Multi-lineage progenitor cells

• US 20050255592A1
• Filed: 04/20/2005
• Published: 11/17/2005
• Est. Priority Date: 04/23/2004
• Status: Active Grant

• Current Assignee: BioE LLC
• Original Assignee: BioE, Inc.
• Inventors: Collins, Daniel P., Tigges, Barbara M., Sprague, Stacey L.
• Granted Patent: US 7,670,596 B2
• Time in Patent Office: Days
• US Class Current: 435/372
• CPC Class Codes: C12N 5/0607 Non-embryonic pluripotent stem cells, e.g. MASC induced pluripotent stem cells C12N5/0696

Abstract

Fetal blood multi-lineage progenitor cells that are capable of a wide spectrum of transdifferentiation are described.

Claims

1. A purified population of human fetal blood multi-lineage progenitor cells (MLPC), wherein said MLPC are positive for CD9 and CD45.

2. The purified population of MLPC of claim 1, wherein said MLPC display a leukocyte morphology.

3. The purified population of MLPC of claim 1, wherein said MLPC are obtained from cord blood.

4. The purified population of MLPC of claim 1, wherein said MLPC are further positive for SSEA-4.

5. The purified population of MLPC of claim 1, wherein said MLPC are further positive for CD34.

6. The purified population of MLPC of claim 1, wherein said MLPC are further positive for CD133, CD41, CD44, CD105, CD29, CD73, CD90, stem cell factor, SSEA-3, and CD13.

7. The purified population of MLPC of claim 1, wherein said MLPC are negative for CD15, CD38, glycophorin-A, CD2, CD3, CD8, CD19, CD20, CD22, CD5, CD7, CD10, CD14, CD4, HLA-DR, CD16, CD33, and CD61.

8. The purified population of MLPC of claim 1, wherein said MLPC attain a fibroblast-like morphology over time in culture.

9. The purified population of MLPC of claim 1, wherein said MLPC adhere to a plastic surface when cultured.

10. The purified population of MLPC of claim 1, wherein said MLPC are capable of differentiating into cells from all three embryonic germ layers.

11. The purified population of MLPC of claim 1, wherein said MLPC are capable of differentiating into cells having an osteocytic phenotype, cells having an adipocytic phenotype, cells having a neural stem cell phenotype, cells having a myocytic phenotype, cells having an endothelial phenotype, cells having a hepatocytic phenotype, and cells having a pancreatic phenotype.

12. The purified population of MLPC of claim 1, wherein said MLPC comprise an exogenous nucleic acid.

13. The purified population of MLPC of claim 12, wherein said exogenous nucleic acid encodes a polypeptide.

38. A composition comprising the purified population of MLPC of claim 1, the purified population of MLPC of claim 14, or the clonal line of claim 24 and a culture medium.

39. The composition of claim 38, wherein said composition further comprises a cryopreservative.

40. The composition of claim 39, wherein said cryopreservative is dimethylsulfoxide (DMSO).

41. The composition of claim 40, wherein said cryopreservative is 1 to 10% DMSO.

42. The composition of claim 39, wherein said cryopreservative is fetal bovine serum, human serum, or human serum albumin in combination with one or more of the following:

DMSO, trehalose, and dextran.

43. The composition of claim 42, wherein said cryopreservative is human serum, DMSO, and trehalose.

44. The composition of claim 42, wherein said cryopreservative is fetal bovine serum and DMSO.

45. An article of manufacture comprising the purified population of MLPC of claim 1, the purified population of MLPC of claim 14, or the clonal line of claim 24.

46. The article of manufacture of claim 45, wherein said purified population of MLPC or said clonal line is housed within a container.

47. The article of manufacture of claim 46, wherein said container is a vial.

48. The article of manufacture of claim 46, wherein said container is a bag.

49. The article of manufacture of claim 46, wherein said container further comprises a cryopreservative.

60. A method for cryopreserving MLPC, said method comprising:

a) contacting said purified population of MLPC of claim 1, said purified population of MLPC of claim 14, or said clonal line of claim 24 with a cryopreservative; and

b) freezing said purified population of MLPC or said clonal line.

