Methods for inducing differentiation of embryonic stem cells and uses thereof

• US 20040014210A1
• Filed: 07/16/2002
• Published: 01/22/2004
• Est. Priority Date: 07/16/2002
• Status: Active Grant

• Current Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Original Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Inventors: Jessell, Thomas M., Wichterle, Hynek, Lieberam, Ivo
• Granted Patent: US 7,390,659 B2
• Time in Patent Office: Days
• US Class Current: 435/368
• 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: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/02 for peripheral neuropathies
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: C12N 2501/385 of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
• CPC Class Codes: C12N 2501/41 Hedgehog proteins; Cyclopamine (inhibitor)
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0619 Neurons
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The present invention provides a method for inducing differentiation of an embryonic stem cell into a differentiated neural cell. The present invention further provides a method for producing differentiated neural cells, and a population of cells comprising the differentiated neural cells. Additionally, the present invention provides a method for repopulating a spinal cord in a subject, and a method for treating nervous tissue degeneration in a subject in need of treatment. The present invention further provides neural progenitor cells, differentiated neural cells, and uses of same. Also provided is a transgenic non-human animal containing the differentiated neural cells. The present invention is further directed to a method for isolating a population of differentiated neural cells. Finally, the present invention provides a method for identifying an agent for use in treating a condition associated with neuron degeneration.

Claims

1. A method for inducing differentiation of an embryonic stem cell into a differentiated neural cell, comprising contacting the embryonic stem cell with amounts of a rostralizing and/or caudalizing embryonic signalling factor and a dorsalizing or ventralizing embryonic signalling factor effective to produce a differentiated neural cell.

2. The method of claim 1, wherein the embryonic stem cell is a murine embryonic stem cell.

3. The method of claim 1, wherein the embryonic stem cell is a human embryonic stem cell.

4. The method of claim 1, wherein the rostralizing embryonic signalling factor is retinoic acid or Wnt.

5. The method of claim 1, wherein the caudalizing embryonic signalling factor is retinoic acid or Wnt.

6. The method of claim 1, wherein the dorsalizing embryonic signalling factor is BMP or Wnt.

7. The method of claim 1, wherein the ventralizing embryonic signalling factor is an activator of a Hedgehog signalling pathway.

8. The method of claim 7, wherein the activator of a Hedgehog signalling pathway is a Hedgehog protein or an agonist of Hedgehog signalling.

9. The method of claim 7, wherein the activator of a Hedgehog signalling pathway is SHh or Hh-Ag1.3.

10. The method of claim 1, wherein the embryonic stem cell is contacted with the rostralizing and/or caudalizing embryonic signalling factor prior to contact with the dorsalizing or ventralizing embryonic signalling factor.

11. The method of claim 10, further comprising contacting the embryonic stem cell with at least one neurotrophic factor following contact with the dorsalizing or ventralizing embryonic signalling factor.

12. The method of claim 1, wherein the differentiated neural cell is selected from the group consisting of astrocyte, neuron, oligodendrocyte, and Schwann cell.

13. The method of claim 12, wherein the differentiated neural cell is a motor neuron.

14. The method of claim 13, wherein the motor neuron is a spinal motor neuron.

15. The method of claim 1, wherein the differentiated neural cell expresses enhanced green fluorescent protein (eGFP).

16. The method of claim 1, wherein the contacting is effected in vivo in a subject.

17. The method of claim 16, wherein the contacting is effected in vivo in a subject by administering the embryonic signalling factors to the subject.

18. The method of claim 1, wherein the contacting is effected in vitro.

19. The method of claim 18, further comprising the step of transplanting the differentiated neural cell into a subject.

20. The method of claim 19, wherein the differentiated neural cell is transplanted into the spinal cord of the subject.

21. The method of claim 19, wherein the subject is an embryo.

22. The method of claim 19, wherein the subject is a human.

23. The method of claim 19, wherein the subject has nervous tissue degeneration.

24. The method of claim 23, wherein the nervous tissue degeneration is a peripheral neuropathy or a neurodegenerative disease.

25. The method of claim 24, wherein the peripheral neuropathy is associated with a condition selected from the group consisting of amyotrophic lateral sclerosis (ALS), neural trauma, paraneoplastic syndrome, polio, postpolio syndrome, progressive bulbar palsy, and spinal muscular atrophy (SMA).

