Novel methods for the in-vitro identification, isolation and differentiation of vasculogenic progenitor cells

• US 20030194802A1
• Filed: 08/05/2002
• Published: 10/16/2003
• Est. Priority Date: 04/16/2002
• Status: Active Grant

• Current Assignee: TECHNION RESEARCH AND DEVELOPMENT FOUNDATION, LTD. (Technion-Israel Institute Of Technology)
• Original Assignee: TECHNION RESEARCH AND DEVELOPMENT FOUNDATION, LTD. (Technion-Israel Institute Of Technology)
• Inventors: Gerecht-Nir, Sharon, Itskovitz-Eldor, Joseph
• Granted Patent: US 7,247,477 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: A61K 45/00 Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/02 Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/06 Animal cells or tissues; Human cells or tissues preservation of living cells or tissues A01N1/02
• CPC Class Codes: C12N 5/0692 Stem cells; Progenitor cells; Precursor cells
• CPC Class Codes: C12Q 1/00 Measuring or testing processes involving enzymes, nucleic acids or microorganisms measuring or testing apparatus with condition measuring or sensing means, e.g. colony counters, C12M1/34; Compositions therefor; Processes of preparing such compositions

Abstract

There is provided a simplified and inexpensive method for the in-vitro identification, isolation and culture of human vasculogenic progenitor cells. The method and the progenitor cells isolated thereby can be used for in-vitro vascular engineering, treatment of congenital and acquired vascular and hematological abnormalities, for evaluation and development of drugs affecting vasculo- and angiogenic processes, and for further investigation into tissue differentiation and development.

Claims

1. A method of preparing vasculogenic progenitor cells from undifferentiated ES cells, the method comprising:

(a) culturing individual undifferentiated ES cells in a manner suitable for inducing differentiation of the undifferentiated ES cells into vasculogenic progenitor cells, thereby obtaining a mixed population of cells; and

(b) isolating cells smaller than 50 μ

m from said mixed population of cells, said cells smaller than 50 μ

m being vasculogenic progenitor cells.

2. The method of claim 1, wherein step (a) is effected by subjecting the undifferentiated ES cells to at least one condition selected from a group consisting of avoiding aggregation of ES cells, growth on collagen, cell seeding concentration between 2×

10⁴ and 1×

10⁵ cells/cm² and presence of differentiation medium.

3. The method of claim 1, wherein said cells smaller than 50 μ

m are isolated via filtration, morphometry and/or densitometry.

4. The method of claim 3, wherein said filtration is effected via a filter having a pore size smaller than 50 μ

m.

5. The method of claim 1, wherein said undifferentiated ES cells are human ES cells.

6. A method of preparing epithelial progenitor cells from undifferentiated ES cells, the method comprising:

(a) culturing individual undifferentiated ES cells in a manner suitable for inducing differentiation of the undifferentiated ES cells into vasculogenic progenitor cells thereby obtaining a mixed population of cells; and

(b) isolating cells larger than 50 μ

m from said mixed population of cells, said cells larger than 50 μ

m being epithelial progenitor cells.

7. The method of claim 6, wherein step (a) is effected by subjecting the undifferentiated ES cells to at least one condition selected from a group consisting of avoiding aggregation of ES cells, growth on collagen, cell seeding concentration between 2×

10⁴ and 1×

10⁵ cells/cm² and presence of differentiation medium.

8. The method of claim 6, wherein said cells larger than 50 μ

m are isolated by filtration, morphometry and/or densitometry.

9. The method of claim 8, wherein said isolation by filtration is effected via a filter having a pore size smaller than 50 μ

m.

10. The method of claim 6, wherein said undifferentiated ES cells are human ES cells.

11. A method of preparing somatic cells from a population of vasculogenic progenitor cells, the method comprising:

(a) obtaining a population of vasculogenic progenitor cells; and

(b) culturing the population of vasculogenic progenitor cells in the presence of at least one growth factor suitable for inducing somatic cell differentiation, thereby preparing the somatic cells.

12. The method of claim 11, wherein step (a) is effected by:

(a) culturing individual undifferentiated ES cells in a manner suitable for inducing differentiation of the undifferentiated ES cells into vasculogenic progenitor cells thereby obtaining a mixed population of cells; and

(b) isolating cells smaller than 50 μ

m from said mixed population of cells, said cells smaller than 50 μ

m being vasculogenic progenitor cells.

13. The method of claim 12, wherein step (a) is effected by subjecting the undifferentiated ES cells to at least one condition being selected from a group consisting of avoiding aggregation of ES cells, growth on collagen, cell seeding concentration between 2×

10⁴ and 1×

10⁵ cells/cm² and presence of differentiation medium.

