Immortalized mesenchymal cells and utilization thereof

• US 20050084959A1
• Filed: 10/31/2002
• Published: 04/21/2005
• Est. Priority Date: 10/31/2001
• Status: Abandoned Application

• Current Assignee: Renomedix Institute, Inc.
• Original Assignee: Renomedix Institute, Inc.
• Inventors: Ito, Yoshinori, Tsuda, Hajime, Kawano, Yutaka, Tanooka, Atsushi, Kobune, Masayoshi, Oka, Shin-ichi, Kato, Junji, Hamada, Hirofumi, Honmou, Osamu, Niitsu, Yoshiro, Nakamura, Kiminori, Sasaki, Katsunori, Matsunaga, Takuya
• Application Number:
  US10/493,868
• Publication Number:
  US 20050084959A1
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: A61K 2035/124 the cells being hematopoietic, bone marrow derived or blood cells
• CPC Class Codes: A61K 35/18 Erythrocytes haemoglobin A61K38/42
• CPC Class Codes: A61K 35/28 Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
• CPC Class Codes: A61K 35/32 Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
• CPC Class Codes: C12N 2503/02 Drug screening
• CPC Class Codes: C12N 2506/1353 from bone marrow mesenchymal stem cells (BM-MSC)
• CPC Class Codes: C12N 2510/04 Immortalised cells
• CPC Class Codes: C12N 5/0618 Cells of the nervous system
• CPC Class Codes: C12N 5/0641 Erythrocytes
• CPC Class Codes: C12N 5/0663 Bone marrow mesenchymal stem cells (BM-MSC)

Abstract

The method developed herein is for expanding cord blood-derived hematopoietic stem cells to a degree that is sufficiently safe for clinical application, such as the transplantation of hematopoietic stem cells into adult patients. Further, to prepare a number of mesenchymal stem cells or mesenchymal cells that have conventionally been available only in an extremely small number, an immortalizing gene such as that of telomerase is introduced alone into mesenchymal stem cells, mesenchymal cells or the like, so as to induce the differentiation of the expanded mesenchymal stem cells.

Claims

1. An immortalized mesenchymal system-related cell, which is a mesenchymal system-related cell that is selected from mesenchymal stem cells, mesenchymal precursor cells, mesenchymal cells and cells derived from mesenchymal cells, and is immortalized by high expression or activation of an immortalizing gene.

2. The cell according to claim 1, wherein the immortalizing gene is any one of a telomerase gene, a gene derived from telomerase, and a gene regulating the expression or activity of telomerase.

3. The cell according to claim 1, wherein the immortalizing gene can be specifically deleted or is already deleted by gene deletion.

4. The cell according to claim 3, wherein the deletion of the immortalizing gene is performed by placing the immortalizing gene between loxP sequences or loxP-like sequences, and then treating the gene with a recombinase such as Cre recombinase.

5. The cell according to claim 1, wherein the immortalizing gene is highly expressed by introducing the immortalizing gene into a cell by gene introduction.

6. The cell according to claim 1, wherein the mesenchymal system-related cell that is selected from mesenchymal stem cells, mesenchymal precursor cells, mesenchymal cells and cells derived from mesenchymal cells is derived from any one of bone marrow, peripheral blood, cord blood, skin, hair root and muscle tissue.

7. The cell according to claim 1, wherein the mesenchymal system-related cell that is selected from mesenchymal stem cells, mesenchymal precursor cells, mesenchymal cells and cells derived from mesenchymal cells is derived from either human ES cells (fetal stem cells) or cells derived from ES cells (fetal stem cells).

8. The cell according to claim 1, wherein the mesenchymal system-related cell is a mesenchymal cell.

9. The cell according to claim 8, wherein the mesenchymal cell is a bone marrow stromal cell that can support the growth of hematopoietic system cells.

10. The cell according to claim 1, wherein the mesenchymal system-related cell is a cell derived from mesenchymal cells.

11. The cell according to claim 10, wherein the cell derived from mesenchymal cells is a cell of the cardiovascular system such as an endothelial cell or cardiac muscle cell, or a precursor cell of the cells of the cardiovascular system.

12. The cell according to claim 10, wherein the cell derived from mesenchymal cells is a cell or a precursor cell of any one of bone, cartilage, tendon, skeletal muscle and adipose tissue.

13. The cell according to claim 10, wherein the cell derived from mesenchymal cells is a nervous system cell or a precursor cell of a nervous system cell.

14. The cell according to claim 10, wherein the cell derived from mesenchymal cells is an endocrine cell or a precursor cell of an endocrine cell.

15. The cell according to claim 10, wherein the cell derived from mesenchymal cells is a hematopoietic cell or a precursor cell of a hematopoietic cell.

16. The cell according to claim 10, wherein the cell derived from mesenchymal cells is a hepatocyte or a precursor cell of a hepatocyte.

17. Artificial bone marrow having differentiation potency and expansion potency, which is obtained by culturing the cells of claim 9 with hematopoietic system stem cells.

18. The artificial bone marrow according to claim 17, which is obtained by further allowing the presence of immortalized stromal cells or a substance derived from the immortalized stromal cells.

19. The artificial bone marrow according to claim 18, wherein the substance derived from the immortalized stromal cells is a substance that is selected from soluble cytokines contained in a conditioned medium, adhesive molecules supplied by the contact with cells, and insoluble cytokine ligands.

20. The artificial bone marrow according to claim 17, which contains erythrocytes or precursor cells of erythrocytes.

21. The artificial bone marrow according to claim 17, which contains blood platelets or precursor cells of blood platelets such as megakaryocytes.

