Isolation and Cultivation of Stem/Progenitor Cells From the Amniotic Membrane of Umbilical Cord and Uses of Cells Differentiated Therefrom

• US 20080248005A1
• Filed: 10/11/2006
• Published: 10/09/2008
• Est. Priority Date: 10/21/2005
• Status: Active Grant

• Current Assignee: CellResearch Corporation Pte Ltd (CellResearch Group of Cos.)
• Original Assignee: CellResearch Corporation Pte Ltd (CellResearch Group of Cos.)
• Inventors: Phan, Toan-Thang
• Granted Patent: US 8,287,854 B2
• Time in Patent Office: Days
• US Class Current: 424/93.7
• CPC Class Codes: A61K 35/32 Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
• CPC Class Codes: A61L 27/18 obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
• CPC Class Codes: A61L 27/3633 Extracellular matrix [ECM]
• CPC Class Codes: A61L 27/3804 characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
• CPC Class Codes: A61L 27/3839 characterised by the site of application in the body materials for artificial blood vessels A61L27/507; materials for use in artificial skin A61L27/60
• CPC Class Codes: A61L 27/56 Porous materials, e.g. foams or sponges
• CPC Class Codes: A61L 27/60 Materials for use in artificial skin
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 19/08 for bone diseases, e.g. rachitism, Paget's disease
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C08L 69/00 Compositions of polycarbonates; Compositions of derivatives of polycarbonates
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2502/094 keratinocytes
• CPC Class Codes: C12N 2502/1323 Adult fibroblasts
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2506/1369 from blood-borne mesenchymal stem cells, e.g. MSC from umbilical blood
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• CPC Class Codes: C12N 5/0605 : Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
• CPC Class Codes: C12N 5/0629 : Keratinocytes; Whole skin
• CPC Class Codes: C12N 5/0655 : Chondrocytes; Cartilage
• CPC Class Codes: C12N 5/0668 : Mesenchymal stem cells from other natural sources
• CPC Class Codes: C12N 5/0676 : Pancreatic cells
• CPC Class Codes: C12N 5/0698 : Skin equivalents

Abstract

The present invention relates to a skin equivalent and a method for producing the same, wherein the skin equivalent comprises a scaffold and stem/progenitor cells isolated from the amniotic membrane of umbilical cord. These stem/progenitor cells may be mesenchymal (UCMC) and/or epithelial (UCEC) stem cells, which may then be further differentiated to fibroblast and keratinocytes. Further described is a method for isolating stem/progenitor cells from the amniotic membrane of umbilical cord, wherein the method comprises separating the amniotic membrane from the other components of the umbilical cord in vitro, culturing the amniotic membrane tissue under conditions allowing cell proliferation, and isolating the stem/progenitor cells from the tissue cultures. The invention also refers to therapeutic uses of these skin equivalents. Another aspect of the invention relates to the generation of a mucin-producing cell using stem/progenitor cells obtained from the amniotic membrane of umbilical cord and therapeutic uses of such mucin-producing cells. In yet another aspect, the invention relates to a method for generating an insulin-producing cell using stem/progenitor cells isolated from the amniotic membrane of umbilical cord and therapeutic uses thereof. The invention further refers to a method of treating a bone or cartilage disorder using UCMC. Furthermore, the invention refers to a method of generating a dopamin and tyrosin hydroxylase as well as a HLA-G and hepatocytes using UCMC and/or UCEC. The present invention also refers to a method of inducing proliferation of aged keratinocytes using UCMC.

Claims

1. A skin equivalent comprising:

a scaffold comprising cells derived from stem/progenitor cells isolated from the amniotic membrane of umbilical cord.

2. A skin equivalent according to claim 1, wherein said cells derived from stem/progenitor cells are mesenchymal stem cells (UCMC) or epithelial stem cells (UCEC).

3. A skin equivalent according to claim 1, wherein said cells are selected from the group consisting of autologous cells, xenogeneic cells and allogeneic cells.