26. The method of claim 25, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer'"'"'s disease, amyotrophic lateral sclerosis (Lou Gehrig'"'"'s Disease), Binswanger'"'"'s disease, Huntington'"'"'s chorea, multiple sclerosis, myasthenia gravis, Parkinson'"'"'s disease, and Pick'"'"'s disease.

27. A differentiated neural cell produced by the method of claim 1.

28. The differentiated neural cell of claim 27, which expresses enhanced green fluorescent protein (eGFP).

29. A method for producing differentiated neural cells, comprising the steps of:

(a) obtaining or generating a culture of embryonic stem cells;

(b) contacting the culture of embryonic stem cells with an amount of a rostralizing and/or caudalizing embryonic signalling factor effective to produce neural progenitor cells;

(c) contacting the neural progenitor cells with an amount of a dorsalizing or ventralizing embryonic signalling factor effective to produce differentiated neural cells; and

(d) optionally, contacting the differentiated neural cells with at least one neurotrophic factor.

30. The method of claim 29, wherein the embryonic stem cells are murine embryonic stem cells.

31. The method of claim 29, wherein the embryonic stem cells are human embryonic stem cells.

32. The method of claim 29, wherein the rostralizing embryonic signalling factor is retinoic acid or Wnt.

33. The method of claim 29, wherein the caudalizing embryonic signalling factor is retinoic acid or Wnt.

34. The method of claim 29, wherein the dorsalizing embryonic signalling factor is BMP or Wnt.

35. The method of claim 29, wherein the ventralizing embryonic signalling factor is an activator of a Hedgehog signalling pathway.

36. The method of claim 35, wherein the activator of a Hedgehog signalling pathway is a Hedgehog protein or an agonist of Hedgehog signalling.

37. The method of claim 35, wherein the activator of a Hedgehog signalling pathway is SHh or Hh-Ag1.3.

38. The method of claim 29, wherein the neural progenitor cells are selected from the group consisting of progenitor cells of DRG neurons, progenitor cells of motor neurons, progenitor cells of peripheral neurons, progenitor cells of spinal cord neurons, and progenitor cells of ventral interneurons.

39. The method of claim 29, wherein the differentiated neural cells are selected from the group consisting of astrocytes, neurons, oligodendrocytes, and Schwann cells.

40. The method of claim 39, wherein the differentiated neural cells are motor neurons.

41. The method of claim 40, wherein the motor neurons are post-mitotic.

42. The method of claim 40, wherein the motor neurons are spinal motor neurons.

43. The method of claim 29, wherein the differentiated neural cells express enhanced green fluorescent protein (eGFP).

44. The method of claim 29, wherein steps (b)-(d) are performed in vivo in a subject.

45. The method of claim 29, wherein one or more of steps (b), (c), and (d) are performed in vitro.

46. The method of claim 45, further comprising the step of transplanting the differentiated neural cells into a subject.

47. The method of claim 46, wherein the differentiated neural cells are transplanted into the spinal cord of the subject.

48. The method of claim 45, further comprising an additional step selected from the group consisting of transplanting the neural progenitor cells into a subject and transplanting the differentiated neural cells into a subject.

49. The method of claim 48, wherein the subject is an embryo.

50. The method of claim 48, wherein the subject is a human.

51. The method of claim 48, wherein the subject has nervous tissue degeneration.

52. The method of claim 51, wherein the nervous tissue degeneration is a peripheral neuropathy or a neurodegenerative disease.

53. The method of claim 52, wherein the peripheral neuropathy is associated with a condition selected from the group consisting of amyotrophic lateral sclerosis (ALS), neural trauma, paraneoplastic syndrome, polio, postpolio syndrome, progressive bulbar palsy, and spinal muscular atrophy (SMA).

54. The method of claim 52, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer'"'"'s disease, amyotrophic lateral sclerosis (Lou Gehrig'"'"'s Disease), Binswanger'"'"'s disease, Huntington'"'"'s chorea, multiple sclerosis, myasthenia gravis, Parkinson'"'"'s disease, and Pick'"'"'s disease.

55. A population of cells, comprising the differentiated neural cells produced by the method of claim 29.

56. The population of claim 55, wherein some or all of the differentiated neural cells express enhanced green fluorescent protein (eGFP).

