Alternative compositions and methods for the culture of stem cells

• US 20050037488A1
• Filed: 08/06/2002
• Published: 02/17/2005
• Est. Priority Date: 08/06/2001
• Status: Active Grant

• Current Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Original Assignee: Bresagen, Inc. (Vertex Pharmaceuticals Incorporated)
• Inventors: Mitalipova, Maisam, Lyons, Ian
• Granted Patent: US 7,432,104 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• 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: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2502/1323 Adult fibroblasts
• CPC Class Codes: C12N 2502/1335 Skeletal muscle cells, myocytes, myoblasts, myotubes
• CPC Class Codes: C12N 2502/1394 Bone marrow stromal cells; whole marrow
• CPC Class Codes: C12N 2502/243 Cells of the female genital tract, non-germinal ovarian cells
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696

Abstract

Methods and cell culture medium for the generation of human pluripotent embryonic stem cells are disclosed. Human embryonic stem cells are cultured with human granulosa feeder cells, muscle cells, Fallopian ductal epithelial cells, bone marrow stromal cells, and skin fibroblasts and the embryonic stem cells maintain their pluripotent phenotype. The human pluripotent embryonic stem cells can be cultured without feeder cells, and in the presence of supplemental growth factors. The human pluripotent embryonic stem cells can be alternatively cultured with conditioned medium obtained from a cell culture capable of maintaining human embryonic stem cells in a pluripotent state, wherein the cell culture is a human granulosa cell culture.

Claims

1. A human pluripotent stem cell culture, comprising a human pluripotent stem cell and a human feeder cell.

2. The human pluripotent stem cell culture of claim 1, wherein the human pluripotent stem cell is an embryonic stem cell.

3. The human pluripotent embryonic stem cell culture of claim 2, wherein the human feeder cell is selected from the group consisting of a skeletal muscle cell, a Fallopian ductal epithelial cell, a bone marrow stromal cell, a granulosa cell or a fibroblast cell.

4. The human pluripotent embryonic stem cell culture of claim 1, wherein the fibroblast cell is a skin keloid fibroblast cell.

5. The human pluripotent embryonic stem cell culture of claim 4, wherein the skin keloid fibroblast cell has the identifying characteristic of ATCC CRL-1762.

6. The human pluripotent stem cell culture of claim 1, wherein the fibroblast cell is a fetal skin fibroblast cell.

7. The human pluripotent embryonic stem cell culture of claim 1, wherein the human feeder cell is a bone marrow stromal cell

8. The human pluripotent embryonic stem cell culture of claim 7, wherein the bone marrow stromal cell has the identifying characteristic of ATCC CRL-11882.

9. The human pluripotent embryonic stem cell culture of claim 1, wherein the human feeder cell is a granulosa cell.

10. The human pluripotent embryonic stem cell culture of claim 1, wherein the human feeder cell is a Fallopian ductal epithelial cell.

11. The human pluripotent embryonic stem cell culture of claim 1, wherein the human feeder cell is a skeletal muscle cell.

12. The human pluripotent embryonic stem cell culture of claim 11, wherein the skeletal muscle cell is a fetal skeletal muscle cell.

13. The human pluripotent embryonic stem cell culture of claim 1, wherein the human feeder cell maintains embryonic stem cells and expresses IL-6 and signals through the gp130 pathway.

14. The human pluripotent embryonic stem cell culture of any of claims 1, 3, 4 or 7, wherein the human feeder cell is mitotically inactivated.

28. The human pluripotent stem cell culture of claim 1 or 15, wherein the human pluripotent embryonic stem cell culture further comprises one or more supplemental growth factors.

29. The human pluripotent stem cell culture of claim 28, wherein one or more supplemental growth factors are selected from the group consisting of SCF, OSM, CNTF, IL-6, IL-6R, FGF, BMP, TNF, and GM-CSF.

32. A tissue generated from the cell culture of claim 1 or 15.

65. A method of treating a disease using the cell or cell culture of any of claims 1, 15, 33, 46, 51, 55, and 59, wherein the disease is selected from the group consisting of Parkinson'"'"'s, Alzheimer'"'"'s, Multiple Sclerosis, spinal cord injuries, stroke, macular degeneration, burns, liver failure, heart disease, diabetes, Duchenne'"'"'s muscular dystrophy, osteogenesis imperfecta, osteoarthritis, rheumatoid arthritis, anemia, leukemia, breast cancer, solid tumors, and AIDS.

