Neural colony forming assay
A neural colony forming cell (NCFC) assay is described. The assay allows one to distinguish neural stem cells from neural progenitor cells. In one embodiment, the present invention provides a method for identifying neural stem cells or neural progenitor cells comprising: (a) suspending neural cells in a semi-solid medium which supports the growth of neural cells; (b) plating the cells in the semi-solid medium at a density that allows for the production of colonies; (c) culturing the plated cells until size differences can be discerned between the colonies; and (d) estimating colony size wherein the larger colonies are likely produced by neural stem cells and wherein the small colonies are likely produced by neural progenitor cells In alternate embodiments, NSC can be distinguished from neural progenitor cells by determining the morphology or antigen expression of the colonies.
1. A method for identifying neural stem cells or neural progenitor cells comprising:
(a) suspending neural cells in a semi-solid medium which supports the growth of neural cells;
(b) plating the cells in the semi-solid medium at a cell density that allows for the production of colonies;
(c) culturing the plated cells until size differences can be discerned between the colonies;
(d) estimating colony size; and
(e) identiying neural stem cells or neural progenitor cells based on the colony size wherein the larger colonies are produced by neural stem cells and wherein the smaller colonies are produced by neural progenitor cells.
2. A method according claim 1 wherein the neural cells are mammalian.
3. A method according to claim 2 wherein the neural cells are from human, rat or mouse.
4. A method according to claim 1 wherein the neural cells are from primary CNS tissue or cultured neurospheres.
5. A method according to claim 1 wherein the semi-solid medium is collagen based.
6. A method according to claim 1 wherein the semi-solid medium is methylcellulose based.
7. A method according to claim 1 wherein the neural cells are diluted in a culture medium prior to step (a).
8. A method according to claim 7 wherein epidermal growth factor (EGF) is added to the medium.
9. A method according to claim 7 wherein basic fibroblast growth factor (bFGF) is added to the medium.
10. A method according to claim 9 wherein the culture medium is serum free.
11. A method according to clam 1 wherein the cells are plated in step (b) at a density of about 1000 to 25,000 cells per 35 mm culture dish.
12. A method according to clam 1 wherein the cells are cultured in step (c) for about 10 to about 28 days.
13. A method according to claim 1 wherein in step (d) a colony size of greater than 2.0 mm indicates neural stem cells.
14. A method according to claim 1 wherein in step (d) a colony size of less than or equal to 2.0 mm indicates neural progenitor cells.