormone producing pancreatic cell into stem cells.

26. The method of claim 25, wherein said stem cells proliferate.

27. The method of claim 25, wherein the differentiated non-hormone producing pancreatic cells comprise pancreatic acinar cells.

28. The method of claim 27, wherein the acinar cells are in a pancreatic cell mixture.

29. The method of claim 25, wherein said differentiated non-hormone producing pancreatic cells are cultured with a culture mode selected from the group consisting of:

adherent, suspension and matrix.

30. The method of claim 29, wherein said culture mode is an adherent culture mode that comprises culturing the pancreatic cell mixture directly on a surface of a tissue culture container or on a surface of a tissue culture container which is coated with at least one compound selected from the group consisting of collagen, fibronectin, laminin, and hyaluronic acid.

31. The method of claim 25, wherein said cells are seeded at a density of 10⁵ to 10⁷ cells/cm².

32. The method of claim 29, wherein said culture mode is a suspension culture mode that comprises culturing said pancreatic cell mixture in suspension in said culture medium.

33. The method of claim 29, wherein said culture mode is a matrix culture mode that comprises culturing said pancreatic cell mixture interspersed in a crosslinked polymerizable gel.

34. The method of claim 33, wherein said pancreatic cell mixture is seeded at a density of 10⁴ to 10⁸ cells/ml in a hydrogel.

35. The method of claim 34, wherein said hydrogel is alginate.

36. The method of claim 25, wherein said culture medium comprises serum and a basal medium selected from the group consisting of Dulbecco'"'"'s Modified Eagle'"'"'s medium (DMEM), Medium 199 (M199), Ham'"'"'s F12 Nutrient Mixture (Ham'"'"'s F12), RPMI 1640 Medium, CMRL medium, and mixtures thereof.

37. The method of claim 36, wherein said culture medium in the first step further comprises at least three compounds selected from the group consisting of insulin, transferrin, selenium, zinc sulphate, glutathione, ethanolamine, cyclodextrin, biotin, alpha Tocopherol, calcium pantothenate, myoinositol, nicotinamide, IGF1, Prolactin, exendin, EGF, VEGF, KGF, and HGF.

38. The method of claim 25, wherein said culture medium in the first step comprises a basal medium without serum selected from the group consisting of Dulbecco'"'"'s Modified Eagle'"'"'s medium (DMEM), Medium 199 (M199), Ham'"'"'s F12 Nutrient Mixture (Ham'"'"'s F12), RPMI 1640 Medium, CMRL medium, and mixtures thereof.

39. The method of claim 38, wherein said culture medium in the first step further comprises at least three compounds selected from the group consisting of insulin, transferrin, selenium, zinc sulphate, glutathione, ethanolamine, cyclodextrin, biotin, alpha Tocopherol, calcium pantothenate, myoinositol, nicotinamide, IGF1, Prolactin, exendin, EGF, VEGF, KGF, and HGF.

40. A method of converting stem cells into differentiated hormone-producing cells, comprising culturing the stem cells in a cell culture system with a cell culture medium whereby said stem cells are differentiated into hormone-producing cells wherein said culture medium comprises basal medium without serum and at least three compounds selected from the group consisting of glutathione, ethanolamine, biotin, alpha Tocopherol (Vitamin E), albumin (human or bovine), L-carnitine, corticosterone, D(+) galactose, linoleic acid, linolenic acid, progesterone, putrescine, retinly acetate, triodo-1-thyronin (T3), superoxide dismutase, apotransfeitin and bFGF.

41. The method of claim 40, wherein said stem cells proliferate.

42. The method of claim 40, wherein said hormone-producing cells produce insulin.

43. The method of claim 40, wherein said hormone-producing cells produce glucagon.

44. The method of claim 40, wherein said stem cell is cultured with a culture mode selected from the group consisting of:

adherent, suspension and matrix.

45. The method of claim 44, wherein said culture mode is an adherent culture mode that comprises culturing stem cells directly on a surface of a tissue culture container or a surface of a tissue culture container which is coated with at least one compound selected from the group consisting of collagen, fibronectin, laminin, and hyaluronic acid.

46. The method of claim 40, wherein said stem cells are seeded at a density of 10⁵ to 10⁷ cells/cm².

47. The method of claim 44, wherein said culture mode is a suspension culture mode that comprises culturing said stem cells in suspension in said culture medium.

48. The method of claim 44, wherein said culture mode is a matrix culture mode that comprises culturing the stem cells interspersed in a crosslinked polymerizable gel.

49. The method of claim 48, wherein said stem cells are seeded at a density of 10⁴ to 10⁸ cells/ml in a hydrogel.

50. The method of claim 49, wherein said hydrogel is alginate.

51. The method of claim 40, wherein said basal medium without serum is selected from the group consisting of Dulbecco'"'"'s Modified Eagle'"'"'s medium (DMEM), Medium 199 (MI 99), Ham'"'"'s F12 Nutrient Mixture (Ham'"'"'s F12), RPMI 1640 Medium, CMRL medium, neurobasal medium, Johe'"'"'s N2 medium, and mixtures thereof.

53. The method of claim 51, wherein said culture medium comprises at least two compounds selected from the group consisting of glutathione, ethanolamine, biotin, alpha Tocopherol (Vitamin E), and albumin (human or bovine).

55. The method of claim 51, wherein said culture medium further comprises at least two compounds selected from the group consisting of L-carnitine, corticosterone, D(+) galactose, linoleic acid, linolenic acid, progesterone, putrescine, retinly acetate, triodo-1-thyronin (T3), α

-tocopherol, catalase, superoxide dismutase, apotransferrin and bFGF.