66. The method of claim 65, wherein the disease is Parkinson'"'"'s or Alzheimer'"'"'s.

15. A human pluripotent stem cell culture comprising a human pluripotent stem cell and a human feeder cell conditioned medium.

16. The human pluripotent stem cell culture of claim 15, wherein the human pluripotent stem cell is an embryonic stem cell.

17. The human pluripotent stem cell culture of claim 16, wherein the human feeder cell is a skeletal muscle cell, Fallopian ductal epithelial cell, a granulosa cell, a bone marrow stromal cell, or a fibroblast cell.

18. The human pluripotent stem cell culture of claim 15, wherein the human feeder cell maintains embryonic stem cells and expresses IL-6 and signals through the gp130 pathway.

19. The human pluripotent stem cell culture of claim 17, wherein the fibroblast cell is a skin keloid fibroblast.

20. The human pluripotent embryonic stem cell culture of claim 19, wherein the skin keloid fibroblast cell has the identifying characteristic of ATCC CRL-1762.

21. The human pluripotent stem cell culture of claim 17, wherein the fibroblast cell is a fetal skin fibroblast cell.

22. The human pluripotent embryonic stem cell culture of claim 15, wherein the human feeder cell is a bone marrow stromal cell.

23. The human pluripotent embryonic stem cell culture of claim 22, wherein the bone marrow stromal cell has the identifying characteristic of ATCC CRL-11882.

24. The human pluripotent embryonic stem cell culture of claim 15, wherein the human feeder cell is a granulosa cell.

25. The human pluripotent embryonic stem cell culture of claim 15, wherein the human feeder cell is a Fallopian ductal epithelial cell.

26. The human pluripotent embryonic stem cell culture of claim 15, wherein the human feeder cell is a skeletal muscle cell.

27. The human pluripotent embryonic stem cell culture of claim 26, wherein the skeletal muscle cell is a fetal skeletal muscle cell.

30. (Canceled)

31. (Canceled)

33. A method of maintaining a human pluripotent stem cell culture, comprising culturing a human stem cell on a human feeder cell layer.

34. The method of claim 33, wherein the human stem cell is an embryonic stem cell.

35. The method of claim 33, wherein the human feeder cell layer is a skeletal muscle cell, a granulosa cell, a bone marrow stromal cell, or a fibroblast cell.

36. The method of claim 35, wherein the human fibroblast cell is a skin keloid fibroblast cell.

37. The method of claim 36, wherein the skin keloid fibroblast cell has the identifying characteristic of ATCC CRL-1762.

38. The method of claim 35, wherein the human fibroblast cell is a fetal skin fibroblast cell.

39. The method of claim 33, wherein the human feeder cell is a bone marrow stromal cell.

40. The method of claim 39, wherein the bone marrow stromal cell has the identifying characteristic of ATCC CRL-11882.

41. The method of claim 33, wherein the human feeder cell is a granulosa cell.

42. The method of claim 33, wherein the human feeder cell is a Fallopian ductal epithelial cell.

43. The method of claim 33, wherein the human feeder cell is a skeletal muscle cell.

44. The method of claim 43, wherein the skeletal muscle cell is a fetal skeletal muscle cell.

45. The method of claim 33, wherein the human feeder cell maintains embryonic stem cells and expresses IL-6 and signals through the gp130 pathway.

61. The method of any of claims 33, 46, 51, 55, and 59, further comprising one or more supplemental growth factors.

62. The method of claim 61, wherein one or more supplemental growth factors are selected from the group consisting of SCF, OSM, CNTF, IL-6, IL-6R, FGF, BMP, TNF, and GM-CSF.

63. A human pluripotent embryonic stem cell developed by the method of any of claims 33, 46, 51, 55, and 59.

64. A tissue generated from the cell culture of any of claims 33, 46, 51, 55, and 59.

46. A method of maintaining a human pluripotent embryonic stem cell culture, comprising the steps of:

(a) isolating cells from the inner cell mass of a blastocyst;

(b) plating the inner cell mass cells, wherein inner cell mass-derived cell masses are formed; and

(c) re-plating and maintaining the human embryonic stem cell colony on a human feeder cell layer to thereby maintain a human pluripotent embryonic stem cell.

