Cell reprogramming

• US 20040072343A1
• Filed: 11/03/2003
• Published: 04/15/2004
• Est. Priority Date: 11/09/2000
• Status: Abandoned Application

• Current Assignee: Bresagen, Inc. (Vertex Pharmaceuticals Incorporated)
• Original Assignee: Bresagen, Inc. (Vertex Pharmaceuticals Incorporated)
• Inventors: Pralong, Danielle Jacqueline, Rathjen, Peter David, Verma, Paul John
• Application Number:
  US10/416,361
• Publication Number:
  US 20040072343A1
• 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: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: C12N 2506/08 from cells of the nervous system
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 5/0622 Glial cells, e.g. astrocytes, oligodendrocytes; Schwann cells
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

Abstract

A method for reprogramming differentiated or partially differentiated cells to a less differentiated or dedifferentiated state, which method includes providing a karyoplast of a differentiated or partially differentiated cell; and a source of multinucleate, aneuploid, euploid or polyploid multipotent or pluripotent cells, or morula or early embryos, or derivatives thereof; and placing the karyoplast in intimate contact with the multipotent or pluripotent cells, or morula or early embryos or derivatives thereof under conditions sufficient to permit at least partial reprogramming of the cells.

Claims

1. A method for reprogramming differentiated or partially differentiated cells to a less differentiated or dedifferentiated state, which method includes

providing

a karyoplast of a differentiated or partially differentiated cell;

and

a source of multinucleate, aneuploid, euploid or polyploid multipotent or pluripotent cells, or morula or early embryos, or derivatives thereof; and

placing the karyoplast in intimate contact with the multipotent or pluripotent cells, or morula or early embryos or derivatives thereof under conditions sufficient to permit at least partial reprogramming of the cells.

2. A method according to claim 1, wherein the differentiated or partially differentiated cells are autologous.

3. A method according to claim 2, wherein the differentiated or partially differentiated cells are selected from the group consisting of blood (including bone marrow), skin, muscle, adipocyte or neural cells.

4. A method according to claim 3, wherein the multipotent or pluripotent cell source is derived from cells selected from the group consisting of embryonic stem cells (ES cells), early primitive ectoderm-like cells (EPL cells), primordial germ cells (PG cells) and embryonic carcinoma cells (EC cells);

or derivatives thereof or mixtures thereof.

5. A method according to claim 4, wherein the multipotent or pluripotent cell source is derived from embryoid bodies derived from ES or EPL cells in vitro.

6. A method according to claim 5, wherein the karyoplast is incorporated within the embryoid body or aggregated with cells of the embryoid body.

7. A method according to claim 1 wherein the reprogramming is conducted utilising a cytoplast derived from the polyploid, aneuploid or multinucleate multipotent or pluripotent cell source.

8. A method according to claim 7, wherein the multipotent or pluripotent cell source includes cytoplasts derived from ES, EPL, PG or EC cells.

9. A method according to claim 1, wherein, when multipotent or pluripotent cells are used, the cell contact step further includes

subjecting the cells to a cell fusion step.

10. A method according to claim 9, wherein the cell fusion step includes subjecting the cells to an electrical pulse or exposure to polyethylene glycol (PEG) or a combination thereof.

11. A method according to claim 1, wherein the multipotent or pluripotent cell source is a multinucleate or polyploid pluripotent cell.

12. A method according to claim 11, wherein the multinucleate or polyploid pluripotent cell is a polyploid ES or EPL cell;

and

the differentiated or partially differentiated cells are blood or neural cells.

13. A method according to claim 1 wherein, when the cells are partially reprogrammed,

isolating the partially reprogrammed cells;

and

repeating the contact step until reprogramming is complete.

38. A reprogrammed or partially reprogrammed cell, when produced according to any one of the preceding claims.

59. A method for identifying dedifferentiation of differentiated or partially differentiated cells, which method includes

providing a cell mixture including reprogrammed or partially reprogrammed cells, produced according to any one of claims 1 to 37

the reprogrammed cells being optionally modified to include a pluripotent marker;

and

screening the cell mixture for the presence or absence of pluripotent activity.

60. A method according to claim 59, wherein, when the reprogrammed cells are modified to include a pluripotent marker,

subsequently screening the cell mixture for the presence or absence of pluripotent marker activity.

61. A method according to claim 59, wherein the reprogramming step includes

placing karyoplasts of differentiated or partially differentiated cells in intimate contact with multipotent or pluripotent cells or morula or early embryos under conditions sufficient to permit at least partial reprogramming of the cells.

62. A method according to claim 61, wherein the multipotent or pluripotent cells are multinucleate, aneuploid, euploid or polyploid multipotent or pluripotent cells derived from fusion of two or more diploid cells.