61. The method of claim 60, wherein said cryopreservative is DMSO.

62. The method of claim 60, wherein said cryopreservative is fetal bovine serum, human serum, or human serum albumin in combination with one or more of the following:

DMSO, trehalose, and dextran.

63. The method of claim 62, wherein said cryopreservative is human serum, DMSO, and trehalose.

64. The method of claim 62, wherein said cryopreservative is fetal bovine serum and DMSO.

65. The method of claim 60, wherein said purified population of MLPC or said clonal line is suspended in said cryopreservative at a concentration between 1×

10⁵ and 5×

10⁷ cells/mL.

66. The method of claim 60, wherein said purified population of MLPC or said clonal line is frozen at a controlled rate.

67. The method of claim 66, wherein the freezing rate is controlled electronically.

68. The method of claim 60, wherein said purified population of MLPC or said clonal line is frozen by placement in an ethanol bath in the vapor phase of a liquid nitrogen cryogenic storage tank.

69. A method of producing a population of differentiated cells, said method comprising culturing the purified population of MLPC of claim 1, the purified population of MLPC of claim 14, or the clonal line of claim 24 with an agent effective to induce differentiation of said MLPC.

70. The method of claim 69, wherein said agent comprises insulin, glutamine, dexamethasone, indomethacin, and 3-isobutyl-1-methyl-xanthine.

71. The method of claim 69, wherein said agent comprises dexamethasone, glutamine, ascorbate, and β

-glycerophosphate.

72. The method of claim 69, wherein said agent comprises epithelial growth factor, insulin, fetuin, dexamethasone, and fibroblast growth factor-basic.

73. The method of claim 69, wherein said agent comprises fibroblast growth factor-basic, epidermal growth factor, NSF-1, and retinoic acid.

74. The method of claim 69, wherein said agent comprises heparin, bovine brain extract, epithelial growth factor, and hydrocortisone.

75. The method of claim 69, wherein said agent comprises ascorbic acid, hydrocortisone, transferrin, insulin, epidermal growth factor, hepatocyte growth factor, fibroblast growth factor-basic, fibroblast growth factor-4, and stem cell factor.

14. A purified population of human fetal blood MLPC, wherein said MLPC are positive for CD9, negative for CD45, negative for CD34, and negative for SSEA-4.

15. The purified population of MLPC of claim 14, wherein said MLPC display a fibroblast morphology.

16. The purified population of MLPC of claim 14, wherein said MLPC are obtained from cord blood.

17. The purified population of MLPC of claim 14, wherein said MLPC are further positive for CD13, CD29, CD44, CD73, CD90, and CD105.

18. The purified population of MLPC of claim 14, wherein said MLPC are further negative for CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD14, CD15, CD16, CD19, CD20, CD22, CD33, CD36, CD38, CD41, CD61, CD62E, CD133, glycophorin-A, stem cell factor, SSEA-3, and HLA-DR.

19. The purified population of MLPC of claim 14, wherein said MLPC adhere to a plastic surface when cultured.

20. The purified population of MLPC of claim 14, wherein said MLPC are capable of differentiating into cells from all three embryonic germ layers.

21. The purified population of MLPC of claim 14, wherein said MLPC are capable of differentiating into cells having an osteocytic phenotype, cells having an adipocytic phenotype, cells having a neural stem cell phenotype, cells having a myocytic phenotype, cells having an endothelial phenotype, cells having a hepatocytic phenotype, and cells having a pancreatic phenotype.

22. The purified population of MLPC of claim 14, wherein said MLPC comprise an exogenous nucleic acid.

23. The purified population of MLPC of claim 22, wherein said exogenous nucleic acid encodes a polypeptide.

24. A clonal line of human fetal blood MLPC, wherein said MLPC are positive for CD9, negative for CD45, negative for CD34, and negative for SSEA-4.