6. A skin equivalent according to claim 1, wherein the origin of said cells is selected from the group consisting of mammalian cells and human cells.

8. A skin equivalent according to claim 1, wherein said scaffold further comprises at least one cell line, which is able to differentiate into blood vessels or glands.

9. A skin equivalent according to claim 8, wherein said scaffold comprises vessel endothelial cells or dermal microvascular endothelial cells (DMEC).

10. A skin equivalent according to claim 9, wherein said vessel endothelial cells are derived from the umbilical cord.

11. A skin equivalent according to claim 10, wherein said umbilical vessel endothelial cells are of human origin.

12. A skin equivalent according to claim 1, wherein said scaffold further comprises cells of aged keratinocytes.

13. A skin equivalent according to claim 1, wherein the scaffold comprises a biodegradable material.

14. A skin equivalent according to claim 1, wherein the scaffold comprises a material selected from the group consisting of agarose, polycaprolactone, niobium coated carbon, chitosan, collagen, hyaluronic acid, calcium phosphate, starch, hydroxyapatite, fibrin, alginate, poly-glycolic acid, carbon nano fibres, porous polycarbonate, polytetrafluoroethylene, polylactide and mixtures thereof.

15. A skin equivalent according to claim 14, wherein the scaffold material is porous polycarbonate.

16. A skin equivalent according to claim 1, wherein said scaffold comprises at least one extracellular matrix component.

17. A skin equivalent according to claim 16, wherein said extracellular matrix component is selected from the group consisting of collagen, elastin, intercellular adhesion molecules, laminin, heparin, fibronectin, proteoglycans, tenascin, fibrillin and mixtures thereof.

18. A skin equivalent according to claim 17, wherein said extracellular matrix component is collagen type I.

19. A skin equivalent according to claim 1, wherein said cells are able to proliferate and further differentiate into fibroblasts and keratinocytes.

57. A cell bank comprising a skin equivalent selected from the group consisting of skin equivalents of claim 1, 19, 20 and 54.

4. (canceled)

5. (canceled)

7. (canceled)

20. A method of producing a skin equivalent comprising:

providing a scaffold;

placing cells derived from stem/progenitor cells isolated from the amniotic membrane of umbilical cord in or onto said scaffold; and

incubating said scaffold in a first medium, which allows said cells to proliferate and further differentiate.

21. A method according to claim 20, wherein said cells derived from stem/progenitor cells are selected from the group consisting of mesenchymal stem cells (UCMC) and epithelial stem cells (UCEC).

23. A method according to claim 20, further comprising:

placing an extracellular matrix in or onto said scaffold before placing said cells in or onto the scaffold.

24. A method according to claim 21, wherein the method comprises:

providing a scaffold;

placing UCMC in or onto said scaffold;

incubating said scaffold in a first medium adapted for the cultivation of fibroblast, which allows said UCMC to proliferate and further differentiate;

placing UCEC in or onto said scaffold; and

incubating said scaffold in a second medium which allows said UCEC to proliferate and further differentiate.

25. A method according to claim 20, wherein said cells are selected from the group consisting of autologous cells, xenogeneic cells and allogeneic cells.

28. A method according to claim 24, wherein said cells are derived from the group consisting of mammallian cells and human cells.

30. A method according to claim 24, wherein said scaffold further comprises at least one cell line, which is able to differentiate into blood vessels or glands.

31. A method according to claim 30, wherein said scaffold comprises vessel endothelial cells or dermal microvascular endothelial cells (DMEC).

32. A method according to claim 31, wherein said vessel endothelial cells are derived from the umbilical cord.

33. A method according to claim 32, wherein said umbilical vessel endothelial cells are of human origin.

34. A method according to claim 21, wherein said first medium comprises a medium adapted for the cultivation of fibroblast when said scaffold comprises UCMC in order to proliferate and differentiate said UCMC in fibroblasts.

35. A method according to claim 24, wherein said first medium comprises a fibroblast growth medium together with fetal serum, such as fetal calf serum (FCS) or fetal bovine serum (FBS) in order to proliferate and differentiate said UCMC in fibroblasts.

