Neural progenitor cell populations

• US 9,803,174 B2
• Filed: 07/25/2012
• Issued: 10/31/2017
• Est. Priority Date: 05/17/2000
• Status: Expired due to Fees

• Current Assignee: Asterias Biotherapeutics Incorporated (Lineage Cell Therapeutics, Inc.)
• Original Assignee: Asterias Biotherapeutics Incorporated (Lineage Cell Therapeutics, Inc.)
• Inventors: Carpenter, Melissa K.
• Primary Examiner(s): WEHBE, ANNE MARIE SABRINA
• Application Number:
  US13/558,078
• Publication Number:
  US 20120308534A1
• Time in Patent Office: 1,924 Days
• Field of Search: 424 937, 435325, 435366, 435377, 435375, 435405
• 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 25/08 Antiepileptics; Anticonvulsants
• CPC Class Codes: A61P 25/14 for treating abnormal movements, e.g. chorea, dyskinesia
• 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 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/08 Vasodilators for multiple indications
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• 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 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 5/0618 : Cells of the nervous system
• 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
• CPC Class Codes: C12Q 2600/158 : Expression markers

Abstract

This invention provides populations of neural progenitor cells, differentiated neurons, glial cells, and astrocytes. The populations are obtained by culturing stem cell populations (such as embryonic stem cells) in a cocktail of growth conditions that initiates differentiation, and establishes the neural progenitor population. The progenitors can be further differentiated in culture into a variety of different neural phenotypes, including dopaminergic neurons. The differentiated cell populations or the neural progenitors can be generated in large quantities for use in drug screening and the treatment of neurological disorders.

Claims

1. A method of producing a culture comprising dopaminergic neurons, comprising:

a) initiating differentiation of human embryonic stem (hES) cells;

b) culturing the initiated cells from a) in a medium comprising basic fibroblast growth factor (bFGF), optionally further comprising one or more factors selected from neurotrophin-3 (NT-3), brain-derived neurotrophic factor (BDNF), sonic hedgehog (SHH) and fibroblast growth factor 8(FGF8) for a first culture period; and

c) culturing the cells from b) in a medium comprising neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF), or alternatively comprising cyclic adenosine monophosphate (cAMP) and ascorbic acid for a second culture period sufficient to produce a culture comprising dopaminergic neurons.

2. The method of claim 1, wherein the hES cells express SSEA3, SSEA4, Tra-1-60, and Tra-1-81.

3. The method of claim 1, wherein the first culture period is for about six days.

4. The method of claim 1, wherein the medium used for the second culture period comprises NT-3 and BDNF.

5. The method of claim 1, wherein the medium used for the second culture period comprises cAMP and ascorbic acid.

6. The method of claim 1, wherein the medium used for the second culture period does not comprise an added mitogen.

7. The method of claim 1, wherein the second culture period is for about six days.

8. The method of claim 1, wherein the culture comprising dopaminergic neurons comprises MAP-2 positive cells.

9. The method of claim 8, wherein the culture comprising dopaminergic neurons comprises at least 21% MAP-2 positive cells.

10. The method of claim 9, wherein the culture comprising dopaminergic neurons comprises from 21% to 37% MAP-2 positive cells.

11. The method of claim 8, wherein the MAP-2 positive cells comprise TH positive cells.

12. The method of claim 11, wherein at least 3.5% of the MAP-2 positive cells are TH positive.

13. The method of claim 12, wherein from 3.5% to 8.7% of the MAP-2 positive cells are TH positive.