Making neural cells for human therapy or drug screening from human embryonic stem cells

• US 6,833,269 B2
• Filed: 05/31/2001
• Issued: 12/21/2004
• 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: Carpenter, Melissa K.
• Primary Examiner(s): FALK, ANNE MARIE
• Application Number:
  US09/872,183
• Publication Number:
  US 20020039724A1
• Time in Patent Office: 1,300 Days
• Field of Search: 435/325, 435/366, 435/368, 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 25/08 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 for producing a neural cell population from human embryonic stem (hES) cells, comprising culturing hES cells in a medium containing one or more added neurotrophins and one or more added nitrogens, thereby producing a population in which at least 60% of the cells express A2B5, polyslalylated NCAM, MAP-2, or Nestin.

2. The method of claim 1, wherein the added neurotrophins include neurotrophin 3 (NT-3) or brain-derived neurotrophic factor (BDNF).

3. The method of claim 2, wherein the added neurotrophins include both NT-3 and BDNF.

4. The method at claim 1, wherein the added mitogens 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).

5. The method of claim 4, wherein the added mitogens include both PDGF and IGF-1.

6. The method of claim 1, wherein the hES cells are cultured on fibronectin.

7. The method of claim 1, comprising selecting and propagating cells that are positive for polyslalylated NCAM.

8. The method of claim 1, wherein the produced cell population is at least 60% positive for polystalylated NCAM.

9. The method of claim 1, wherein the produced cell population is at least 90% positive for polystalylated NCAM.

10. The method of claim 1, wherein the produced cell population is at least 38% β

-tubulin III positive.

11. The method of claim 1, wherein the produced cell population has the characteristic that culturing it in the presence of neurotrophins maintains capacity of the cells to proliferate and to form tyrosine hydroxylase positive neurons upon differentiation.

12. The method of claim 1, wherein the produced cell population is essentially free of undifferentiated hES cells.

13. The method of claim 1, further comprising differentiating the cells into a population that contains at least 30% neurons.

14. The method of claim 1, further comprising differentiating the cells into a population that contains at least 30% MAP-2 positive cells.

15. The method of claim 14, wherein at least 3% of the differentiated cells positive for MAP-2 are also positive for tyrosine hydroxylase.

16. The method of claim 14, wherein at least 8% of the differentiated cells positive for MAP-2 are also positive for tyrosine hydroxylase.

17. The method of claim 14, wherein the differentiating comprises culturing the cells in a medium containing one or more factors selected from neurotrophins, cAMP, and ascorbic acid for at least 3 days, in the absence of added mitogens.

18. The method of claim 1, wherein the differentiated cell population comprises sensory or motor neurons.

19. The method of claim 1, wherein the differentiated cell population comprises oligodendrocytes or astrocytes.

20. The method of claim 1, further comprising combining the produced cell population with a pharmaceutically compatible excipient.

21. A method for producing a neural cell population from human embryonic stem (hES) cells, comprising initialing differentiation of the hES cells into a heterogeneous population of precursor cells, then culturing the initiated cells in a medium containing one or more added neurotrophins and one or more added mitogens, thereby producing a population in which at least 60% of the cells express A2B5, polysialylated NCAM, MAP-2, or Nestin.

22. The method of claim 21, wherein the added neurotrophins include neurotrophin 3 (NT-3) or brain-derived neurotrophic factor (BDNF).

23. The method of claim 22, wherein the added neurotrophins include both NT-3 and BDNF.

24. The method of claim 21, wherein the added mitogens 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).

25. The method of claim 24, wherein the added mitogens include both PDGF and IGF-1.

26. The method of claim 21, wherein the precursor cells are cultured on fibronectin.

27. The method of claim 21, wherein differentiation is initiated by culturing the hES cells in a medium containing retinoic acid before culturing with the neurotrophins and mitogens.

28. The method of claim 21, wherein differentiation is initiated by culturing the cells as embryold bodies before culturing with the neurotrophins and mitogens.

29. The method of claim 21, wherein differentiation is initiated by culturing the cells as cell aggregates before culturing with the neurotrophins and mitogens.

30. The method of claim 21, comprising selecting and propagating cells that are positive for polysialylated NCAM.

31. The method of claim 21, wherein the produced cell population is at least 60% positive for polysialytated NCAM.

32. The method of claim 21, wherein the produced cell population is at least 90% positive for polysialylated NCAM.

33. The method of claim 21, wherein the produced cell population is at least 38% β

-tubulin III positive.

34. The method of claim 21, wherein the produced cell population has the characteristic that culturing it in the presence of neurotrophins maintains capacity of the cells to proliferate and to form tyrosine hydroxylase positive neurons upon differentiation.

35. The method of claim 21, wherein the produced cell population is essentially free of undifferentiated hES cells.

36. The method of claim 21, further comprising differentiating the cells into a population that contains at least 30% neurons.

37. The method of claim 21, further comprising differentiating the cells into a population that contains at least 30% MAP-2 positive cells.

38. The method of claim 37, wherein at least 3% of the differentiated cells positive for MAP-2 are also positive for tyrosine hydroxylase.

39. The method of claim 37, wherein at least 8% of the differentiated cells positive for MAP-2 are also positive for tyrosine hydroxylase.

40. The method of claim 37, wherein the differentiating comprises culturing the cell in a medium containing one or more factors selected from neurotrophins, cAMP, and ascorbic acid for at least 3 days, in the absence of added mitogens.

41. The method of claim 21, wherein the differentiated cell population comprises sensory or motor neurons.

42. The method of claim 21, wherein the differentiated cell population comprises oligodendrocytes or astrocytes.

43. The method of claim 21, further comprising combining the produced cell population with a pharmaceutically compatible excipient.