Amplification of cell populations from embryonic stem cells

• US 20070042491A1
• Filed: 08/17/2006
• Published: 02/22/2007
• Est. Priority Date: 08/18/2005
• Status: Abandoned Application

• Current Assignee: Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: Langer, Robert, Khademhosseini, Ali, Karp, Jeffrey, Ferreira, Lino
• Application Number:
  US11/505,683
• Publication Number:
  US 20070042491A1
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: C12N 2500/42 Organic phosphate, e.g. beta glycerophosphate
• CPC Class Codes: C12N 2501/39 Steroid hormones
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0654 Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth

Abstract

Culturing embryonic stem cells without the use of embryoid bodies leads to a increase in the frequency of predetermined cell types.

Claims

1. A method of culturing embryonic stem cells, comprising:

providing a plurality of embryonic stem cells (ESC);

seeding the ESC on a substrate or suspending them in a culture medium as a single cell culture; and

incubating the seeded or suspended ESC under conditions in which they can differentiate to obtain a population of cells, wherein

the ESC are not stimulated to form embryoid bodies, and

wherein the proportion of a predetermined differentiated cell type in the population of cells is at least 2 times greater than the proportion of the predetermined differentiated cell type in a population obtained in the same manner except that the ESC are stimulated to form embryoid bodies.

2. The method of claim 1, wherein the proportion is at least 3 times greater.

3. The method of claim 1, wherein the proportion is at least 4 times greater.

4. The method of claim 1, wherein the proportion is at least 5 times greater.

5. The method of claim 1, wherein the proportion is at least 6 times greater.

6. The method of claim 1, wherein the proportion is at least 7 times greater.

7. The method of claim 1, wherein providing comprises culturing a population of ESC over a feeder layer.

8. The method of claim 7, further comprising separating the cells into individual cells.

9. The method of claim 1, wherein the plurality of embryonic stem cells comprising aggregates of cells, and wherein these aggregates are seeded.

10. The method of claim 1, wherein the plurality of embryonic stem cells comprising aggregates of cells, and wherein providing comprises separating the aggregates into smaller aggregates, and wherein at least a portion of the smaller aggregates are seeded.

11. The method of claim 1, wherein providing comprises separating undifferentiated ESC into single cells.

12. The method of claim 1, wherein providing comprises separating OCT-4/alkaline phosphatase positive ESC into single cells.

13. The method of claim 1, wherein providing does not comprise disposing the ESC over a feeder layer.

14. The method of claim 1, wherein seeding comprises seeding the cells on a two-dimensional substrate or a three-dimensional substrate.

15. The method of claim 1, wherein suspending comprises encapsulating the ESC in a hydrogel and placing the encapsulated ESC in a culture medium.

16. The method of claim 1, wherein suspending comprises placing the ESC in a bioreactor and wherein incubating occurs in the bioreactor.

17. The method of claim 1, wherein the ESC are human ESC.

18. The method of claim 1, further comprising culturing the seeded or suspended cells in the presence of at least one growth factor or cytokine.

19. The method of claim 18, wherein the at least one growth factor or cytokine is selected from dexamethasone, leptin, sortilin, transglutaminase, prostaglandin E, 1,25-dihydroxyvitamin D3, ascorbic acid, β

-glycerophosphate, TAK-778, statins, interleukins such as IL-3 and IL-6, growth hormone, steel factor (SF), activin A (ACT), retinoic acid (RA), epidermal growth factor, bone morphogenetic proteins (BMP), platelet derived growth factor (PDGF), hepatocyte growth factor, insulin-like growth factors (IGF) I and II, hematopoietic growth factors, peptide growth factors, erythropoietin, interleukins, tumor necrosis factors, interferons, colony stimulating factors, heparin binding growth factor (HBGF), alpha or beta transforming growth factor (α

or β

-TGF), fibroblastic growth factors, epidermal growth factor (EGF), vascular endothelium growth factor (VEGF), nerve growth factor (NGF) and muscle morphogenic factor (MMP).

20. The method of claim 19, wherein seeded ESC are cultured in the presence of dexamethasone, ascorbic acid, and β

-glycerophosphate.

21. The method of claim 18, wherein the growth factor is TGF-beta and the predetermined cell type produces cartilage tissue.

22. The method of claim 18, wherein the growth factor is selected from activin A and IGF and the predetermined cell type is liver progentor cells.

23. The method of claim 18, wherein the growth factor is retinoic acid and the predetermined cell type is neuronal cells.

24. The method of claim 18, wherein the growth factor is selected from bone morphogenetic protein, colony stimulating factor, and PDGF and the predetermined cell type produces collagen.

25. The method of claim 18, wherein the at least one growth factor is dexamethasone and insulin and the predetermined cell type is adipocytes.