36. A method according to claim 35, wherein said fibroblast growth medium is selected from the group consisting of KGM®

-Keratinocyte Medium (Cambrex), MEGM-Mammary Epithelial Cell Medium (Cambrex), EpiLife®

medium (Cascade Biologics), Green'"'"'s Medium, CMRL1066 (Mediatech, Inc.), M171 (Cascade Biologics), L-15 medium, Dulbecco'"'"'s Modified Eagle Medium (DMEM), DMEM-F12 and RPMI media.

37. A method according to claim 34, wherein said first medium is selected from a group consisting of a medium comprising about 90 to about 95% (v/v) fibroblast growth medium together with about 5 to about 10% fetal calf serum (FCS) or fetal bovine serum (FBS).

38. A method according to claim 37, wherein said first medium comprises about 90 to about 95% (v/v) CMRL1066 (Mediatech, Inc.) and about 5 to about 10% fetal ecalf serum (FCS).

46. A method according to claim 24, wherein said second medium comprises a medium adapted for the cultivation of keratinocytes as claimed in claim 38.

47. A method according to claim 20, wherein the scaffold comprises a biodegradable material.

48. A method according to claim 20, wherein the scaffold comprises a material selected from the group consisting of agarose, polycaprolactone, niobium coated carbon, chitosan, collagen, hyaluronic acid, calcium phosphate, starch, hydroxyapatite, fibrin, alginate, poly-glycolic acid, carbon nano fibres, porous polycarbonate, polytetrafluoroethylene, polylactide and mixtures thereof.

49. A method according to claim 48, wherein the scaffold material is porous polycarbonate.

50. A method according to claim 20, wherein in or onto said scaffold an extracellular matrix component is placed together with said cells.

51. A method according to claim 23, wherein said extracellular matrix component is selected from the group consisting of collagen, elastin, intercellular adhesion molecules, laminin, heparin, fibronectin, proteoglycans, tenascin, fibrillin and mixtures thereof.

52. A method according to claim 51, wherein said extracellular matrix component is collagen type I.

22. (canceled)

39. A method according to claim 22, wherein said first medium comprises a medium adapted for the cultivation of keratinocyte when said scaffold comprises UCEC in order to proliferate and differentiate said UCEC in keratinocytes.

40. A method according to claim 39, wherein said first medium comprises a keratinocyte growth medium, a growth factor, insulin, transferrin and selenous acid.

41. A method according to claim 40, wherein said growth factor is selected from the group consisting of epidermal growth factor (EGF), insulin-like growth factor-1, platelet-derived growth factor-BB (PDGFb), transforming growth factory, keratinocyte growth factor (KGF), TGF-α

and amphiregulin.

42. A method according to claim 40, wherein said keratinocyte growth medium is selected from the group consisting of KGM®

-Keratinocyte Medium (Cambrex), MEGM-Mammary Epithelial Cell Medium (Cambrex), EpiLife®

medium (Cascade Biologics), Green'"'"'s Medium, CMRL1066 (Mediatech, Inc.), M171 (Cascade Biologics), L-15 medium, Dulbecco'"'"'s Modified Eagle Medium (DMEM), DMEM-F12 and RPMI media.

43. A method according to claim 39, wherein said first medium is selected from a group consisting of a medium comprising a kerationocyte growth medium, epidermal growth factor (EGF), insulin, transferrin and selenous acid;

a medium comprising EpiLife®

medium (Cascade Biologics), epidermal growth factor (EGF), insulin, transferrin and selenous acid; and

a medium comprising about 98,8 to about 99,4% (v/v) EpiLife®

medium (Cascade Boilogics), about 0.2 to about 0.4 (v/v) insulin, about 0.2 to about 0.4% (v/v) transferrin, about 0.2 to about 0.4% (v/v) selenous acid and about 10 ng/ml epidermal growth factor (EGF).