63. A method according to claim 62, wherein the reprogrammed cell construct includes an ES or EPL cell line genetically modified to express a marker gene.

64. A method according to claim 63, wherein the cell line is an ES cell line transfected with an Oct 4 TK-GFP construct from which a karyoplast is derived.

65. A method for the production of differentiated or partially differentiated cells, which method includes:

providing

a source of reprogrammed multipotent or pluripotent cells according to claim 38;

a suitable medium; and

culturing the reprogrammed cells, optionally in the presence of a selected growth factor, for a period sufficient to permit partial or terminal differentiation.

66. A method according to claim 65, wherein the reprogrammed cells are derived from autologous cells.

67. A differentiated or partially differentiated cell when produced according to the method according to claim 65.

68. A method for the production of a chimeric animal, which method includes animal, which method includes

providing

a source of reprogrammed multipotent or pluripotent cells according to claim 38;

a pregastrulation embryo;

introducing the multipotent or pluripotent cells into the pregastrulation embryo; and

monitoring chimera forming ability.

69. Use of reprogrammed cells according to claim 38, or their differentiated or partially differentiated progeny, for use in human cell therapy or transgenic animal production.

70. Use of reprogrammed cells according to claim 38, or their differentiated or partially differentiated progeny, for use in human or animal gene therapy.

14. A method for reprogramming differentiated or partially differentiated cells to a less differentiated state, or de-differentiated state, which method includes

providing

a karyoplast of a differentiated or partially differentiated cell;

a source of mammalian morula or early embryos or derivatives thereof; and

placing the karyoplast in intimate contact with morula or early embryos or derivatives thereof for a period sufficient to permit at least partial reprogramming of the cells.

15. A method according to claim 14 wherein a mammalian karyoplast is placed in the perivitelline space of an early embryo.

16. A method according to claim 14, which method further includes separating the reprogrammed cells from other cells.

17. A method according to claim 16, which method includes

providing a source of differentiated or partially differentiated cells genetically modified to include a selectable marker;

and

selecting the reprogrammed cells utilising the selectable marker.

18. A method according to claim 16, wherein the selectable marker is a marker expressed in pluripotent or multipotent cells.

19. A method according to claim 18, wherein the selectable marker contains a gene construct encoding Green Fluorescent Protein (GFP) linked to an Oct 4 promoter.

20. A method according to claim 14, wherein the differentiated or partially differentiated cells are blood (including bone marrow), skin, muscle, adipocyte or neural cells.

21. A method according to claim 14 wherein, when the cells are partially reprogrammed,

isolating the partially reprogrammed cells;

and

repeating the contact step until reprogramming is complete.

22. A method for reprogramming differentiated or partially differentiated cells to a less differentiated, or dedifferential state, by formation of a cell hybrid, which method includes

providing:

a source of karyoplasts of differentiated or partially differentiated cells;

a source of multipotent or pluripotent cells, morula or early embryos or derivatives thereof;

placing the karyoplasts in intimate contact with the multipotent or pluripotent cells;

subjecting the cells to a cell fusion step to form a cell hybrid;

subsequently removing from said reprogrammed cells, the multipotent or pluripotent cell nuclei; and

culturing the reprogrammed cells to permit proliferation thereof.

23. A method according to claim 22, wherein the differentiated or partially differentiated cell source is a somatic cell.

24. A method according to claim 23, wherein the differentiated or partially differentiated cells are selected from the group consisting of blood (including bone marrow), skin, muscle, adipocyte or neural cells.

25. A method according to claim 22, wherein the multipotent or pluripotent cells are multinucleate, aneuploid, euploid or polypoid multipotent or pluripotent cells.

26. A method according to claim 22 wherein the cell fusion step includes subjecting the cells to an electrical pulse or exposure to polyethylene glycol (PEG) or a combination thereof.

27. A method according to claim 22, wherein the cell fusion step is such that the nuclei of the cell components remain separated;

and

subsequently removing from said reprogrammed cells, the multipotent or pluripotent cell nuclei.

28. A method according to claim 22 wherein

the somatic cell nucleus and the nucleus of multipotent or pluripotent cells are maintained as separate nuclei by maintaining the cells at low temperature and/or by utilising a cell cycle arrester, and/or a cytoskeletal inhibitor.

29. A method according to claim 22 wherein the multipotent or pluripotent cell nucleus is removed by differential centrifugation.

30. A method according to claim 22, wherein the reprogrammed cells exhibit spontaneous removal of the multipotent or pluripotent cell nuclei.

