Embryonic or stem-like cell lines produced by cross species nuclear transplantation and methods for enhancing embryonic development by genetic alteration of donor cells or by tissue culture conditions

• US 20030229908A1
• Filed: 12/27/2002
• Published: 12/11/2003
• Est. Priority Date: 08/19/1996
• Status: Active Grant

• Current Assignee: Advanced Cell Technology Incorporated (Astellas Pharma Incorporated)
• Original Assignee: Advanced Cell Technology Incorporated (Astellas Pharma Incorporated)
• Inventors: West, Michael D., Cibelli, Jose
• Granted Patent: US 7,696,404 B2
• Time in Patent Office: Days
• US Class Current: 800/14
• CPC Class Codes: A01K 2217/075 inducing loss of function, i.e. knock out
• 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 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C12N 15/87 Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• CPC Class Codes: C12N 15/873 Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
• CPC Class Codes: C12N 15/877 Techniques for producing new mammalian cloned embryos
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2517/04 Cells produced using nuclear transfer

Abstract

An improved method of nuclear transfer involving the transplantation of differentiated donor cell nuclei into enucleated oocytes of a species different from the donor cell is provided. The resultant nuclear transfer units are useful for the production of isogenic embryonic stem cells, in particular human isogenic embryonic or stem cells. These embryonic or stem-like cells are useful for producing desired differentiated cells and for introduction, removal or modification, of desired genes, e.g., at specific sites of the genome of such cells by homologous recombination. These cells, which may contain a heterologous gene, are especially useful in cell transplantation therapies and for in vitro study of cell differentiation. Also, methods for improving nuclear transfer efficiency by genetically altering donor cells to inhibit apoptosis, select for a specific cell cycle and/or enhance embryonic growth and development are provided.

Claims

1. A method of producing embryonic or stem-like cells comprising the following steps:

(i) inserting a desired differentiated human or mammalian cell or cell nucleus into an enucleated animal oocyte, wherein such oocyte is derived from a different animal species than the human or mammalian cell under conditions suitable for the formation of a nuclear transfer (NT) unit;

(ii) activating the resultant nuclear transfer unit;

(iii) culturing said activated nuclear transfer unit until greater than the 2-cell developmental stage; and

(iv) culturing cells obtained from said cultured NT units to obtain embryonic or stem-like cells.

2. The method of claim 1, wherein the cell inserted into the enucleated animal oocyte is a human cell.

3. The method of claim 2, wherein said human cell is an adult cell.

4. The method of claim 2, wherein said human cell is an epithelial cell, keratinocyte, lymphocyte or fibroblast.

5. The method of claim 2, wherein the oocytes are obtained from a mammal.

6. The method of claim 5, wherein the animal oocyte is obtained from an ungulate.

7. The method of claim 6, wherein said ungulate is selected from the group consisting of bovine, ovine, porcine, equine, caprine, and buffalo.

8. The method of claim 1, wherein the enucleated oocyte is matured priorto enucleation.

9. The method of claim 1, wherein the fused nuclear transfer units are activated in vitro.

10. The method of claim 1, wherein the activated nuclear transfer units are cultured on a feeder layer culture.

11. The method of claim 10, wherein the feeder layer comprises fibroblasts.

12. The method of claim 1, wherein in step (iv) cells from a NT unit having 16 cells or more are cultured on a feeder cell layer.

13. The method of claim 12, wherein said feeder cell layer comprises fibroblasts.

14. The method of claim 13, wherein said fibroblasts comprise mouse embryonic fibroblasts.

15. The method of claim 1, wherein the resultant embryonic or stem-like cells are induced to differentiate.

16. The method of claim 2, wherein the resultant embryonic or stem-like cells are induced to differentiate.

17. The method of claim 1, wherein fusion is effected by electrofusion.

18. Embryonic or stem-like cells obtained according to the method of claim 1.

19. Human embryonic or stem-like cells obtained according to the method of claim 2.

20. Human embryonic or stem-like cells obtained according to the method of claim 3.

21. Human embryonic or stem-like cells obtained according to the method of claim 4.

22. Human embryonic or stem-like cells obtained according to the method of claim 6.

23. Human embryonic or stem-like cells obtained according to the method of claim 7.

24. Differentiated human cells obtained by the method of claim 16.

25. The differentiated human cells of claim 24, which are selected from the group consisting of neural cells, hematopoietic cells, pancreatic cells, muscle cells, cartilage cells, urinary cells, liver cells, spleen cells, reproductive cells, skin cells, intestinal cells, and stomach cells.

26. A method of therapy which comprises administering to a patient in need of cell transplantation therapy isogenic differentiated human cells according to claim 24.

27. The method of claim 26, wherein said cell transplantation therapy is effected to treat a disease or condition selected from the group consisting of Parkinson'"'"'s disease, Huntington'"'"'s disease, Alzheimer'"'"'s disease, ALS, spinal cord defects or injuries, multiple sclerosis, muscular dystrophy, cystic fibrosis, liver disease, diabetes, heart disease, cartilage defects or injuries, burns, foot ulcers, vascular disease, urinary tract disease, AIDS and cancer.

28. The method of claim 26, wherein the differentiated human cells are hematopoietic cells or neural cells.

29. The method of claim 26, wherein the therapy is for treatment of Parkinson'"'"'s disease and the differentiated cells are neural cells.

30. The method of claim 26, wherein the therapy is for the treatment of cancer and the differentiated cells are hematopoietic cells.