26. The method of claim 18, wherein the growth factor is 5-azacytidine and the predetermined cell type is skeletal muscle cells.

27. The method of claim 18, wherein the growth factor is PDGF and the predetermined cell type is smooth muscle cells.

28. The method of claim 18, wherein the growth factor is b-FGF and the predetermined cell type is cardiac muscle cells.

29. The method of claim 18, wherein the growth factor is ascorbic acid and the predetermined cell type is osteoclasts.

67. The method of claim 18, wherein the growth factor is ascorbic acid and the predetermined cell type is osteoclasts.

30. A single cell population of undifferentiated embryonic stem cells (ESC) in contact with a growth factor or cytokine.

31. The single cell population of claim 30, wherein the predetermined lineage is mesodermal.

32. The single cell population of claim 30, wherein the predetermined lineage is osteogenic.

33. The single cell population of claim 30, wherein the ESC are hESC.

34. The single cell population of claim 33, wherein at least 90% of the cells express one or more of OCT-4, ALP, nanog, TRA-1-60, and TRA-1-81 and do not express SSEA-1.

35. The single cell population of claim 30, wherein the ESC are mESC.

36. The single cell population of claim 35, wherein express one or more of OCT-4, ALP, nanog, and SSEA-1 and do not express SSEA-3 or SSEA-4.

37. The single cell population of claim 30, wherein the population is capable of differentiating along a predetermined lineage with a frequency that is at least two times greater than the frequency for a population of human embryonic stem cells recovered from embryoid bodies.

38. The single cell population of claim 30, wherein the population is capable of differentiating to express a predetermined phenotype within an elapsed time (“

the first elapsed time”

) that is less than an elapsed time (“

the second elapsed time”

) before expression of the phenotype by a population of cells that are stimulated to form embryoid bodies.

39. The single cell population of claim 30, wherein the at least one growth factor or cytokine is selected from dexamethasone, leptin, sortilin, transglutaminase, prostaglandin E, 1,25-dihydroxyvitamin D3, ascorbic acid, β

-glycerophosphate, TAK-778, statins, interleukins such as IL-3 and IL-6, growth hormone, steel factor (SF), activin A (ACT), retinoic acid (RA), epidermal growth factor, bone morphogenetic proteins (BMP), platelet derived growth factor (PDGF), hepatocyte growth factor, insulin-like growth factors (IGF) I and II, hematopoietic growth factors, peptide growth factors, erythropoietin, interleukins, tumor necrosis factors, interferons, colony stimulating factors, heparin binding growth factor (HBGF), alpha or beta transforming growth factor (α

or β

-TGF), fibroblastic growth factors, epidermal growth factor (EGF), vascular endothelium growth factor (VEGF), nerve growth factor (NGF) and muscle morphogenic factor (MMP).

40. A method of culturing embryonic stem cells, comprising:

providing a plurality of embryonic stem cells (ESC);

seeding the ESC on a substrate or suspending them in a culture medium as a single cell culture; and

incubating the seeded or suspended ESC under conditions in which they can differentiate to obtain a population of cells, wherein

the ESC are not stimulated to form embryoid bodies, and

wherein at least a portion of the cells express a predetermined phenotype during the step of incubating, and wherein the elapsed time (“

the first elapsed time”

) before the predetermined phenotype is expressed is less than an elapsed time (“

the second elapsed time”

) for a population of cells obtained in the same manner except that the ESC are stimulated to form embryoid bodies.

41. The method of claim 40, wherein the first elapsed time is at least 20% less than the second elapsed time.

42. The method of claim 40, wherein the first elapsed time is at least 30% less than the second elapsed time.

43. The method of claim 40, wherein the first elapsed time is at least 40% less than the second elapsed time.

44. The method of claim 40, wherein the first elapsed time is at least 50% less than the second elapsed time.

45. The method of claim 40, wherein the first elapsed time is at least 60% less than the second elapsed time.

46. The method of claim 40, wherein providing comprises culturing a population of ESC over a feeder layer.

47. The method of claim 40, further comprising separating the cells into individual cells.

48. The method of claim 40, wherein the plurality of embryonic stem cells comprising aggregates of cells, and wherein these aggregates are seeded.

49. The method of claim 40, wherein the plurality of embryonic stem cells comprising aggregates of cells, and wherein providing comprises separating the aggregates into smaller aggregates, and wherein at least a portion of the smaller aggregates are seeded.

50. The method of claim 40, wherein providing comprises separating ac4-alkaline phosphatase positive ESC into single cells.

51. The method of claim 40, wherein providing does not comprise disposing the ESC over a feeder layer.

52. The method of claim 40, wherein seeding comprises seeding the cells on a two-dimensional substrate or a three-dimensional substrate.

