Cultures of GFAP+ nestin+ cells that differentiate to neurons

• US 7,651,853 B2
• Filed: 10/31/2007
• Issued: 01/26/2010
• Est. Priority Date: 10/25/1999
• Status: Expired due to Term

• Current Assignee: BOCO Silicon Valley, Inc.
• Original Assignee: NsGene A/S
• Inventors: Eriksson, Cecilia, Wictorin, Klas, Campbell, Kenneth, Skogh, Charlotta, Wahlberg, Lars
• Primary Examiner(s): Davis; Ruth A
• Application Number:
  US11/932,349
• Publication Number:
  US 20080138895A1
• Time in Patent Office: 818 Days
• Field of Search: 435/325, 435/366, 435/368, 435/378
• US Class Current: 435/325
• 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 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• 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

Abstract

Cultures of cells immunoreactive for glial fibrillary acidic protein (GFAP), as well as for the intermediate filament marker nestin were grown in a medium including epidermal growth factor (EGF) and serum. The cultured cells had the morphology of astroglial cells. The cells can be proliferated in adherent or suspension cultures. Depending on the culture conditions, the cells can be induced to differentiate to neurons or glial cells. The cultures can be expanded over a large number of passages during several months, and survive, express an astroglial phenotype and integrate well after transplantation into both neonatal and adult rat forebrain.

Claims

1. An in vitro cell culture of RC-2⁺ cells, wherein

a) one or more cells in the culture have the capacity to differentiate into neurons;

b) the cell culture divides in a culture medium containing at least one proliferation-inducing growth factor; and

c) one or more cells in the culture differentiate into neurons upon withdrawal of the proliferation-inducing growth factor.

2. The cell culture of claim 1, wherein the cells are RC2⁺ nestin⁺ cells.

3. The cell culture of claim 1, wherein the cells are derived from the central nervous system of a mammal.

4. The cell culture of claim 1, wherein the cell culture differentiates into at least 10% neurons under differentiation-inducing culture conditions.

5. The cell culture of claim 1, wherein, under differentiation-inducing culture conditions, the majority of differentiated neuronal cells have a GABA-ergic phenotype.

6. The cell culture of claim 1, wherein the culture is capable of at least 6 doublings.

7. The cell culture of claim 1, wherein the cells are derived from the lateral ganglionic eminence (LGE) or medial ganglionic eminence (MGE) of a mammal.

8. The cell culture of claim 1, wherein the doubling rate of the culture is faster than seven days.

9. The cell culture of claim 1, wherein the cells in the culture are murine.

10. The cell culture of claim 1, wherein the cells in the culture are human.

11. The cell culture of claim 1, wherein fewer than 5% of the cells in the culture are β

-tubulin III immunoreactive (β

-tubulin III⁺) under proliferation-promoting culture conditions and between 10-40% of the cells in the culture are β

-tubulin III immunoreactive (β

-tubulin III⁺) under differentiation-inducing culture conditions.

12. The cell culture of claim 11, wherein the glia are both RC-2⁺ and vimentin positive.

13. The cell culture of claim 11, wherein the morphology of the glia is:

(a) bipolar;

(b) elongated; and

(c) non-fibrillary.

14. The cell culture of claim 1, wherein the proliferation-inducing growth factor is selected from the group consisting of epidermal growth factor, amphiregulin, basic fibroblast growth factor, acidic fibroblast growth factor, transforming growth factor alpha, leukemia inhibitor factor, ciliary neurotrophic factor and combinations thereof.

15. The cell culture of claim 1, wherein one or more of the cells in culture differentiate into glia in the absence of the proliferation-inducing growth factor from the culture medium.

16. The cell culture of claim 1, wherein one or more of the cells in culture, under differentiation-inducing culture conditions, differentiate into neurons that exhibit:

(a) axon-dendrite polarity,

(b) synaptic terminals, and

(c) localization of proteins involved in synaptogenesis and synaptic activity.

17. The culture of claim 1 wherein the majority of differentiated neuronal cells are immunoreactive with striatal neuronal markers.

18. The culture of claim 1 wherein the majority of differentiated neuronal cells are not immunoreactive with cortical neuronal markers.

19. The culture of claim 1 wherein the majority of differentiated neuronal cells are not immunoreactive with neuronal markers of the medial ganglionic eminence.

20. The cell culture of claim 1, wherein the majority of cells are immunoreactive to RC-2 and nestin.

21. The cell culture of claim 20, wherein the RC-2⁺ nestin⁺ cells show glial morphology.

22. The cell culture of claim 1, wherein at least about 75% of cells are immunoreactive to RC-2 and nestin.

23. The cell culture of claim 22, wherein the RC-2⁺ nestin⁺ cells show glial morphology.

24. The cell culture of claim 1, wherein at least about 90% of cells are immunoreactive to RC-2 and nestin.

25. The cell culture of claim 24, wherein the RC-2⁺ nestin⁺ cells show glial morphology.

26. An in vitro adhesion cell culture of GFAP⁺ cells, wherein

a. one or more cells in the culture have the capacity to differentiate into neurons;

b. the cell culture divides in a culture medium containing serum and at least one proliferation-inducing growth factor; and

c. one or more cells in the culture differentiate into neurons upon withdrawal of both serum and the proliferation inducing growth factor.

27. The cell culture of claim 26, herein the majority of cells in the culture are nestin⁺ under proliferation-promoting culture conditions.