22. An artificial cell construct of the cardiovascular system, which can be obtained by culturing the precursor cells of cardiovascular cells together with the cell of claim 1, has expansion potency, and whose differentiation can be regulated.

23. Artificial bone, artificial cartilage, artificial tendon, artificial skeletal muscle or artificial adipose tissue, which can be obtained by culturing cells having properties of the precursor cells of the cells of any one of bone, cartilage, tendon, skeletal muscle and adipose tissue together with the cell of claim 1, has expansion potency, and whose differentiation can be regulated.

24. A cell group that is part of the nervous system or a cell group co-existing in the nervous system, which can be obtained by culturing the precursor cells of the nervous system together with the cell of claim 1, or substances derived from these cells, and whose expansion or differentiation can be regulated.

25. A cell group that is part of the endocrine tissue or a cell group coexisting in the endocrine tissue, which can be obtained by culturing the precursor cells of the endocrine system together with the cell of claim 1, or with substances derived from these cells, and whose expansion or differentiation can be regulated.

26. A nerve stem cell, which is induced from the cell of claim 8 by differentiation-inducing treatment.

27. An examination method, which is for evaluating the drug efficacy of a neuroactive agent using the cell of claim 8.

28. A bone marrow stromal cell, which is made to differentiate from the cell of claim 8 by differentiation-inducing treatment.

29. An examination method for evaluating the drug efficacy of an agent, which uses the cell of claim 28.

30. An erythrocyte, which is induced by differentiating the artificial bone marrow of claim 17.

31. A method for immortalizing and proliferating mesenchymal system-related cells, which comprises causing high expression of or activating an immortalizing gene in a mesenchymal system-related cell that is selected from mesenchymal precursor cells, mesenchymal stem cells, mesenchymal cells and cells derived from mesenchymal cells.

32. The method according to claim 31, wherein the immortalizing gene is a telomerase.

33. The method according to claim 31, wherein high expression is caused by introducing telomerase using a retrovirus vector into a cell derived from mesenchymal stem cells.

34. The method according to claim 33, wherein the retrovirus vector is pBabe.

35. The method according to claim 31, wherein an oncogene is not introduced into a cell derived from mesenchymal stem cells.

36. The method according to claim 31, wherein the mesenchymal system-related cell is a bone marrow stromal cell.

37. A method for producing artificial bone marrow, which comprises culturing immortalized and expanded bone marrow stromal cells together with hematopoietic stem cells.

38. A bone morrow stromal cell that is immortalized by high expression or activation of an immortalizing gene selected from any one of a telomerase gene, a gene derived from telomerase, and a gene regulating the expression or activity of telomerase.

39. The bone marrow stromal cell according to claim 38, wherein the immortalizing gene can be specifically deleted or is already deleted by gene deletion.

40. The bone marrow stromal cell according to claim 39, wherein the deletion of the immortalizing gene is performed by placing the immortalizing gene between IxoP-like sequences, and then treating the gene with a recombinase such a Cre recombinase.

41. The bone marrow stromal cell according to claim 38, wherein the bone marrow cell(s) is derived from either human ES cells (Fetal stem cells) or cells derived from ES cells (fetal stem cells).

42. The bone marrow stromal cell according to claim 38, wherein the bone marrow stromal cell can support the growth of hematopoietic system cells.

43. Artificial bone marrow having differentiation potency and expansion potency, which is obtained by culturing the bone marrow stromal cells of claim 42 with hematopoietic system stem cells.

44. The artificial bone marrow according to claim 43, which is obtained by further allowing the presence of immortalized bone marrow stromal cells or a substance derived from the immortalized stromal cells.

45. The artificial bone marrow according to claim 44, wherein the substance derived from the immortalized bone marrow stromal cells is a substance that is selected from soluble cytokines contained in a conditioned medium, adhesive molecules supplied by the contact with cells, and insoluble cytokine ligands.

46. The artificial bone marrow according to claim 43, which contains erythrocytes or precursor cells of erythrocytes.

47. The artificial bone marrow according to claim 43, which contains blood platelets or precursor cells of blood platelets such as megakaryocytes.

48. An erythrocyte, which is induced by differentiating the artificial bone marrow of claim 43.

55. The artificial bone marrow of claim 44, which contains blood platelets or precursor cells of blood platelets such as megakaryocytes.

56. The artificial bone marrow of claim 45, which contains blood platelets or precursor cells of blood platelets such as megakaryocytes.

57. The artificial bone marrow of claim 44, which contains erythrocytes or precursor cells of erythrocytes.

58. The artificial bone marrow of claim 45, which contains erythrocytes or precursor cells of erythrocytes.

59. The bone marrow stromal cell according to claim 39, wherein the bone marrow cell(s) is derived from either human ES cells (Fetal stem cells) or cells derived from ES cells (fetal stem cells).

60. The bone marrow stromal cell according to claim 40, wherein the bone marrow cell(s) is derived from either human ES cells (Fetal stem cells) or cells derived from ES cells (fetal stem cells).

49. A method for immortalizing and proliferating bone marrow stromal cell, which comprises causing high expression of or activating an immortalizing gene in a bone marrow stromal cell.

50. The method according to claim 49, wherein the immortalizing gene is a telomerase.

51. The method according to claim 49, wherein high expression is caused by introducing telomerase using a retrovirus vector into a cell derived from mesenchymal stem cells.

52. The method according to claim 51, wherein the retrovirus vector is pBabe.

53. The method according to claim 49, wherein an oncogene is not introduced into a bone marrow stromal cell.

54. A method for producing artificial bone marrow, which comprises culturing immortalized and expanded bone marrow stromal cells together with hematopoietic stem cells.