Multi-lineage progenitor cells

• US 20060040392A1
• Filed: 08/22/2005
• Published: 02/23/2006
• Est. Priority Date: 04/23/2004
• Status: Active Grant

• Current Assignee: BioE LLC
• Original Assignee: BioE LLC
• Inventors: Collins, Daniel P., Tigges, Barbara M., Sprague, Stacey L.
• Granted Patent: US 7,622,108 B2
• Time in Patent Office: Days
• US Class Current: 435/455
• CPC Class Codes: C12N 2506/03 from non-embryonic pluripotent stem cells
• 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) or undifferentiated progeny thereof, wherein said MLPC have enhanced expression of mRNA for CXCR4, FLT3, and CD133 relative to that of a population of mesenchymal stem cells (MSC).

2. The purified population of MLPC of claim 1, wherein said MLPC further have an enhanced expression of mRNA for TERT, KIT, and POU5F relative to said population of MSC.

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 further have an enhanced expression of mRNA for CD34 relative to said population of MSC.

5. The purified population of MLPC of claim 1, wherein said MLPC are positive for CD9, negative for CD45, negative for CD34, and negative for SSEA-4.

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

7. 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.

8. The purified population of MLPC of claim 1, wherein said MLPC further have an enhanced expression of mRNA for CD24, CD44, CD45, CD58, CD68, CD33, CD37, and CD38 relative to said population of MSC.

9. The purified population of MLPC of claim 1, wherein said MLPC further have an enhanced expression of mRNA for ICAM2, ITGAX, TFRC, KIT, IL6R, IL7R, ITGAM, FLT3, PDGFRB, SELE, SELL, TFRC, ITGAL, ITGB2, PECAM1, ITGA2B, ITGA3, ITGA4, ITGA6, and ICAM1 relative to said population of MSC.

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

11. The purified population of MLPC of claim 10, wherein said exogenous nucleic acid encodes a polypeptide.

12. Differentiated progeny of said purified population of MLPC of claim 1.

13. The differentiated progeny of claim 12, wherein said progeny have an osteocytic phenotype, an adipocytic phenotype, a neural stem cell phenotype, a myocytic phenotype, an endothelial phenotype, a hepatocytic phenotype, or a pancreatic phenotype.

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

28. The composition of claim 27, wherein said composition further comprises a cryopreservative.

29. The composition of claim 28, wherein said cryopreservative is dimethylsulfoxide (DMSO).

30. The composition of claim 29, wherein said cryopreservative is 1 to 10% DMSO.

31. The composition of claim 28, 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.

32. The composition of claim 31, wherein said cryopreservative is human serum, DMSO, and trehalose.

33. The composition of claim 31, wherein said cryopreservative is fetal bovine serum and DMSO.

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

35. The article of manufacture of claim 34, said article of manufacture further comprising a label indicating that said MLPC have enhanced expression of mRNA for CXCR4, FLT3, and CD133 relative to that of a population of MSC.

36. The article of manufacture of claim 34, wherein said purified population of MLPC or said clonal line is housed within a container.

37. The article of manufacture of claim 36, wherein said container is a vial.

38. The article of manufacture of claim 36, wherein said container is a bag.

39. The article of manufacture of claim 36, wherein said container further comprises a cryopreservative.

40. The article of manufacture of claim 34, said article of manufacture further comprising a reagent for characterizing said population or clonal line of MLPC, wherein said reagent is selected from the group consisting of a nucleic acid probe for detecting expression of CXCR4, a nucleic acid probe for detecting expression of FLT3, a nucleic acid probe for detecting expression of CD133, a nucleic acid probe for detecting expression of CD34, a nucleic acid probe for detecting expression of TERT, a nucleic acid probe for detecting expression of KIT, a nucleic acid probe for detecting expression of POU5F, an antibody having specific binding affinity for CD9, an antibody having specific binding affinity for CD34, an antibody having specific binding affinity for CD45, and an antibody having specific binding affinity for SSEA-4.

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

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

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

49. The method of claim 48, wherein said cryopreservative is DMSO.

50. The method of claim 49, 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.

51. The method of claim 50, wherein said cryopreservative is human serum, DMSO, and trehalose.

52. The method of claim 50, wherein said cryopreservative is fetal bovine serum and DMSO.

53. The method of claim 48, 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.

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

55. The method of claim 54, wherein said agent comprises insulin, glutamine, dexamethasone, indomethacin, and 3-isobutyl-1-methyl-xanthine.

56. The method of claim 54, wherein said agent comprises dexamethasone, glutamine, ascorbate, and β

-glycerophosphate.