57. A method for repopulating a spinal cord in a subject, comprising the steps of:

(a) obtaining or generating a culture of embryonic stem cells;

(b) contacting the culture of embryonic stem cells with an amount of a rostralizing and/or caudalizing embryonic signalling factor effective to produce neural progenitor cells;

(c) contacting the neural progenitor cells with an amount of a dorsalizing or ventralizing embryonic signalling factor effective to produce differentiated neural cells;

(d) optionally, contacting the differentiated neural cells with at least one neurotrophic factor; and

(e) transplanting the differentiated neural cells into a spinal cord of the subject.

58. The method of claim 57, wherein the embryonic stem cells are murine embryonic stem cells.

59. The method of claim 57, wherein the embryonic stem cells are human embryonic stem cells.

60. The method of claim 57, wherein the caudalizing embryonic signalling factor is retinoic acid.

61. The method of claim 57, wherein the ventralizing embryonic signalling factor is an activator of a Hedgehog signalling pathway.

62. The method of claim 61, wherein the activator of a Hedgehog signalling pathway is a Hedgehog protein or an agonist of Hedgehog signalling.

63. The method of claim 61, wherein the activator of a Hedgehog signalling pathway is SHh or Hh-Ag1.3.

64. The method of claim 57, wherein the neural progenitor cells are selected from the group consisting of progenitor cells of DRG neurons, progenitor cells of motor neurons, progenitor cells of peripheral neurons, progenitor cells of spinal cord neurons, and progenitor cells of ventral interneurons.

65. The method of claim 57, wherein the differentiated neural cells are selected from the group consisting of astrocytes, neurons, oligodendrocytes, and Schwann cells.

66. The method of claim 65, wherein the differentiated neural cells are motor neurons.

67. The method of claim 66, wherein the motor neurons are post-mitotic.

68. The method of claim 66, wherein the motor neurons are spinal motor neurons.

69. The method of claim 57, wherein the differentiated neural cells express enhanced green fluorescent protein (eGFP).

70. The method of claim 57, wherein the subject is an embryo.

71. The method of claim 57, wherein the subject is a human.

72. The method of claim 57, wherein the subject has nervous tissue degeneration.

73. The method of claim 72, wherein the nervous tissue degeneration is a peripheral neuropathy.

74. The method of claim 73, wherein the peripheral neuropathy is associated with a condition selected from the group consisting of amyotrophic lateral sclerosis (ALS), neural trauma, paraneoplastic syndrome, polio, postpolio syndrome, progressive bulbar palsy, and spinal muscular atrophy (SMA).

75. The method of claim 74, wherein the peripheral neuropathy is ALS or SMA.

76. A method for treating nervous tissue degeneration in a subject in need of treatment, comprising the steps of:

(a) obtaining or generating a culture of embryonic stem cells;

(b) contacting the culture of embryonic stem cells with an amount of a rostralizing and/or caudalizing embryonic signalling factor effective to produce neural progenitor cells;

(c) contacting the neural progenitor cells with an amount of a dorsalizing or ventralizing embryonic signalling factor effective to produce differentiated neural cells;

(d) optionally, contacting the differentiated neural cells with at least one neurotrophic factor; and

(e) transplanting the differentiated neural cells into the subject in an amount effective to treat the nervous tissue degeneration.

77. The method of claim 76, wherein the nervous tissue degeneration is a peripheral neuropathy or a neurodegenerative disease.

78. The method of claim 77, wherein the peripheral neuropathy is associated with a condition selected from the group consisting of amyotrophic lateral sclerosis (ALS), neural trauma, paraneoplastic syndrome, polio, postpolio syndrome, progressive bulbar palsy, and spinal muscular atrophy (SMA).

79. The method of claim 77, wherein the neurodegenerative disease is Alzheimer'"'"'s disease, amyotrophic lateral sclerosis (Lou Gehrig'"'"'s Disease), Binswanger'"'"'s disease, Huntington'"'"'s chorea, multiple sclerosis, myasthenia gravis, Parkinson'"'"'s disease, and Pick'"'"'s disease.

