Cultures of GFAP + nestin + cells that differentiate to neurons

• US 8,501,467 B2
• Filed: 12/23/2009
• Issued: 08/06/2013
• Est. Priority Date: 10/25/1999
• Status: Active Grant

• Current Assignee: BOCO Silicon Valley, Inc.
• Original Assignee: Stemcells California Incorporated (Microbot Medical, Inc.)
• Inventors: Wahlberg, Lars, Campbell, Kenneth, Skogh, Charlotta, Eriksson, Cecilia, Wictorin, Klas
• Primary Examiner(s): DAVIS, RUTH A
• Application Number:
  US12/646,557
• Publication Number:
  US 20110003358A1
• Time in Patent Office: 1,322 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. A suspension culture of GFAP⁺ nestin⁺ cells or GFAP⁺ RC2⁺ cells, wherein said cells are substantially formed into cell aggregates, and wherein the cell aggregates are maintained in a culture medium containing a proliferation-inducing growth factor.

2. The culture of claim 1, wherein the aggregated cells differentiate into neurons upon withdrawal of the proliferation-inducing growth factor.

3. The 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.

4. The culture of claim 2, wherein the aggregated cells differentiate into neurons when plated on an adherent substrate.

5. The culture of claim 1, wherein the proliferation-inducing growth factor is epidermal growth factor (EGF) and/or basic fibroblast growth factor (FGF-2).

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

7. The culture of claim 1, wherein the cells are murine.

8. The culture of claim 1, wherein the cells are human.

9. The 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.

10. The culture of claim 1, wherein at least about 75% of cells are immunoreactive to GFAP⁺ and nestin⁺.

11. The culture of claim 1, wherein at least about 90% of cells are immunoreactive to GFAP⁺ and nestin⁺.

12. The culture of claim 1, wherein the culture medium further contains a serum.

13. A composition comprising GFAP⁺ nestin⁺ cells or GFAP⁺ RC2⁺ cells, wherein said cells are substantially formed into cell aggregates.

14. The composition of claim 13, wherein the cell aggregates are maintained in a culture medium containing a proliferation-inducing growth factor.

15. The composition of claim 14, wherein the aggregated cells differentiate into neurons upon withdrawal of the proliferation-inducing growth factor.

16. The composition of claim 15, wherein the aggregated cells differentiate into neurons when plated on an adherent substrate.

17. The composition of claim 14, wherein the culture medium further contains a serum.

18. The composition of claim 13, 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.

19. The composition of claim 13, wherein the proliferation-inducing growth factor is epidermal growth factor (EGF) and/or basic fibroblast growth factor (FGF-2).

20. The composition of claim 13, wherein the cells are derived from the lateral ganglionic eminence (LGE) or medial ganglionic eminence (MGE) of a mammal.

21. The composition of claim 13, wherein the cells are murine.

22. The composition of claim 13, wherein the cells are human.

23. The composition of claim 13, 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.

24. The composition of claim 13, wherein at least about 75% of cells are immunoreactive to GFAP⁺ and nestin⁺ or GFAP⁺ and RC2⁺.

25. The composition of claim 13, wherein at least about 90% of cells are immunoreactive to GFAP⁺ and nestin⁺ or GFAP⁺ and RC2⁺.