25. The clonal line of claim 24, wherein said MLPC display a fibroblast morphology.

26. The clonal line of claim 24, wherein said MLPC are obtained from cord blood.

27. The clonal line of claim 24, wherein said MLPC are further positive for CD13, CD29, CD44, CD73, CD90, and CD105.

28. The clonal line of claim 24, wherein said MLPC are further negative for CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD14, CD15, CD16, CD19, CD20, CD22, CD33, CD36, CD38, CD41, CD61, CD62E, CD133, glycophorin-A, stem cell factor, SSEA-3, and HLA-DR.

29. The clonal line of claim 24, wherein said MLPC adhere to a plastic surface when cultured.

30. The clonal line of claim 24, wherein said MLPC are capable of differentiating into cells from all three embryonic germ layers.

31. The clonal line of claim 24, wherein said MLPC are capable of differentiating into cells having an osteocytic phenotype, cells having an adipocytic phenotype, cells having a neural stem cell phenotype, cells having a myocytic phenotype, cells having an endothelial phenotype, cells having a hepatocytic phenotype, and cells having a pancreatic phenotype.

32. The clonal line of claim 24, wherein said MLPC comprise an exogenous nucleic acid.

33. The clonal line of claim 32, wherein said exogenous nucleic acid encodes a polypeptide.

34. The clonal line of claim 24, wherein said MLPC have undergone at least 5 doublings in culture.

35. The clonal line of claim 24, wherein said MLPC have undergone at least 8 doublings in culture.

36. The clonal line of claim 24, wherein said MLPC have undergone at least 10 doublings in culture.

37. The clonal line of claim 24, wherein said MLPC have undergone at least 25 doublings in culture.

50. A method for purifying a population of MLPC from human fetal blood, said method comprising:

a) contacting a human fetal blood sample with a composition, said composition comprising;

i) dextran;

ii) anti-glycophorin A antibody;

iii) anti-CD15 antibody; and

iv) anti-CD9 antibody;

b) allowing said sample to partition into an agglutinate and a supernatant phase;

c) recovering cells from said supernatant phase; and

d) purifying MLPC from the recovered cells by adherence to a solid substrate, wherein said MLPC are positive for CD9 and positive for CD45.

51. The method of claim 50, wherein said MLPC are further positive for CD34, CD133, CD41, CD44, CD105, CD29, CD73, CD90, stem cell factor, SSEA-3, SSEA-4, and CD13.

52. The method of claim 50, wherein said MLPC are negative for CD15, CD38, glycophorin-A, CD2, CD3, CD8, CD19, CD20, CD22, CD5, CD7, CD10, CD14, CD4, HLA-DR, CD16, CD33, and CD61.

53. The method of claim 50, wherein said fetal blood sample is cord blood.

54. The method of claim 50, wherein said solid substrate is a plastic substrate.

55. The method of claim 50, said method further comprising culturing said MLPC such that said MLPC obtain a fibroblast morphology, wherein said MLPC, after obtaining said fibroblast morphology, are positive for CD9, negative for CD45, negative for CD34, and negative for SSEA-4.

56. The method of claim 55, wherein said MLPC, after obtaining said fibroblast morphology, are further positive for CD13, CD29, CD44, CD73, CD90, and CD105.

57. The method of claim 55, wherein said MLPC, after obtaining said fibroblast morphology, are further negative for CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD14, CD15, CD16, CD19, CD20, CD22, CD33, CD36, CD38, CD41, CD61, CD62E, CD133, glycophorin-A, stem cell factor, SSEA-3, and HLA-DR.

58. The method of claim 50 or claim 55, further comprising testing said MLPC for CD9.

59. The method of claim 58, further comprising testing said MLPC for CD29, CD45, CD73, and CD90.