80. The method of claim 76, wherein the embryonic stem cells are murine embryonic stem cells.

81. The method of claim 76, wherein the embryonic stem cells are human embryonic stem cells.

82. The method of claim 76, wherein the rostralizing embryonic signalling factor is retinoic acid or Wnt.

83. The method of claim 76, wherein the caudalizing embryonic signalling factor is retinoic acid or Wnt.

84. The method of claim 76, wherein the dorsalizing embryonic signalling factor is BMP or Wnt.

85. The method of claim 76, wherein the ventralizing embryonic signalling factor is an activator of a Hedgehog signalling pathway.

86. The method of claim 85, wherein the activator of a Hedgehog signalling pathway is a Hedgehog protein or an agonist of Hedgehog signalling.

87. The method of claim 85, wherein the activator of a Hedgehog signalling pathway is SHh or Hh-Ag1.3.

88. The method of claim 76, wherein the neural progenitor cells are selected from the group consisting of progenitor cells of DRG neurons, progenitor cells of motor neurons, progenitor cells of peripheral neurons, progenitor cells of spinal cord neurons, and progenitor cells of ventral interneurons.

89. The method of claim 76, wherein the differentiated neural cells are selected from the group consisting of astrocytes, neurons, oligodendrocytes, and Schwann cells.

90. The method of claim 89, wherein the differentiated neural cells are motor neurons.

91. The method of claim 90, wherein the motor neurons are post-mitotic.

92. The method of claim 90, wherein the motor neurons are spinal motor neurons.

93. The method of claim 76, wherein the subject is an embryo.

94. The method of claim 76, wherein the subject is a human.

95. Neural progenitor cells produced by a method comprising the steps of:

(a) obtaining or generating a culture of embryonic stem cells;

(b) contacting the culture of embryonic stem cells with an amount of a rostralizing and/or caudalizing embryonic signalling factor effective to produce neural progenitor cells; and

(c) optionally, contacting the neural progenitor cells with a dorsalizing or ventralizing embryonic signalling factor.

96. The neural progenitor cells of claim 95, which express enhanced green fluorescent protein (eGFP).

97. Use of the neural progenitor cells of claim 95 in analyzing neuron development, function, and death, or in monitoring synaptic differentiation.

98. Differentiated neural cells produced by a method comprising the steps of:

(a) obtaining or generating a culture of embryonic stem cells;

(b) contacting the culture of embryonic stem cells with an amount of a rostralizing and/or caudalizing embryonic signalling factor effective to produce neural progenitor cells;

(c) contacting the neural progenitor cells with an amount of a dorsalizing or ventralizing embryonic signalling factor effective to produce differentiated neural cells; and

(d) optionally, contacting the differentiated neural cells with at least one neurotrophic factor.

99. The differentiated neural cells of claim 98, which express enhanced green fluorescent protein (eGFP).

100. Use of the differentiated neural cells of claim 98 in analyzing neuron development, function, and death, or in monitoring synaptic differentiation.

101. A transgenic non-human animal line containing the embryonic stem cells of claim 98, wherein the cells express enhanced green fluorescent protein (eGFP).

102. A method for isolating a population of differentiated neural cells, comprising the steps of:

(a) obtaining or generating a culture of embryonic stem cells that express enhanced green fluorescent protein (eGFP);

(b) contacting the culture of embryonic stem cells with an amount of a rostralizing and/or caudalizing embryonic signalling factor effective to produce neural progenitor cells that express eGFP;

(c) contacting the neural progenitor cells with an amount of a dorsalizing or ventralizing embryonic signalling factor effective to produce differentiated neural cells that express eGFP;

(d) optionally, contacting the differentiated neural cells with at least one neurotrophic factor;

(e) detecting expression of eGFP in the differentiated neural cells; and

(f) isolating the differentiated neural cells that express eGFP.

103. A method for identifying an agent for use in treating a condition associated with motor neuron degeneration, comprising the steps of:

(a) obtaining or generating a culture of embryonic stem cells;

(b) contacting the culture of embryonic stem cells with an amount of retinoic acid effective to produce neural progenitor cells;

(c) activating a Hedgehog signalling pathway in the neural progenitor cells, to produce motor neurons, wherein some or all of the motor neurons are degenerated;

(d) contacting the degenerated motor neurons with a candidate agent; and

(e) determining if the agent enhances regeneration of some or all of the degenerated motor neurons.

104. The method of claim 103, wherein genomic DNA in the degenerated neurons carries a mutation in an SOD or SMN gene.

105. The method of claim 104, wherein the degenerated neurons express enhanced green fluorescent protein (eGFP).