26. (canceled)

27. (canceled)

29. (canceled)

44. (canceled)

45. (canceled)

53. (canceled)

54. (canceled)

55. (canceled)

56. (canceled)

58. A method of generating a mucin-producing cell comprising:

placing umbilical cord amniotic membrane epithelial stem cells (UCEC) or umbilical cord amniotic membrane mesenchymal stem cells (UCMC) in a container; and

incubating said cells in a medium adapted for the cultivation of a mucin-producing cell.

59. A method according to claim 58, wherein said medium comprises a growth medium for the cultivation of mucin-producing cells, insulin, transferrin, selenous acid and a growth factor.

60. A method according to claim 59, wherein said growth factor is selected from the group consisting of epidermal growth factor (EGF), insulin-like growth factor-1, platelet-derived growth factor-BB (PDGFb), transforming growth factor-62 , keratinocyte growth factor (KGF), TGF-α

and amphiregulin.

61. A method according to claim 59, wherein said growth medium for the cultivation of mucin-producing cells is selected from the group consisting of KGM®

-Keratinocyte Medium (Cambrex), MEGM-Mammary Epithelial Cell Medium (Cambrex), EpiLife®

medium (Cascade Biologics), Green'"'"'s Medium, CMRL1066 (Mediatech, Inc.), M171 (Cascade Biologics), L-15 medium, Dulbecco'"'"'s Modified Eagle Medium (DMEM), DMEM-F12 and RPMI media.

62. A method according to claim 58, wherein said medium adapted for the cultivation of a mucin-producing cell comprises a growth medium for the cultivation of mucin-producing cells, epidermal growth factor (EGF), insulin, transferrin and selenous acid.

63. A method according to claim 62, wherein said medium adapted for the cultivation of a mucin-producing cell comprises about 98.8 to about 99.4% (v/v) of a growth medium for the cultivation of mucin-producing cells, about 0.2 to about 0.4% (v/v) insulin, about 0.2 to about 0.4% (v/v) transferrin, about 0.2 to about 0.4% (v/v) selenous acid and about 10 ng/ml epidermal growth factor (EGF).

64. A method according to claim 63, wherein said medium adapted for the cultivation of a mucin-producing cell is selected from the group consisting of a medium comprising about 98.8 to about 99.4% (v/v) CMRL1066 (Mediatech, Inc.), about 0.2 to about 0.4% (v/v) insulin, about 0.2 to about 0.4% (v/v) transferrin, about 0.2 to about 0.4% (v/v) selenous acid and about 10 ng/ml epidermal growth factor (EGF) and a medium comprising about 98.8 to about 99.4% (v/v) M171 (Cascade Boilogics), about 0.2 to about 0.4% (v/v) insulin, about 0.2 to about 0.4% (v/v) transferrin, about 0.2 to about 0.4% (v/v) selenous acid and about 10 ng epidermal growth factor (EGF).

66. A method of treating a cell affected by smoke, comprising contacting tissue comprising cells affected by smoke with a mucin producing cell generated as defined in claim 58.

67. A method according to claim 66, wherein the affected cells are cells of the respiratory tract or the ocular surface.

68. A method of treating a synovial cell sarcom, a smoke inhalation injury or ocular surface injury using mucin-producing cells obtained by the method of claim 58.

65. (canceled)

69. (canceled)

70. A method of generating an insulin-producing cell, comprising

cultivating cells derived from stem/progenitor cells isolated from the amniotic membrane of umbilical cord;

and

proliferating and differentiating said cells in a suitable cultivation medium into β

-islet cells.

71. A method according to claim 70, further comprising

isolating insulin produced by said β

-islet cells.

72. A method according to claim 70, wherein said cells derived from stem/progenitor cells are mesenchymal stem cells (UCMC) or epithelial stem cells (UCEC).

73. A method according to claim 70, wherein said suitable cultivation medium comprises nicotinamide.

74. A method according to claim 73, wherein said cultivation medium further comprises a growth medium for the cultivation of β

-islet cells.

