Caudal motor neuron derived from embryonic stem cells under conditions essentially free of any retinoid

• US 8,969,081 B2
• Filed: 12/09/2009
• Issued: 03/03/2015
• 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: Wichterle, Hynek, Jessell, Thomas M., Peljto, Mirza
• Primary Examiner(s): Crouch, Deborah
• Application Number:
  US12/653,217
• Publication Number:
  US 20100196332A1
• Time in Patent Office: 1,910 Days
• US Class Current: 435/377
• 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. A method for producing a caudal brachial motor neuron from an embryonic stem cell, comprising

a) culturing the embryonic stem cell in a composition essentially free of any retinoid and comprising Advanced Dulbecco'"'"'s Modified Eagle'"'"'s Medium/F12 and Neurobasal medium that has been supplemented with 10% Knockout-Serum Replacement (ADFNK medium) wherein the amount of the composition is effective to generate the caudal brachial motor neuron under conditions such that the caudal brachial motor neuron is produced, and

b) recovering the caudal brachial motor neuron so produced.

2. The method of claim 1, wherein during culturing the embryonic stem cell is contacted with at least one neurotrophic factor.

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

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

5. The method of claim 1, wherein the caudal brachial motor neuron is transfected so that it expresses enhanced green fluorescent protein (eGFP).

6. The method of claim 1, wherein the culturing is effected in vivo in a subject.

7. The method of claim 1, wherein the culturing is effected in vitro.

8. A method for producing a caudal motor neuron comprising

a) culturing an embryonic stem cell in a serum-free neural induction composition which is essentially free of any retinoid and comprises an isotonic salt solution, wherein the amount of the composition is effective to generate the caudal motor neuron under condition such that the caudal motor neuron is produced, and

b) recovering the caudal motor neuron so produced.

9. The method of claim 8, wherein during culturing the embryonic stem cell is contacted with at least one neurotrophic factor.

10. The method of claim 8, wherein the embryonic stem cell is a murine embryonic stem cell.

11. The method of claim 8, wherein the embryonic stem cell is a human embryonic stem cell.

12. The method of claim 8, wherein the caudal motor neuron is transfected so that it expresses enhanced green fluorescent protein (eGFP).

13. The method of claim 8, wherein the culturing is effected in vivo in a subject.

14. The method of claim 8, wherein the culturing is effected in vitro.

15. The method of claim 8, wherein composition further comprises an amount of Fibroblast Growth Factor-2 (FGF-2).

16. The method of claim 8, wherein composition further comprises an amount of Growth differentiation factor 11 (Gdf11).

17. The method of claim 8, wherein composition further comprises an amount of a Wnt factor.

18. The method of claim 8, 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.