31. The differentiated human cells of claim 24, which contain and express an inserted gene.

32. The method of claim 1, wherein a desired gene is inserted, removed or modified in said embryonic or stem-like cells.

33. The method of claim 32, wherein the desired gene encodes a therapeutic enzyme, a growth factor or a cytokine.

34. The method of claim 32, wherein said embryonic or stem-like cells are human embryonic or stem-like cells.

35. The method of claim 32, wherein the desired gene is removed, modified or deleted by homologous recombination.

36. The method of claim 1, wherein the donor cell is genetically modified to impair the development of at least one of endoderm, ectoderm and mesoderm.

37. The method of claim 1, wherein the donor cell is genetically modified to increase differentiation efficiency.

38. The method of claim 36, wherein the cultured nuclear transfer unit is cultured in a media containing at least one capsase inhibitor.

39. The method of claim 1, wherein the donor cell expresses a detectable label that is indicative of the expression of a particular cyclin.

40. The method of claim 36, wherein the donor cell has been modified to alter the expression of a gene selected from the group consisting of SRF, MESP-1, HNF4, beta-1, integrin, MSD, GATA-6, GATA-4, RNA helicase A, and H beta 58.

41. The method of claim 37, wherein said donor cell has been genetically modified to introduce a DNA that provides for expression of the Q7 and/or Q9 genes.

42. The method of claim 41, wherein said gene or genes are operably linked to a regulatable promoter.

43. The method of claim 1, wherein the donor cell has been genetically modified to inhibit apoptosis.

44. The method of claim 43, wherein reduced apoptosis is provided by altering expression of one or more genes selected from the group consisting of Bad, Bok, BH3, Bik, BIk, Hrk, BNIP3, GimL, Bid, EGL-1, Bcl-XL, Bcl-w, McI-1, A1, Nr-13, BHRF-1, LMW5-HL, ORF16, Ks-Bcl-2, E1B-19K, and CED-9.

45. The method of claim 44, wherein at least one of said genes is operably linked to an inducible promoter.

46. A mammalian somatic cell that expresses a DNA that encodes a detectable marker, the expression of which is linked to a particular cyclin.

47. The cell of claim 46, wherein the cyclin is selected from the group consisting of cyclin D1, D2, D3, B1, B2, E, A and H.

48. The cell of claim 46, wherein the detectable marker is a fluorescent polypeptide.

49. The cell of claim 48, wherein said mammalian cell is selected from the group consisting of human, primate, rodent, ungulate, canine, and feline cells.

50. The cell of claim 48, wherein said cell is a human, bovine or primate cell.

51. A method of producing human embryonic stem-like cells, comprising:

(i) inserting a human cell or cell nucleus into a recipient oocyte that is derived from a non-human mammal, under conditions suitable for the formation of a nuclear transfer (NT) unit;

(ii) activating the resultant NT unit;

(iii) culturing the activated NT unit until greater than the 2-cell developmental stage; and

(iv) culturing cells obtained from the cultured NT unit to obtain human embryonic stem-like cells.

52. The method of claim 51, wherein the recipient oocyte is enucleated prior to inserting the human cell or cell nucleus.

53. The method of claim 51, wherein the oocyte comprises a protein or other substance that improves reprogramming efficiency or limits the differentiation potential of the NT unit.

54. The method of claim 52, wherein the recipient oocyte is a rabbit oocyte.

55. The method of claim 54, wherein the recipient oocyte is spiked with rabbit ooplasm.

56. The method of claim 55, wherein the oocyte comprises a protein or other substance that improves reprogramming efficiency or limits the differentiation potential of the nuclear transfer unit.

57. The method of claim 51, wherein the activated NT unit is cultured until the blastocyst stage.

58. A pluripotent human embryonic stem-like cell made by the method of claim 51.

59. The pluripotent human embryonic stem-like cell of claim 58, which cell contains mitochondrial DNA of the recipient oocyte.

60. A pluripotent human embryonic stem-like cell made by the method of claim 54.

61. The pluripotent human embryonic stem-like cell of claim 60, which cell contains rabbit mitochondrial DNA.

62. A method for producing a nuclear transfer human embryo by cross-species nuclear transfer comprising:

(i) transferring a human cell or human nucleus into a rabbit oocyte which is enucleated before, simultaneous or after said human cell or nuclear transfer; and

(ii) the resultant fusion is permitted to develop into a nuclear transfer embryo.

63. The method of claim 62 which comprises transferring the pronucleus that results after step (ii) into another oocyte which is enucleated before, simultaneous or after transfer.

64. The method of claim 62 which further comprises transferring additional ooplasm from another rabbit oocyte into the resultant fusion.

65. The method of claim 62 wherein the resultant fusion is activated simultaneous to fusion to induce embryonic development.

66. The method of claim 62 wherein the rabbit oocyte is activated prior to transferral of said human cell or nucleus therein.

67. The method of claim 62 which includes an activation step effected after said human cell or human nuclear transfer.

68. The method of claim 65, 66 or 67 wherein activation includes the use of DMAP and cycloheximide.

69. The method of claim 62 wherein the nuclear transfer embryo produced by step (ii) is cultured on a feeder layer.

70. The method of claim 69 wherein said culture gives rises to human pluripotent cells.

71. The method of claim 70 wherein said pluripotent cells are permitted to differentiate into different cell types.

72. The method of claim 69 wherein the embryo is cultured on a fibroblast feeder layer.

76. A cross-species blastula or morula that results from the process of claim 62 or claim 73.

77. Differentiated cells derived from the morula or blastocyst of claim 76.

73. A method for enhancing the development of a cross-species nuclear transfer unit produced-by transferral of a human cell or nucleus into a non-human oocyte, comprising introducing into said oocyte or said human cell prior to transferral ooplasm from a rabbit oocyte.

74. The method of claim 73 wherein said non-human oocyte is an ungulate oocyte.

75. The method of claim 74 wherein said ungulate oocyte is a bovine oocyte.