Generation of brachial, thoracic and lumbar spinal motor neurons from embryonic stem cells in the absence of all-trans retinoic acid supplement

• US 20100196332A1
• Filed: 12/09/2009
• Published: 08/05/2010
• Est. Priority Date: 12/10/2008
• 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: Peljto, Mirza, Jessell, Thomas M., Wichterle, Hynek
• Granted Patent: US 8,969,081 B2
• Time in Patent Office: Days
• US Class Current: 424/93.7
• CPC Class Codes: A61K 35/30 Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/13 Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/19 Growth and differentiation factors [GDF]
• 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 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0619 Neurons
• CPC Class Codes: C12N 5/0623 Stem cells
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

Abstract

Disclosed are methods for generating a neuron expressing Hoxc8 transcription factor or a caudal motor neuron comprising culturing an embryonic stem cell in a composition which is essentially free of retinoids and comprises an isotonic salt solution, so as to generate the neuron which expresses Hoxc8 transcription factor or the caudal motor neuron. Disclosed are also methods for generating a caudal brachial motor neuron, a thoracic motor neuron, or a lumbar motor neuron from an embryonic stem cell in a composition essentially free of retinoids and comprising ADFNK medium, an amount of FGF-2, or Gdf11 respectively. Disclosed are also methods of transplanting a motor neuron into a subject comprising generating the motor neuron and transplanting the motor neuron into the subject. Disclosed is also a population of motor neuron cells enriched for motor neuron cells expressing Foxp1 and expressing a gene associated with Spinal Muscular Atrophy (SMA) or Amyotrophic Lateral Sclerosis (ALS).

Claims

1-9. -9. (canceled)

10. A method for generating a caudal brachial motor neuron from an embryonic stem cell, comprising culturing the embryonic stem cell in a composition essentially free of retinoids and comprising an amount of Advanced Dulbecco'"'"'s Modified Eagle'"'"'s Medium/F12 and Neurobasal medium that has been supplemented with 10% Knockout-Serum Replacement (ADFNK medium) effective to produce the caudal brachial motor neuron.

11. The method of claim 10, wherein the method further comprises contacting the caudal brachial motor neuron with at least one neurotrophic factor.

12. The method of claim 10, wherein the caudal brachial motor neuron expresses Hoxc8 transcription factor.

13. The method of claim 10, wherein the caudal brachial motor neuron expresses Hox6 transcription factor.

14. The method of claim 10, wherein the embryonic stem cell is a murine embryonic stem cell.

15. The method of claim 10, wherein the embryonic stem cell is a human embryonic stem cell.

16. The method of claim 10, wherein the caudal brachial motor neuron is transfected so that it expresses enhanced green fluorescent protein (eGFP).

17. The method of claim 10, wherein the culturing is effected in vivo in a subject.

18. The method of claim 10, wherein the culturing is effected in vitro.

32. A caudal brachial motor neuron produced by the method of claim 10.

33. The caudal brachial motor neuron of claim 32, which express enhanced green fluorescent protein (eGFP).

34. A population of cells, comprising the caudal brachial motor neuron produced by the method of claim 10.

35. The population of claim 34, wherein some or all of the caudal brachial motor neurons express enhanced green fluorescent protein (eGFP).

19-22. -22. (canceled)

23. A method of transplanting a motor neuron into a subject comprising:

a) generating a motor neuron by culturing an embryonic stem cell in a composition essentially free of retinoids and comprising an isotonic salt solution, so as to generate the motor neuron; and

b) transplanting the motor neuron into the subject.

24. The method of claim 23, wherein the motor neuron is a caudal brachial motor neuron.

25. The method of claim 24, wherein the caudal brachial motor neuron is transplanted into a spinal cord of the subject.

26. The method of claim 23, wherein the subject is an adult.

27. The method of claim 23, wherein the subject is a human.

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

29. The method of claim 28, wherein the nervous tissue degeneration is a peripheral neuropathy or a neurodegenerative disease.

30. The method of claim 29, wherein the peripheral neuropathy is a symptom of amyotrophic lateral sclerosis (ALS), neural trauma, paraneoplastic syndrome, polio, postpolio syndrome, progressive bulbar palsy, or spinal muscular atrophy (SMA).

31. The method of claim 29, 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, or Pick'"'"'s disease.

36-87. -87. (canceled)

88. A population of motor neuron cells enriched for motor neuron cells expressing Foxp1 and expressing a gene associated with Spinal Muscular Atrophy (SMA) or Amyotrophic Lateral Sclerosis (ALS).

89. The population of claim 88, wherein the gene associated with ALS is a mutated SOD1 gene.

90. The population of claim 88, wherein the gene associated with SMA is a mutated SMN1 gene.

91. A method for generating a caudal motor neuron comprising culturing an embryonic stem cell in a composition which is essentially free of retinoids and comprises an isotonic salt solution, so as to generate the caudal motor neuron.

92. The method of claim 91, wherein the method further comprises contacting the caudal motor neuron with at least one neurotrophic factor.

93. The method of claim 91, wherein the caudal motor neuron expresses Hoxc8 transcription factor.

94. The method of claim 91, wherein the caudal motor neuron expresses Hox6 transcription factor.

95. The method of claim 91, wherein the embryonic stem cell is a murine embryonic stem cell.

96. The method of claim 91, wherein the embryonic stem cell is a human embryonic stem cell.

97. The method of claim 91, wherein the caudal motor neuron is transfected so that it expresses enhanced green fluorescent protein (eGFP).

98. The method of claim 91, wherein the culturing is effected in vivo in a subject.

99. The method of claim 91, wherein the culturing is effected in vitro.

100. The method of claim 91, wherein composition further comprises an amount of Fibroblast Growth Factor-2 (FGF-2).

101. The method of claim 91, wherein composition further comprises an amount of Growth differentiation factor 11 (Gdf11).

102. The method of claim 91, wherein composition further comprises an amount of a Wnt factor.

103. The method of claim 91, wherein composition further comprises an amount of Fibroblast Growth Factor-2 (FGF-2), an amount of Growth differentiation factor 11 (Gdf11), and an amount of a Wnt factor.