31. A method for reprogramming differentiated or partially differentiated cells to a less differentiated or dedifferentiated state, which method includes

providing

a source of cytoplasm from a multinucleate, aneuploid, euploid or polypoid multipotent or pluripotent cell;

a karyoplast derived from a partially or terminally differentiated cell differing from the cytoplasm source;

a suitable culture medium

combining the cytoplasm source and karyoplast to form a reconstructed cell; and

maintaining the reconstructed cell in the culture medium for a time sufficient to permit at least partial reprogramming of the karyoplast in the combined cell.

32. A method according to claim 31, wherein the source of cytoplasm is cytoplasm removed from ES or EPL cells or derivatives thereof, or cytoplasm derived by at least partial enucleation of said cells.

33. A method according to claim 31, wherein the karyoplast is derived from blood (including bone marrow), skin, muscle, adipocyte or neural cells.

34. A method according to claim 31, wherein the cell reconstruction step includes fusion of the karyoplast and cytoplast so that the cytoplast content interacts with the karyoplast and induces at least partial reprogramming of the nuclear material.

35. A method according to claim 34, wherein the fusion step includes subjecting the cell components to an electrical pulse or exposure to polyethylene glycol (PEG) or a combination thereof.

36. A method according to claim 31, wherein, when the cells are partially reprogrammed,

isolating the partially reprogrammed cells;

and

repeating the reconstruction and maintenance steps until reprogramming is complete.

37. A method according to claim 31, wherein the multinucleate or polyploid pluripotent cell is a polyploid ES or EPL cell;

and

the differentiated or partially differtiated cells are blood or neural cells.

39. A method for preparing a cytoplast, which method includes

providing a multinucleate, aneuploid, euploid or polyploid multipotent or pluripotent cell;

subjecting the multipotent or pluripotent cell to an enucleation step; and

harvesting the cytoplast so formed.

40. A method according to claim 39, where the cytoplast is obtained by micromanipulation, density gravity centrifugation or centrifugal enucleation optionally in the presence of cytochalasin B.

41. A method for preparing a karyoplast which method includes

providing a partially or terminally differentiated cell;

subjecting the partially or terminally differentiated cell to an enucleation step; and

harvesting the karyoplast so formed.

42. A method according to claim 41, wherein the partially or terminally differentiated cell is a partially or terminally differentiated somatic cell.

43. A method according to claim 42, wherein the somatic cell is selected from the group consisting of blood (including bone marrow), skin, muscle, adipocyte or neural cell.

44. A method according to claim 41, wherein the karyoplast is obtained by micromanipulation, density gravity centrifugation or centrifugal enucleation, optionally in the presence of cytochalasin B.

45. A method for preparing a multinucleate, aneuploid, euploid or polyploid multipotent or pluripotent cell, which method includes

providing a source of multipotent and/or pluripotent cells;

and

subjecting the cells to a fusion step to produce a multinucleate, aneuploid, euploid or polyploid cell.

46. A method according to claim 45, wherein the multipotent or pluripotent cell source includes two or more diploid cells.

47. A method according to claim 45, wherein the cell fusion step leads to the production of large multinucleate, aneuploid, euploid or polyploid cells that contain an increased pool of cytoplasm.

48. A method according to claim 45, wherein the pluripotent cells are embryonic stem (ES) cells or early primitive ectoderm-like (EPL) cells.

49. A method according to claim 45, wherein the cell fusion step includes fusion utilising electrofusion or PEG-modified fusion, or a combination thereof.

50. A method according to claim 45, wherein the cell so formed is a multinucleate or polyploid pluripotent cell.

51. A multinucleate, aneuploid, euploid or polyploid multipotent or pluripotent cell derived from fusion of two or more diploid cells.

52. A multinucleate, aneuploid, euploid or polyploid multipotent or pluripotent cell according to claim 51, wherein the cell is a multinucleate or polyploid pluripotent cell.

55. A multinucleate, aneuploid;

euploid or polyploid multipotent or pluripotent cell according to claim 51, wherein the cell is derived from ES, EPL, PG or EC cells.

56. A cytoplast derived from a multinucleate, aneuploid, euploid of polyploid multipotent or pluripotent cell according to claim 51.

57. A cell line formed from the multinucleate, aneuploid, euploid or polyploid multipotent or pluripotent cell according to claim 51.

58. A cell line according to claim 57, which is stable in culture in vitro.

53. A multinucleate, aneuploid, euploid or polyploid multipotent or pluripotent cell capable of expressing at least one multipotent or pluripotent marker.

54. A multinucleate, aneuploid, euploid or polyploid multipotent or pluripotent cell according to claim 53, wherein the multipotent or pluripotent marker is Oct4 or alkaline phosphatase.