53. The method of claim 40, wherein suspending comprises encapsulating the ESC in a hydrogel and placing the encapsulated ESC in a culture medium.

54. The method of claim 40, wherein suspending comprises placing the ESC in a bioreactor and wherein incubating occurs in the bioreactor.

55. The method of claim 40, wherein the ESC are human ESC.

56. The method of claim 40, further comprising culturing the seeded or suspended cells in the presence of at least one growth factor or cytokine.

57. The method of claim 56, wherein the at least one growth factor or cytokine is selected from dexamethasone, leptin, sortilin, transglutaminase, prostaglandin E, 1,25-dihydroxyvitamin D3, ascorbic acid, β

-glycerophosphate, TAK-778, statins, interleukins such as IL-3 and IL-6, growth hormone, steel factor (SF), activin A (ACT), retinoic acid (RA), epidermal growth factor, bone morphogenetic proteins (BMP), platelet derived growth factor (PDGF), hepatocyte growth factor, insulin-like growth factors (IGF) I and II, hematopoietic growth factors, peptide growth factors, erythropoietin, interleukins, tumor necrosis factors, interferons, colony stimulating factors, heparin binding growth factor (HBGF), alpha or beta transforming growth factor (α

or β

-TGF), fibroblastic growth factors, epidermal growth factor (EGF), vascular endothelium growth factor (VEGF), nerve growth factor (NGF) and muscle morphogenic factor (MMP).

58. The method of claim 56, wherein seeded ESC are cultured in the presence of dexamethasone, ascorbic acid, and β

-glycerophosphate.

59. The method of claim 56, wherein the growth factor is TGF-beta and the predetermined phenotype is production of cartilage tissue.

60. The method of claim 56, wherein the growth factor is selected from activin A and IGF and the predetermined phenotype is production of proteins characteristic of developing liver.

61. The method of claim 56, wherein the growth factor is retinoic acid and the predetermined phenotype is production of ectodermal structures.

62. The method of claim 56, wherein the growth factor is selected from bone morphogenetic protein, colony stimulating factor, and PDGF and the predetermined phenotype is production of at least bone extracellular matrix component.

63. The method of claim 56, wherein the at least one growth factor is dexamethasone and insulin and the predetermined phenotype is characteristic of adipocytes.

64. The method of claim 56, wherein the growth factor is 5-azacytidine and the predetermined phenotype is characteristic of skeletal muscle cells.

65. The method of claim 56, wherein the growth factor is PDGF and the predetermined cell type is characteristic of smooth muscle cells.

66. The method of claim 56, wherein the growth factor is b-FGF and the predetermined cell type is characteristic of cardiac muscle cells.

68. A method of obtaining a population of osteogenic cells, comprising:

providing a population of embryonic stem cells (ESC);

seeding the ESC on a substrate as a single cell culture; and

incubating the seeded ESC in the presence of an osteogenic differentiation factor, wherein

the ESC are not stimulated to form embryoid bodies.

69. The method of claim 68, wherein the osteogenic differentiation factor is dexamethasone.

70. The method of claim 68, wherein incubating further comprises incubating the seeded ESC in the presence of ascorbic acid and beta-glycerophosphate.

71. The method of claim 68, wherein the proportion of osteogenic cells in the incubated ESC is at least 2 times greater than the proportion of osteogenic cells in a population of cells obtained in the same manner except that the ESC are stimulated to form embryoid bodies.

72. The method of claim 71, wherein the proportion is at least 3 times greater.

73. The method of claim 71, wherein the proportion is at least 4 times greater.

74. The method of claim 71, wherein the proportion is at least 5 times greater.

75. The method of claim 71, wherein the proportion is at least 6 times greater.

76. The method of claim 71, wherein the proportion is at least 7 times greater.

77. The method of claim 68, wherein the ESC are human ESC.

78. The method of claim 68, wherein the seeded ESC produce bone nodules during the step of incubating, and wherein the elapsed time (“

the first elapsed time”

) before the bone nodules are produced is less than an elapsed time (“

the second elapsed time”

) for a population of cells obtained in the same manner except that the ESC are stimulated to form embryoid bodies.

79. The method of claim 78, wherein the first elapsed time is at least 20% less than the second elapsed time.

80. The method of claim 78, wherein the first elapsed time is at least 30% less than the second elapsed time.

81. The method of claim 78, wherein the first elapsed time is at least 40% less than the second elapsed time.

82. The method of claim 78, wherein the first elapsed time is at least 50% less than the second elapsed time.

83. The method of claim 78, wherein the first elapsed time is at least 60% less than the second elapsed time.