14. The method of claim 12, wherein said cells smaller than 50 μ

m are isolated by filtration, morphometry and/or densitometry.

15. The method of claim 14, wherein said isolation by filtration is effected via a filter having a pore size smaller than 50 μ

m.

16. The method of claim 11, wherein said undifferentiated ES cells are human ES cells.

17. The method of claim 11, wherein said growth factor is selected from the group consisting of vascular endothelial growth factor (VEGF), angiopoietin (Ang), platelet derived growth factor (PDGF), ephrin (Eph), fibroblast growth factor (FGF), tumor growth factor (TGF), placental growth factor (PlGF), tumor growth factor (TGF), cytokines, erythropoietin, thrombopoietin, transferrin, insulin, stem cell factor (SCF), Granulocyte colony-stimulating factor (G-CSF) and Granulocyte-macrophage colony stimulating factor (GM-CSF).

18. A method of preparing vascular tissue, the method comprising:

(a) obtaining a population of vasculogenic progenitor cells; and

(b) culturing said population of vasculogenic progenitor cells in the presence of at least one vasculogenic and/or angiogenic growth factor, under conditions suitable for inducing vascular tissue differentiation.

19. The method of claim 18, wherein step (a) is effected by:

(a) culturing individual undifferentiated ES cells in a manner suitable for inducing differentiation of the undifferentiated ES cells into vasculogenic progenitor cells thereby obtaining a mixed population of cells; and

(b) isolating cells smaller than 50 μ

m from said mixed population of cells, said cells smaller than 50 μ

m being vasculogenic progenitor cells.

20. The method of claim 19, wherein step (a) is effected by subjecting the undifferentiated ES cells to at least one condition being selected from a group consisting of avoiding aggregation of ES cells, growth on collagen, cell seeding concentration between 2×

10⁴ and 1×

10⁵ cells/cm² and presence of differentiation medium.

21. The method of claim 19, wherein said cells smaller than 50 μ

m are isolated by filtration, morphometry and/or densitometry.

22. The method of claim 21, wherein said isolation by filtration is effected via a filter having a pore size smaller than 50 μ

m.

23. The method of claim 19, wherein said undifferentiated ES cells are human ES cells.

24. The method of claim 18, wherein said population of vasculogenic progenitor cells is cultured in a semi-solid, vascularization-promoting medium.

25. The method of claim 18, wherein said population of vasculogenic progenitor is cultured on a 3-dimensional scaffold.

26. The method of claim 18, wherein said vasculogenic and/or angiogenic factor is selected from the group consisting of vascular endothelial growth factor (VEGF), angiopoietin (Ang), platelet derived growth factor (PDGF), ephrin (Eph), fibroblast growth factor (FGF), tumor growth factor (TGF) and placental growth factor (PlGF).

27. A method of determining an effect of a factor on vascular development, growth and/or modification, the method comprising:

(a) obtaining a population of vasculogenic progenitor cells;

(b) exposing said population of vasculogenic progenitor cells to the factor; and

(c) determining an effect of the factor on said population of vasculogenic progenitor cells to thereby determine the effect thereof on vascular development.

28. The method of claim 27, wherein step (a) is effected by:

(a) culturing individual undifferentiated ES cells in a manner suitable for inducing differentiation of the undifferentiated ES cells into vasculogenic progenitor cells thereby obtaining a mixed population of cells; and

(b) isolating cells smaller than 50 μ

m from said mixed population of cells, said cells smaller than 50 μ

m being vasculogenic progenitor cells.

29. The method of claim 28, wherein step (a) is effected by subjecting the undifferentiated ES cells to at least one condition being selected from a group consisting of avoiding aggregation of ES cells, growth on collagen, cell seeding concentration between 2×

10⁴ and 1'"'"'10⁵ cells/cm² and presence of differentiation medium.

30. The method of claim 28, wherein said cells smaller than 50 μ

m are isolated by filtration, morphometry and/or densitometry.

31. The method of claim 30, wherein said isolation by filtration is effected via a filter having a pore size smaller than 50 μ

m.

32. The method of claim 28, wherein said undifferentiated ES cells are human ES cells.

33. The method of claim 27, wherein the factor is a substance and/or an environmental factor.

34. The method of claim 27, wherein the factor is a putative angiogenesis and/or vasculogenesis downregulator, whereas the method further comprising culturing said population of vasculogenic progenitor cells under conditions suitable for promoting angiogenesis and/or vasculogenesis prior to step (b).

