Screening small molecule drugs using neural cells differentiated from human embryonic stem cells

• US 7,250,294 B2
• Filed: 05/28/2002
• Issued: 07/31/2007
• Est. Priority Date: 05/17/2000
• Status: Expired due to Term

• Current Assignee: Asterias Biotherapeutics Incorporated (Lineage Cell Therapeutics, Inc.)
• Original Assignee: Geron Corporation
• Inventors: Inokuma, Margaret S., Thies, R. Scott, Carpenter, Melissa K., Denham, Jerrod J.
• Primary Examiner(s): FALK, ANNE MARIE
• Application Number:
  US10/157,288
• Publication Number:
  US 20030103949A1
• Time in Patent Office: 1,890 Days
• Field of Search: 435/325, 435/363, 435/366, 435/368, 435/375, 435/377
• 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: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/02 for peripheral neuropathies
• CPC Class Codes: A61P 25/08 Antiepileptics; Anticonvulsants
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• CPC Class Codes: A61P 25/24 Antidepressants
• 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 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12N 2501/01 Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• 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 2501/135 Platelet-derived growth factor [PDGF]
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2502/99 genetically modified cells
• CPC Class Codes: C12N 2503/02 : Drug screening
• CPC Class Codes: C12N 2506/02 : from embryonic cells
• CPC Class Codes: C12N 2510/00 : Genetically modified cells
• CPC Class Codes: C12N 2510/04 : Immortalised 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 5/0618 : Cells of the nervous system
• CPC Class Codes: C12N 5/0619 : Neurons
• CPC Class Codes: C12N 5/0622 : Glial cells, e.g. astrocytes, oligodendrocytes; Schwann cells
• CPC Class Codes: C12N 5/0623 : Stem cells
• CPC Class Codes: C12Q 1/6881 : for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes

Abstract

This invention provides populations of neural progenitor cells and differentiated neurons, obtained by culturing pluripotent cells in special growth cocktails. The technology can be used to produce progenitors that proliferate through at least ˜40 doublings, while maintaining the ability to differentiate into a variety of different neural phenotypes, including dopaminergic neurons. The neural progenitors and terminally differentiated neurons of this invention can be generated in large quantities for use in drug screening and the treatment of neurological disorders.

Claims

1. A method of screening a compound for its effect on neural cells or a neural cell activity, comprising:

a) obtaining from an established line of primate pluripotent stem (pPS) cells a population of cells wherein at least 75% of the cells are characterized as cells of the neuronal lineage by the criterion that they express polysialylated NCAM;

b) combining the cell population with the compound;

c) determining any change to phenotype or activity of cells in the population that results from being combined with the compound; and

d) correlating the change with an effect of the compound on neural cells or a neural cell activity.

2. The method of claim 1, comprising determining whether the compound is toxic to cells in the population.

3. The method of claim 1, comprising determining whether the compound affects ability of cells in the population to be maintained in culture.

4. The method of claim 1, comprising determining whether the compound changes neurotransmitter synthesis, release, or uptake by cells in the population.

5. The method of claim 1, comprising determining whether the compound changes electrophysiology of cells in the population.

6. The method of claim 1, wherein the pPS cells are human embryonic stem cells.

7. The method of claim 1, wherein the cell population was produced by a process that comprised culturing progeny of the pPS cells in a medium containing one or more added TGF-β

superfamily antagonists.

8. The method of claim 7, wherein process comprised initiating differentiation of the pPS cells by plating onto a solid surface without forming embryoid bodies or cell aggregates.

9. The method of claim 7, wherein the process comprised culturing progeny of the pPS cells in a medium containing both noggin and follistatin.

10. The method of claim 1, wherein the cell population was produced by culturing progeny of the pPS cells in a medium containing one or more added mitogens and one or more added neurotrophins.

11. The method of claim 10, wherein the cell population was produced by pre-differentiating pPS cells by forming embryoid bodies before culturing with the neurotrophins and mitogens.

12. The method of claim 10, wherein the cell population was produced by culturing the cells with one or more added mitogens before culturing with the neurotrophins.

13. The method of claim 10, wherein the added mitogen(s) include a mitogen selected from epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and insulin-like growth factor 1 (IGF-1), and the added neurotrophins include neurotrophin 3 (NT-3) or brain-derived neurotrophic factor (BDNF).

14. The method of claim 10, wherein the added mitogen(s) include erythropoietin (EPO).

15. The method of claim 10, wherein the cell population was produced by passaging the cells at least 6 times in a medium comprising an added neurotrophin and an added mitogen.

16. The method of claim 1, wherein the cell population has been terminally differentiated by culturing progeny of the pPS cells in a medium containing one or more factors selected from neurotrophins, cAMP, and ascorbic acid in the absence of added mitogens.