Use of cyclic AMP and ascorbic acid to produce dopaminergic neurons from embryonic stem cells

• US 20050095707A1
• Filed: 12/10/2004
• Published: 05/05/2005
• Est. Priority Date: 05/17/2000
• Status: Active Grant

• Current Assignee: Asterias Biotherapeutics Incorporated (Lineage Cell Therapeutics, Inc.)
• Original Assignee: Malissa K. Carpenter, Thies R. Scott
• Inventors: Carpenter, Malissa K., Thies, R. Scott
• Granted Patent: US 7,763,463 B2
• Time in Patent Office: Days
• US Class Current: 435/368
• 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 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
• 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

This disclosure provides improved methods for obtaining populations of dopaminergic neurons from pluripotent stem cells. The process involves taking a population of neural precursor cells derived from a line of human embryonic stem cells, and culturing the cells in a medium that contains a neurotrophin, either cyclic adenosine monophosphate (cAMP) or a compound that elevates intracellular cAMP levels, and optionally an antioxidant such as ascorbic acid. Cell populations have been obtained that contain a high proportion of cells staining for tyrosine hydroxylase, which is a feature of 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 clinically important neurological disorders, such as Parkinson'"'"'s disease.

Claims

1. A method for making dopaminergic neuronal cells from human embryonic stem (hES) cells, comprising:

a) differentiating hES cells into a neural progenitor cell population in which at least 60% of the cells are Nestin positive;

b) culturing the Nestin positive progenitor cells in a culture medium comprising a neurotrophin, and either cyclic adenosine monophosphate (cAMP) or a compound that elevates intracellular cAMP levels; and

c) harvesting a cell population from the culture medium that comprises neuronal cells expressing tyrosine hydroxylase.

2. The method of claim 1, wherein the culture medium is free of added mitogens.

3. The method of claim 1, wherein the culture medium further comprises an antioxidant.

4. The method of claim 3, wherein the antioxidant is ascorbic acid.

5. The method of claim 1, wherein said neurotrophin in the culture medium is selected from neurotrophin 3 (NT-3), neurotrophin 4 (NT-4), brain-derived neurotrophic factor (BDNF), and nerve growth factor (NGF).

6. The method of claim 1, wherein the culture medium contains both BDNF and NT-3.

7. The method of claim 1, wherein the culture medium contains cAMP.

8. The method of claim 1, wherein the culture medium contains dibutyl cAMP.

9. The method of claim 1, wherein the culture medium contains a compound that elevates intracellular cAMP levels.

10. The method of claim 9, wherein the compound is selected from forskolin, cholera toxin, and isobutylmethylxanthine

11. The method of claim 1, wherein the hES cells are differentiated into said neural progenitor cell population by way of a process that comprises culturing the cells with retinoic acid.

12. The method of claim 1, wherein the hES cells are differentiated into said neural progenitor cell population by way of a process that comprises culturing the cells in a differentiation medium containing one or more neurotrophins and one or more mitogens.

13. The method of claim 12, wherein the differentiation medium contains both NT-3 and BDNF.

14. The method of claim 12, wherein the mitogen(s) are selected from epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), fibroblast growth factor 8 (FGF-8), platelet-derived growth factor (PDGF), and insulin-like growth factor 1 (IGF-1)

15. The method of claim 12, wherein the differentiation medium contains erythropoietin (EPO).

16. The method of claim 1, wherein the hES cells are differentiated into said neural progenitor cell population by way of a process that comprises culturing the cells in a differentiation medium containing sonic hedgehog (SHH) and one or more mitogens.

17. The method of claim 1, wherein the hES cells are differentiated into said neural progenitor cell population by way of a process that comprises culturing the cells in a differentiation medium containing one or more neurotrophins and one or more TGF-β

Superfamily Antagonists.

18. The method of claim 17, wherein the TGF-β

Superfamily Antagonist(s) are selected from noggin and follistatin.

19. The method of claim 17, wherein the differentiation medium further contains a mitogen.

20. The method of claim 1, wherein at least 5% of the cells in the harvested cell population express tyrosine hydroxylase.

21. A method for making dopaminergic neuronal cells from human embryonic stem (hES) cells, comprising:

a) culturing a neural progenitor cell population derived from an hES cell line in which at least 25% of the cells are MAP-2 positive in a culture medium comprising a neurotrophin, and either cyclic adenosine monophosphate (cAMP) or a compound that elevates intracellular cAMP levels; and

b) harvesting a cell population from the culture medium that comprises neuronal cells expressing tyrosine hydroxylase.

22. The method of claim 21, wherein the culture medium further comprises ascorbic acid.

23. A method for making dopaminergic neuronal cells from human embryonic stem (hES) cells, comprising:

a) culturing hES cells in a differentiation medium containing at least one mitogen and a factor selected from neurotrophins, sonic hedge hog, and TGF-β

Superfamily Antagonists;

b) culturing the cells obtained from step a) in a culture medium comprising a neurotrophin, either cyclic adenosine monophosphate (cAMP) or a compound that elevates intracellular cAMP levels, and ascorbic acid; and

c) harvesting a cell population from the culture medium that comprises neuronal cells expressing tyrosine hydroxylase.

24. A method for making dopaminergic neuronal cells from human embryonic stem (hES) cells, comprising:

a) culturing hES cells in a differentiation medium containing at least one mitogen and at least one neurotrophin;

b) culturing the cells obtained from step a) in a culture medium comprising a neurotrophin, cyclic adenosine monophosphate (cAMP), and ascorbic acid; and

c) harvesting a cell population from the culture medium that comprises neuronal cells expressing tyrosine hydroxylase.