35. The method of claim 27, wherein the factor is a putative angiogenesis and/or vasculogenesis upregulator, whereas the method further comprising culturing said population of vasculogenic progenitor cells under conditions limiting angiogenesis and/or vasculogenesis prior to step (b).

36. A cell culture comprising a population of vasculogenic progenitor cells being sustainable in a proliferative state for at least 14 days and being capable of differentiation into smooth muscle, endothelial and/or hematopoietic cells upon exposure to at least one growth factor selected from the group consisting of vascular endothelial growth factor (VEGF), angiopoietin (Ang), platelet derived growth factor (PDGF), ephrin (Eph), fibroblast growth factor (FGF), tumor growth factor (TGF), placental growth factor (PIGF), cytokines, erythropoietin, thrombopoietin, transferrin, insulin, stem cell factor (SCF), Granulocyte colony-stimulating factor (G-CSF) and Granulocyte-macrophage colony stimulating factor (GM-CSF).

37. The cell culture of claim 36, wherein said population of vasculogenic progenitor cells is capable of expressing at least one exogenous polypeptide.

38. The cell culture of claim 37, wherein said exogenous polypeptide is selected from the group consisting of cell-surface markers, cell-surface antigens, angiogenic factors, vasculogenic factors and hematopoietic factors.

39. The cell culture of claim 37, wherein said exogenous polypeptide is expressed in an inducible manner.

40. A composition of matter comprising a substrate and a population of vasculogenic progenitor cells, wherein said vasculogenic progenitor cells are prepared from undifferentiated ES cells by a method comprising the steps:

(a) culturing individual undifferentiated ES cells in a manner suitable for inducing differentiation of the undifferentiated ES cells into vasculogenic progenitor cells thereby obtaining a mixed population of cells; and

(b) isolating cells smaller than 50 μ

m from said mixed population of cells, said cells smaller than 50 μ

m being vasculogenic progenitor cells.

41. The composition of claim 40, wherein step (a) is effected by subjecting the undifferentiated ES cells to at least one condition being selected from a group consisting of avoiding aggregation of ES cells, growth on collagen, cell seeding concentration between 2×

10⁴ and 1×

10⁵ cells/cm² and presence of differentiation medium.

42. The composition of claim 41, wherein said cells smaller than 50 μ

m are isolated by filtration, morphometry and/or densitometry.

43. The composition of claim 42, wherein said isolation by filtration is effected via a filter having a pore size smaller than 50 μ

m.

44. The composition of claim 40, wherein said undifferentiated ES cells are human ES cells.

45. The composition of claim 40, wherein said substrate is selected from the group consisting of matrigel, collagen gel, and polymeric scaffold.

46. The composition of claim 40, wherein said vasculogenic progenitor cells being contacted with said substrate in a manner so as to induce vascular development within said substrate.

47. A method of relieving or preventing a vascular disease or condition in a mammalian subject, the method comprising:

(a) obtaining a population of vasculogenic progenitor cells; and

(b) administering said vasculogenic progenitor cells into the subject under conditions suitable for stimulating differentiation of said vasculogenic progenitor cells into endothelial and smooth muscle cells, thereby alleviating said vascular disease or condition.

48. The method of claim 47, wherein obtaining said population of vasculogenic progenitor cells is effected by:

(a) culturing individual undifferentiated ES cells in a manner suitable for inducing differentiation of the undifferentiated ES cells into vasculogenic progenitor cells;

thereby obtaining a mixed population of cells; and

(b) isolating cells smaller than 50 μ

m from said mixed population of cells, said cells smaller than 50 μ

m being vasculogenic progenitor cells.

49. The method of claim 48, wherein step (a) is effected by subjecting the undifferentiated ES cells to at least one condition being selected from a group consisting of avoiding aggregation of ES cells, growth on collagen, cell seeding concentration between 2×

10⁴ and 1×

10⁵ cells/cm² and presence of differentiation medium.

50. The method of claim 49, wherein said cells smaller than 50 μ

m are isolated by filtration, morphometry and/or densitometry.

51. The method of claim 50, wherein said isolation by filtration is effected via a filter having a pore size smaller than 50 μ

m.

52. The method of claim 47, wherein said undifferentiated ES cells are human ES cells.

53. The method of claim 47, wherein said vascular disease or condition is selected from a group consisting of congenital vascular disorders, acquired vascular disorders and ischemia/reperfusion injury.