75. A method according to claim 74, wherein said cultivation medium further comprises a growth factor or fetal serum of calf or bovine origin.

76. A method according to claim 74, wherein said cultivation medium further comprises insulin, transferrin and selenous acid.

77. A method according to claim 75, wherein said growth factor is selected from the group consisting of epidermal growth factor (EGF), insulin-like growth factor-1, platelet-derived growth factor-BB (PDGFb), transforming growth factor-β

, keratinocyte growth factor (KGF), TGF-α and

amphiregulin.

78. A method according to claim 74, wherein said growth medium for the cultivation of β

-islet cells is selected from the group consisting of KGM®

-Keratinocyte Medium (Cambrex), MEGM-Mammary Epithelial Cell Medium (Cambrex), EpiLife®

medium (Cascade Biologics), Green'"'"'s Medium, CMRL1066 (Mediatech, Inc.), M171 (Cascade Biologics), L-15 medium, Dulbecco'"'"'s Modified Eagle Medium (DMEM), DMEM-F12 and RPMI media.

79. An insulin producing cell obtained by a method as defined in claim 70.

80. A method of treating a disorder associated with an imbalance in the insulin level, comprising administering to a mammal an insulin producing cell obtained by the method as defined in claim 70.

81. The method of claim 80, wherein the disorder is insulin dependent diabetes mellitus (IDDM).

82. (canceled)

83. A method of treating a bone disorder comprising administering osteoblasts which are produced from mesenchymal stem cells (UCMC) isolated from the amniotic membrane of the umbilical cord.

84. A method of treating cartilage disorder comprising administering chondrocytes which are produced from mesenchymal stem cells isolated from the amniotic membrane of the umbilical cord.

85. A method of generating a dopamine and tyrosine hydroxylase producing cell, comprising

cultivating cells derived from stem/progenitor cells isolated from the amniotic membrane of umbilical cord;

and

proliferating and differentiating said cells in a suitable cultivation medium into dopamine and tyrosine hydroxylase producing cells.

86. A method according to claim 85, further comprising

isolating dopamine and/or tyrosine hydroxylase produced by said dopamine and tyrosine hydroxylase producing cells.

87. A method according to claim 85, wherein said cells derived from stem/progenitor cells are mesenchymal stem cells (UCMC).

88. A method for generating a human leukocyte antigen G (HLA-G) producing cell, comprising

cultivating cells derived from stem/progenitor cells isolated from the amniotic membrane of umbilical cord;

and

proliferating and differentiating said cells in a suitable cultivation medium into HLA-G producing cells.

89. A method according to claim 88, further comprising

isolating HLA-G produced by said HLA-G producing cells.

90. A method according to claim 88, wherein said cells derived from stem/progenitor cells are mesenchymal stem cells (UCMC) or epithelial stem cells (UCEC).

91. A method of inducing proliferation of aged keratinocytes comprising

culturing aged keratinocytes in a suitable growth medium;

and

adding mesenchymal stem cells (UCMC) of the amniotic membrane of the umbilical cord to said aged keratinocytes to induce proliferation of said aged keratinocytes.

92. A method according to claim 91, wherein said method further comprises

isolating said proliferated aged keratinocytes;

and

applying said proliferated aged keratinocytes into or onto a scaffold.

93. A method according to claims 91, wherein said aged keratinocytes are isolated from a patient who is as old or older than 35 years, 40 years, 50 years, 60 years or 70 years.

94. A method of generating a hepatocytes, comprising

cultivating cells derived from stem/progenitor cells isolated from the amniotic membrane of umbilical cord;

and

proliferating and differentiating said cells in a suitable cultivation medium into hepatocytes.

95. A method according to claim 94, wherein said cells derived from stem/progenitor cells are epithelial stem cells (UCEC).

96. A method according to claim 94, wherein said suitable cultivation medium comprises oncostatin-M.

97. A method according to claim 96, wherein said cultivation medium further comprises a growth medium for the cultivation of hepatocytes.