Systems and methods for screening for modulators of neural differentiation

• US 7,632,679 B2
• Filed: 02/26/2004
• Issued: 12/15/2009
• Est. Priority Date: 07/16/2002
• Status: Expired due to Fees

• 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: Wilson, Sara, Jessell, Thomas, Wichterle, Hynek
• Primary Examiner(s): Gamett; Daniel C
• Application Number:
  US10/789,308
• Publication Number:
  US 20040224887A1
• Time in Patent Office: 2,119 Days
• Field of Search: None
• US Class Current: 435/377
• 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: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• 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 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2503/00 Use of cells in diagnostics
• CPC Class Codes: C12N 2503/02 Drug screening
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0619 Neurons
• CPC Class Codes: C12N 5/0623 Stem cells
• CPC Class Codes: G01N 2333/51 Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
• CPC Class Codes: G01N 33/5073 Stem cells
• 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 in vitro systems for use in identifying modulators of neural differentiation. Also provided are modulators identified by these systems. The present invention further provides methods for identifying a modulator of neural differentiation, a modulator of a Wnt signalling pathway, a modulator of Wnt-dependent neural differentiation, a modulator of a BMP signalling pathway, a modulator of BMP-dependent neural differentiation, a modulator of a Hh signalling pathway, and a modulator of Hh-dependent neural differentiation. Also provided are modulators identified by these methods.

Claims

1. A method for identifying a modulator of Hh-dependent motor neuron differentiation, comprising the steps of:

(a) providing a first collection of embryonic stem cells and a second collection of embryonic stem cells;

(b) contacting both collections of cells with an effective amount of retinoic acid to induce differentiation of the cells to form spinal progenitor cells;

(c) activating a Hh signalling pathway in both collections of cells;

(d) contacting the first collection of cells with a candidate modulator; and

(e) determining if the candidate modulator in step (d) modulates motor neuron differentiation by comparing the motor neuron phenotypes of the cells in the first and second collections of cells produced by steps (b)-(d) where , if the motor neuron phenotypes of the cells in the first and second collections are different, it indicates that the candidate modulator is a modulator of Hh-dependent motor neuron differentiation.

2. The method of claim 1, where the motor neuron phenotype comprises expression of the motor neuron associated protein HB9.

3. The method of claim 1, where the motor neuron phenotype comprises expression of Green Fluorescent Protein, where expression of Green Fluorescent Protein is controlled by the HB9 promoter.

4. The method of claim 1, where the modulator has an agonistic effect on motor neuron differentiation whereby the rate of motor neuron differentiation in the first collection of cells, contacted with the modulator, is increased relative to the rate of motor neuron differentiation in the second collection of cells, as measured by the presence of one or more feature characteristic of a motor neuron phenotype.

5. A method for identifying a modulator of Hh-dependent motor neuron differentiation, comprising the steps of:

(a) contacting a collection of embryonic stem cells with an effective amount of retinoic acid to induce differentiation of the cells to form spinal progenitor cells;

(b) activating a Hh signalling pathway in the collection of cells;

(c) contacting the collection of cells with a candidate modulator; and

(d) determining if the candidate modulator increases the rate of motor neuron differentiation as measured by the presence of one or more feature characteristic of a motor neuron phenotype, wherein an increase in the rate of motor neuron differentiation indicates that the candidate modulator is an agonist of motor neuron differentiation.

6. The method of claim 5, where the phenotype comprises expression of the motor neuron associated protein HB9.

7. The method of claim 5, where the phenotype comprises expression of Green Fluorescent Protein, where expression of Green Fluorescent Protein is controlled by the HB9 promoter.