47. The method of claim 46, wherein the wherein the human feeder cell is selected from the group consisting of a skeletal muscle cell, a Fallopian ductal epithelial cell, a granulosa cell, a bone marrow stromal cell, a fetal skin cell, and a skin keloid fibroblast cell.

48. The method of claim 46, wherein the inner cell mass-derived cells are dissociated, and re-plated on a mouse embryonic fibroblast cell.

49. The method of claim 46, further comprising the step of selecting a colony with the characteristics of a human embryonic stem cell after re-plating the inner cell mass cells and before re-plating the human embryonic stem cell colony.

50. The method of claim 49, wherein the wherein the human feeder cell is selected from the group consisting of a skeletal muscle cell, a Fallopian ductal epithelial cell, a granulosa cell, a bone marrow stromal cell, a fetal skin fibroblast cell, and a skin keloid fibroblast cell.

51. A method of isolating and maintaining a human pluripotent embryonic stem cell culture, comprising the steps of:

(a) isolating cells from the inner cell mass of a blastocyst;

(b) plating the inner cell mass cells on a human feeder cell, wherein inner cell mass-derived cell masses are formed; and

(c) re-plating and maintaining the human embryonic stem cell colony on a human feeder cell to thereby isolate and maintain a human pluripotent embryonic stem cell.

52. The method of claim 51, wherein the wherein the human feeder cell is selected from the group consisting of a skeletal muscle cell, a Fallopian ductal epithelial cell, a granulosa cell, a bone marrow stromal cell, a fetal skin fibroblast cell, and a skin keloid fibroblast cell.

53. The method of claim 51, wherein the inner cell mass-derived cells are dissociated, and re-plated on a human feeder cell, and a colony is selected with the characteristics of a human embryonic stem cell prior to re-plating the selected human embryonic stem cell colony on a human feeder cell.

54. The method of claim 53, wherein the wherein the human feeder cell is selected from the group consisting of a skeletal muscle cell, a Fallopian ductal epithelial cell, a granulosa cell, a bone marrow stromal cell, a fetal skin fibroblast cell, and a skin keloid fibroblast cell.

55. A method of maintaining a human pluripotent embryonic stem cell culture, comprising culturing a selected embryonic stem cell colony in a human feeder cell conditioned medium.

56. The method of claim 55, wherein the wherein the human feeder cell is selected from the group consisting of a skeletal muscle cell, a Fallopian ductal epithelial cell, a granulosa cell, a bone marrow stromal cell, a fetal skin fibroblast cell, and a skin keloid fibroblast cell.

57. The method of claim 55, comprising the steps of:

(a) isolating cells from the inner cell mass of a blastocyst;

(b) plating the inner cell mass cells on a mouse embryonic fibroblast cell, wherein inner cell mass-derived cell masses are formed;

(c) dissociating the inner cell mass-derived cell masses into dissociated cells;

(d) re-plating the dissociated cells on a mouse embryonic fibroblast cell;

(e) selecting colonies with the characteristics of human ES cells;

(f) re-plating the colonies in the absence of a feeder cell; and

(g) adding a human feeder cell conditioned medium to thereby maintain a human pluripotent embryonic stem cell.

58. The method of claim 57, wherein the wherein the human feeder cell is selected from the group consisting of a skeletal muscle cell, a Fallopian ductal epithelial cell, a granulosa cell, a bone marrow stromal cell, a fetal skin fibroblast cell, and a skin keloid fibroblast cell.

59. A method of isolating and maintaining a human pluripotent embryonic stem cell culture, comprising the steps of:

(a) isolating cells from the inner cell mass of a blastocyst;

(b) plating the inner cell mass cells on a human feeder cell, wherein inner cell mass-derived cell masses are formed;

(c) dissociating the inner cell mass-derived cell masses into dissociated cells;

(d) re-plating the dissociated cells on a human feeder cell;

(e) selecting colonies with the characteristics of human ES cells;

(f) re-plating the colonies in the absence of a feeder cell; and

(g) adding a human feeder cell conditioned medium to thereby isolate and maintain a human pluripotent embryonic stem cell.

60. The method of claim 59, wherein the wherein the human feeder cell is selected from the group consisting of a skeletal muscle cell, a Fallopian ductal epithelial cell, a granulosa cell, a bone marrow stromal cell, a fetal skin fibroblast cell, and a skin keloid fibroblast cell.