57. The method of claim 54, wherein said agent comprises epithelial growth factor, insulin, fetuin, dexamethasone, and fibroblast growth factor-basic.

58. The method of claim 54, wherein said agent comprises fibroblast growth factor-basic, epidermal growth factor, NSF-1, and retinoic acid.

59. The method of claim 54, wherein said agent comprises heparin, bovine brain extract, epithelial growth factor, and hydrocortisone.

60. The method of claim 54, 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.

62. The method of claim 54, wherein expression of mRNA for CXCR4, FLT3, and CD133 is assessed.

63. The method of claim 54, wherein expression of mRNA for TERT, KIT, and POU5F is assessed.

64. The method of claim 54, wherein expression of mRNA for CD34 is assessed.

14. A clonal line of human fetal blood MLPC and undifferentiated progeny thereof, wherein said MLPC have enhanced expression of mRNA for CXCR4, FLT3, and CD 133 relative to that of a population of MSC.

15. The clonal line of claim 14, wherein said MLPC are positive for CD9, negative for CD45, negative for CD34, and negative for SSEA-4.

16. The clonal line of claim 14, wherein said MLPC are obtained from cord blood.

17. The clonal line of claim 14, wherein said MLPC further have an enhanced expression of mRNA for TERT, KIT, and POU5F relative to said population of MSC.

18. The clonal line of claim 14, wherein said MLPC further have an enhanced expression of mRNA for CD34 relative to said population of MSC.

19. The clonal line of claim 14, wherein said MLPC comprise an exogenous nucleic acid.

20. The clonal line of claim 19, wherein said exogenous nucleic acid encodes a polypeptide.

21. The clonal line of claim 14, wherein said clonal line has undergone at least 5 doublings in culture.

22. The clonal line of claim 14, wherein said clonal line has undergone at least 8 doublings in culture.

23. The clonal line of claim 14, wherein said clonal line has undergone at least 10 doublings in culture.

24. The clonal line of claim 14, wherein said clonal line has undergone at least 25 doublings in culture.

25. Differentiated progeny of said clonal line of MLPC of claim 14.

26. The differentiated progeny of claim 25, wherein said progeny have an osteocytic phenotype, an adipocytic phenotype, a neural stem cell phenotype, a myocytic phenotype, an endothelial phenotype, a hepatocytic phenotype, or a pancreatic phenotype.

41. 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-CD 15 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;

d) purifying MLPC from the recovered cells by adherence to a solid substrate; and

e) culturing said MLPC such that said MLPC obtain a fibroblast morphology, wherein said MLPC, after obtaining said fibroblast morphology, have enhanced expression of mRNA for CXCR4, FLT3, and CD133 relative to that of a population of MSC.

42. The method of claim 41, wherein said fetal blood sample is cord blood.

43. The method of claim 41, wherein said solid substrate is a plastic substrate.

44. The method of claim 41, wherein said MLPC further have an enhanced expression of TERT, KIT, and POU5F relative to said population of MSC.

45. The method of claim 41, further comprising test said MLPC for enhanced expression of mRNA for CXCR4, FLT3, and CD133 relative to said population of MSC.

46. The method of claim 41, further comprising testing said MLPC for CD9.

47. The method of claim 46, further comprising testing said MLPC for CD29, CD45, CD73, and CD90.

61. A method of characterizing a population of MLPC, said method comprising a) providing a purified population of MLPC;

and b) assessing expression in said population of MLPC of one or more mRNAs selected from the group consisting of CXCR4, FLT3, CD133, ICAM2, ITGAX, TFRC, KIT, IL6R, IL7R, ITGAM, FLT3, PDGFRB, SELE, SELL, TFRC, ITGAL, ITGB2, PECAM1, ITGA2B, ITGA3, ITGA4, ITGA6, ICAM1, CD24, CD34, CD44, CD45, CD58, CD68, CD33, CD37, CD38, TERT, KIT, and POUF5.

65. A method for characterizing the immaturity of a population of MLPC, said method comprising a) providing a purified population of MLPC;

and b) assessing expression of mRNA for CXCR4, FLT3, and CD133 in said population of MLPC, wherein enhanced expression of CXCR4, FLT3, and CD133 relative to that of a population of MSC is indicative of an immature phenotype.

66. A method of making a clonal line of MLPC, said method comprising

a) providing a population of MLPC,

b) culturing a single MLPC in a culture vessel lacking other cells until a plurality of progeny are produced;

and

c) culturing said progeny to obtain said clonal line.