Primitive neural stem cells and method for differentiation of stem cells to neural cells

• US 20020164791A1
• Filed: 09/28/2001
• Published: 11/07/2002
• Est. Priority Date: 09/29/2000
• Status: Active Grant

• Current Assignee: Derek Van Der Kooy, Vincent Tropepe
• Original Assignee: Derek Van Der Kooy, Vincent Tropepe
• Inventors: Tropepe, Vincent, Van Der Kooy, Derek
• Granted Patent: US 7,211,434 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• 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: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• 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: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2503/00 Use of cells in diagnostics
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0623 Stem cells

Abstract

Described are a novel cell type in the neural lineage, and method of producing the same based on the degree of neural commitment and growth factor responsiveness in vitro and the potential to give rise to neural and non-neural progeny in vivo. The novel veil type of neural lineage and cells derived therefrom have a number of applications including applications regarding tissue engineering, transplantation and gene therapy and drug discovery. Also described are suggested uses of the method and cell type including isolating genes that positively and negatively regulate the transmission from an ES cell to a neural cell and generally for studying ES cell models of mammalian neural development.

Claims

20. A method for producing secondary neural stem cell colonies comprising:

(a) culturing ES cells in low cell density completely defined serum-free media for a time and under conditions sufficient to differentiate the said ES cells;

(b) dissociating and subcloning primary neural cell colonies generated from the said ES cells; and

(c) administering a growth factor to the dissociated neural cells.

21. A method according to claim 20 wherein the growth factor is selected from among the members of the fibroblast growth factor (FGF) family of growth factors.

22. A method according to claim 21 wherein the growth factor is FGF2.

23. A method according to claim 20 wherein a cytokine is administered to the dissociated neural cells.

24. A method according to claim 23 wherein the cytokine is LIF of B27.

26. A cell according to claim 25 wherein the neural precusor marker nestin is expressed.

27. A cell according to claim 25 or 26 wherein the neural-specific mRNA molecule is Fmx2 or HoxB1

37. A method for screening for differentiation factors of cellular development comprising:

(a) culturing the cells of claim 29 in serum free media, in the present or the differentiation factor.

(b) detecting any differentiation of the cells.

43. A method for treating neurodegenerative disorders comprising administering to a patient in need thereof the cells of claim 29.

44. A method for the treatment of any disease or conditions resulting from cell loss or function in the neural system comprising administering the cells of claim 29 to a patient in need thereof.

45. A method of gene therapy, wherein the cell of claim 29 is modified to express a gene of interest and administering said modified cell to a patient in need thereof.

25. One or more cell(s) expressing one or more neural precursor cell marker(s) and/or one or more neural-specific mRNA molecule(s), and having multilineage potential.

30. A primitive neural stem cell produced comprising at least one neural cell marker and is pluripotent.

31. A method of producing a pre-selected cell type derived form a cell of claim 30 comprising, culturing the cells under differentiating conditions that promote formation of the cell type.

32. The method of claim 31 wherein the pre-selected cell type is a neural cell, and the differentiating conditions comprise culturing the cell in a serum free media that comprises FGF2.

33. A method for screening for modulators of cellular differentiation comprising

(a) culturing pluripotent cells in serum-free media under low density conditions in the presence of the potential modulator;

(b) allowing for differentiation of the cells;

(c) detecting any differentiation of the cells and cell types generated, if any.

1. A method for differentiating one or more pluripotent embryonic stem (ES) cells toward one or more neural cells comprising:

(a) culturing the ES cells at low density in the serum-free media; and

(b) allowing said ES cells to differentiate toward the neural cells.

2. The method according to claim 1 for differentiating embryonic stem cells to cells with markers characteristic of neural cells comprising:

(a) culturing the embryonic stem cells in a serum free media at low cell density wherein said density is selected to minimize ES cell aggregation of EB formation;

(b) allowing said cells to differentiate.

3. The method of claim 2 wherein the density is selected as to avoid EB formation.

4. The method of claim 1 wherein said cell density is greater than 0 cells/μ

l to 50 cells/μ

l.

5. The method of claim 4 wherein the cell density is greater than 0 cells/μ

l to 20 cells/μ

l.

6. The method of claim 5 wherein the cell density is greater than 0 cells/μ

l to 10 cells/μ

l.

7. The method of claim 6 wherein the cell density is 10 cells/μ

l.

8. The method of claim 6 wherein there is no EB formation.

9. The method of claim 7 wherein the differentiating ES cells form at least one neuro sphere.

10. The method of claim 1 wherein the differentiating ES cells form at least one neurosphere.

11. The method of claim 1 wherein the serum free media further comprises a cytokine.

12. The method of claim 11 wherein the cytokine is leukemia inhibitory factor (LIF).

13. The method of claim 12 wherein the ES cells differentiate into a primitive neural stem cell, that is pluripotent.

14. The method of claim 1 and 12 wherein the serum free media further comprises a growth factor.

15. The method of claim 14 wherein the growth factor is selected from the members of the fibroblast growth factor (FCF) family of growth factors.

16. The method of claim 15 wherein the growth factor is FGF2.

17. The method according to claim 1 wherein the media comprises an inhibitor of TGF-β

-related signaling.

18. The method of claim 17, wherein the inhibitor is the protein Noggin.

19. The method of claim 18 wherein the inhibitor is selected from the Cerebus family of proteins.

28. A method according to anyone of claims 1 or 12 for analyzing the role of genes in the regulation of neural fate specification.

29. A primitive neural stem cell produced by the method of claim 12 that comprises neural cell markers and is pluripotent.

34. A method in accordance with claim 33 wherein the modulators comprise any culturing conditions that may modulate cellular differentiation.

36. A method of claim 35 for screening for modulators or differentiation factors of neural cell development.

38. The method of claim 37, wherein the media further comprises FGF2.

39. A method according to claim 38 for modulating cellular differentiation.

40. The method of claim 1 for obtaining a homogenous uniform cell base.

41. The method of claim 40 wherein the cell base is a neural cell base.

42. A method for supplying cells for transplantation comprising culturing cells pursuant to the method of claim 1 or 12.

35. A method for screening for differentiation factors of cellular development comprising:

(a) culturing the cells in serum free media at low cell density in the presence of the differentiation factor;

(b) allowing cells to differentiate;

(c) detecting differentiation of the cells, if any.

38-1. A modulator or differentiation factor detected by the methods of claims 33-37.