Method of inducing and maintaining neuronal cells

• US 6,686,198 B1
• Filed: 04/09/1997
• Issued: 02/03/2004
• Est. Priority Date: 10/14/1993
• Status: Expired due to Term

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Melton, Douglas A., Hemmati-Brivanlou, Ali
• Primary Examiner(s): Allen, Marianne P.
• Application Number:
  US08/835,279
• Time in Patent Office: 2,491 Days
• Field of Search: 435/172.1, 435/172.3, 435/6, 435/7.1, 435/325, 435/320.1, 435/375, 435/377, 536/23.5, 536/24.5, 530/324, 530/350, 514/2, 514/12, 514/44
• US Class Current: 435/377
• CPC Class Codes: A61K 2300/00 Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 38/22 Hormones derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin A61K38/33, e.g. corticotropin A61K38/35
• CPC Class Codes: A61K 38/57 from animals; from humans A61K38/553, A61K38/556 take precedence
• CPC Class Codes: C07K 14/46 from vertebrates
• CPC Class Codes: C07K 14/4702 Regulators; Modulating activity
• CPC Class Codes: C07K 14/71 for growth factors; for growth regulators

Abstract

The present invention makes available a method for inducing neuronal differentiation and preventing the death or degeneration of neuronal cells both in vitro and in vivo. The subject method stems from the unexpected finding that, contrary to traditional understanding of neural induction, the default fate of ectodermal tissue is neuronal rather than mesodermal and/or epidermal. In particular, it has been discovered that preventing or antagonizing a signaling pathway in a cell for a growth factor of the TGF-β family can result in neuronal differentiation of that cell.

Claims

1. A method for inducing a stem cell having activin receptors responsive to activin to differentiate to a neuronal cell phenotype, which stem cell is provided in a culture of two or more cells in vitro, comprising providing said cell with

a first agent that antagonizes the biological action of activin selected from follistatin, proteins that include at least one follistatin module, an α

2-macroglobulin, and an inhibin, and

a second agent which agent is a neurotrophic factor that enhances a particular differentiation fate of the cell, wherein said first agent and second agent are provided in amounts sufficient to induce differentiation of said cell to a neuronal cell phenotype.

2. The method of claim 1, wherein said first agent inhibits the biological activity of activin by preventing activin from binding activin receptors on the surface of said cell.

3. The method of claim 2, wherein said antagonizing agent binds activin and sequesters activin such that it cannot bind said activin receptors.

4. The method of claim 3, wherein said antagonizing agent is selected from follistatin, an α

2-macroglobulin, and a protein containing at least one follistatin module.

5. The method of claim 2, wherein said antagonizing agent inhibits binding of activin with said activin receptors via its own binding to said activin receptor.

6. The method of claim 5, wherein said antagonizing agent is an inhibin.

7. A method of claim 1, wherein said neuronal cell phenotype comprises a neural progenitor cell.

8. The method of claim 7, wherein said neural progenitor cell is selected from a melanocyte progenitor cell, a glial progenitor cell, a sensory neuron progenitor cell, a sympatho-adrenal progenitor cell, a parasympathetic progenitor cell, and an enteric progenitor cell.

9. The method of claim 1, wherein said stem cell is selected from an embryonic cell, a fetal cell, and a neonatal cell.

10. The method of claim 1, wherein said neuronal cell phenotype is a glial cell.

11. The method of claim 1, wherein said neuronal cell phenotype is a nerve cell.

12. The method of claim 1, wherein said cell is further contacted with an antimitotic agent in an amount sufficient to reduce the rate of growth of glial cells and favor the growth of nerve cells.

13. The method of claim 1, wherein the second agent is selected from ciliary neurotrophic growth factor, schwannoma-derived growth factor, glial growth factor, striatal-derived neuronotrophic factor, platelet-derived growth factor, scatter factor, a vertebrate hedgehog protein, noggin, and a ligand for a Notch receptor.

14. A method for enhancing survival of at least one neuronal cell having activin receptors responsive to activin, which neuronal cell is provided in a culture of two or more cells in vitro, comprising contacting said cell with an agent, selected from follistatin, proteins that include at least one follistatin module, an α

2-macroglobulin, and inhibin, that antagonizes the biological action of activin, in an amount sufficient to enhance survival of said cell.

15. The method of claim 14, wherein said cell is further contacted with a second agent which agent is a neurotrophic factor which enhances a particular differentiation fate of the cell.

16. The method of claim 15, wherein the second agent is selected from ciliary neurotrophic growth factor, schwannoma-derived growth factor, glial growth factor, striatal-derived neuronotrophic factor, platelet-derived growth factor, scatter factor, a vertebrate hedgehog protein, noggin, and a ligand for a Notch receptor.

17. The method of claim 14, wherein said cell is a stem cell.

18. A method for inducing at least one progenitor cell to differentiate to a neuronal cell phenotype, which progenitor cell is provided in a culture of two or more cells in vitro, comprising contacting said cell with an agent which disrupts an activin signaling pathway in said cell, wherein

said activin signaling pathway normally induces said cell to differentiate to a non-neuronal cell type,

said agent is selected from a soluble growth factor-binding domain of an activin receptor, follistatin, proteins that include at least one follistatin module, an α

2-macroglobulin, and inhibin, and

said agent is provided in an amount sufficient to induce differentiation of said cell to a neuronal cell phenotype.

19. A method for inducing at least one cell having activin receptors responsive to activin to differentiate to a neuronal cell phenotype, which neuronal cell is provided in a culture of two or more cells in vitro, comprising contacting said cell with an agent that antagonizes the biological action of activin, selected from follistatin, proteins that include at least one follistatin module, an α

2-macroglobulin, and inhibin, in an amount sufficient to induce neuronal differentiation, wherein said neuronal cell phenotype is selected from a melanocyte progenitor cell, a glial progenitor cell, a sensory neuron progenitor cell, a sympatho-adrenal progenitor cell, a parasympathetic progenitor cell, and an enteric progenitor cell.

20. The method of claim 19, wherein said cell is further contacted with a second agent which agent is a growth factor having neurotrophic or neural inductive activity.

21. The method of claim 19, wherein said cell is a stem cell.