54. A method of vascularizing a mammalian tissue, the method comprising:

(a) obtaining a population of vasculogenic progenitor cells;

(b) contacting said vasculogenic progenitor cells with said mammalian tissue under conditions suitable for stimulating differentiation of said vasculogenic progenitor cells into endothelial and smooth muscle cells, thereby enriching the vascularity of the tissue.

55. The method of claim 54, wherein obtaining said population of vasculogenic progenitor cells is effected by:

(a) culturing individual undifferentiated ES cells in a manner suitable for inducing differentiation of the undifferentiated ES cells into vasculogenic progenitor cells;

thereby obtaining a mixed population of cells; and

(b) isolating cells smaller than 50 μ

m from said mixed population of cells, said cells smaller than 50 μ

m being vasculogenic progenitor cells.

56. The method of claim 55, wherein step (a) is effected by subjecting the undifferentiated ES cells to at least one condition being selected from a group consisting of avoiding aggregation of ES cells, growth on collagen, cell seeding concentration between 2×

10⁴ and 1×

10⁵ cells/Cm² and presence of differentiation medium.

57. The method of claim 56, wherein said cells smaller than 50 μ

m are isolated by filtration, morphometry and/or densitometry.

58. The method of claim 57, wherein said isolation by filtration is effected via a filter having a pore size smaller than 50 μ

m.

59. The method of claim 54, wherein said undifferentiated ES cells are human ES cells.

60. The method of claim 54, wherein said mammalian tissue is an engineered, non-vascular tissue in need of vascularization.

61. The method of claim 54, wherein said mammalian tissue is an embryonic tissue.

62. The method of claim 54, wherein step (b) is performed in vitro.

63. The method of claim 54, wherein step (b) is performed in vivo.

64. A method of relieving or preventing a hematological disease or condition in a mammalian subject, the method comprising:

(a) obtaining a population of vasculogenic progenitor cells; and

(b) administering said vasculogenic progenitor cells into the subject under conditions suitable for stimulating differentiation of said vasculogenic progenitor cells into endothelial and blood cells, thereby alleviating said hematopoietic disease or condition.

65. The method of claim 64, wherein step (a) is effected by:

(a) culturing individual undifferentiated ES cells in a manner suitable for inducing differentiation of the undifferentiated ES cells into vasculogenic progenitor cells, thereby obtaining a mixed population of cells; and

(b) isolating cells smaller than 50 μ

m from said mixed population of cells, said cells smaller than 50 μ

m being vasculogenic progenitor cells.

66. The method of claim 65, wherein step (a) is effected by subjecting the undifferentiated ES cells to at least one condition being selected from a group consisting of avoiding aggregation of ES cells, growth on collagen, cell seeding concentration between 2×

10⁴ and 1×

10⁵ cells/cm² and presence of differentiation medium.

67. The method of claim 66, wherein said cells smaller than 50 μ

m are isolated by filtration, morphometry and/or densitometry.

68. The method of claim 67, wherein said isolation by filtration is effected via a filter having a pore size smaller than 50 μ

m.

69. The method of claim 64, wherein said undifferentiated ES cells are human ES cells.

70. The method of claim 64, wherein said hematological disease or condition is selected from a group consisting of congenital blood disorders, acquired blood disorders, clotting disorders and neoplastic disease.

71. A method of preparing endothelial cells from vascular tissue, the method comprising:

(a) subjecting the vascular tissue to conditions designed for dissociating cells from the vascular tissue, thereby obtaining a mixed population of dissociated cells; and

(b) isolating cells smaller than 50 μ

m from said mixed population of cells, said cells smaller than 50 μ

m being endothelial cells.

72. The method of claim 71, wherein said cells smaller than 50 μ

m are isolated by filtration, morphometry and/or densitometry.

73. The method of claim 72, wherein said isolation by filtration is effected via a filter having a pore size smaller than 50 μ

m.

74. The method of claim 71, wherein said vascular tissue is human vascular tissue.

75. A method of preparing epithelial cells from vascular tissue, the method comprising:

(a) subjecting the vascular tissue to conditions designed for dissociating cells from the vascular tissue, thereby obtaining a mixed population of dissociated cells, thereby obtaining a mixed population of individual cells; and

(b) isolating cells larger than 50 μ

m from said mixed population of cells, said cells larger than 50 μ

m being epithelial cells.

76. The method of claim 75, wherein said cells larger than 50 μ

m are isolated by filtration, morphometry and/or densitometry.

77. The method of claim 76, wherein said isolation by filtration is effected via a filter having a pore size smaller than 50 μ

m.

78. The method of claim 75, wherein said vascular tissue is human vascular tissue.