Enriched central nervous system stem cell and progenitor cell populations, and methods for identifying, isolating and enriching for such populations
View Patent ↗Enriched neural stem and progenitor cell populations, and methods for identifying, isolating and enriching for neural stem cells using reagent that bind to cell surface markers, are provided.
1. A method of producing a population enriched for human central nervous system stem cells (CNS-SC), which can initiate neurospheres (NS-IC), comprising:
a) contacting neural or neural derived cells with monoclonal antibody 8G1;
b) selecting said neural or neural-derived cells that bind to monoclonal antibody 8G1; and
c) removing said bound cells wherein the remaining cells in the population are enriched for human CNS-SC,
the method further comprising the steps of further enriching a population for CNS-SC by
d) contacting the remaining cells with a second monoclonal antibody selected from the group consisting of AC133 and 5E12, and
e) selecting those cells that bind to said second monoclonal antibody to produce a population enriched for CNS-SC.
2. The method of claim 1 , wherein the monoclonal antibodies are fluorochrome conjugated.
3. The method of claim 1 , wherein the monoclonal antibodies are conjugated to magnetic particles.
4. The method of claim 1 , wherein the selecting is by flow cytometry.
5. The method of claim 1 , wherein the selecting is by fluorescence activated cell sorting or high gradient magnetic selection.
6. The method of claim 1 , wherein the selecting is by attachment to and disattachment from the solid phase.
7. The method of claim 1 , wherein the population containing neural or neural-derived cells is obtained from a neurosphere culture or an adherent culture.
8. The method of claim 1 , wherein the population containing neural or neural-derived cells is obtained from neural tissue.
9. A method for producing a population enriched for human central nervous system stem cells (CNS-SC) which can initiate neurospheres (NS-IC) comprising eliminating from a population of neural or neural-derived cells those cells that are CD24 +, the method further comprising the step of further enriching the population for CNS-SC by selecting from the remaining population of neural or neural-derived cells for cells that bind to monoclonal antibody AC133.
10. A method for producing a population enriched for human central nervous system stem cells (CNS-SC) which can initiate neurospheres (NS-IC) comprising eliminating from a population of neural or neural-derived cells those cells that are CD24 +, the method further comprising the step of further enriching the population for CNS-SC by selecting from the remaining population of neural or neural-derived cells for cells that bind to monoclonal antibody 5E12.
11. The method of claim 9 , further comprising the step of further enriching the population by selecting for cells that bind to monoclonal antibody 5E12.
12. The method of claim 10 , further comprising the step of further enriching the population by selecting for cells that bind to monoclonal antibody AC133.
13. A method for enriching from a population of neural cells for the populations of neurosphere initiating stem cell (NS-IC) fraction, comprising selecting from the neural cells for cells that bind to monoclonal antibody 8G1 and removing said cells from said population, wherein the remaining cells are enriched in the fraction of NS-IC as compared with the population of neural cells, the method further comprising the step of further enriching for the fraction of NS-IC by selecting from the remaining cells for cells that bind to monoclonal antibody AC133 or to monoclonal antibody 5E12.
14. A method for isolating a neurosphere initiating stem cell (NS-IC), comprising:
a) selecting from a population of neural or neural-derived cells for cells that are CD24 + ;
b) eliminating said CD24 + cells from the population;
c) selecting from the remaining population for at least one cell that binds to monoclonal antibody AC133 or to monoclonal antibody 5E12;
d) introducing at least one selected cell to a serum-free culture medium containing one or more growth factors selected from the group consisting of LIF, EGF, bFGF, and combinations thereof; and
e) proliferating at least one selected cell in the culture medium.
15. A method for producing a population enriched for human central nervous system stem cells (CNS-SC) which can initiate neurospheres (NS-IC) comprising selecting from a population of neural or neural-derived cells for cells that bind to monoclonal antibody AC133.
16. A method for producing a population enriched for human central nervous system stem cells (CNS-SC) which can initiate neurospheres (NS-IC) comprising selecting from neural or neural-derived cells for cells that bind to monoclonal antibody AC133 or to monoclonal antibody 5E12, to produce a population enriched for CNS-SC, wherein the selecting is by attachment to and disattachment from solid phase.
17. A method for the enrichment of human central nervous system neural stem cells (CNS-SC) which can initiate neurospheres (NS-IC), said method comprising:
a) combining a population of neural or neural-derived cells with an antibody that binds to the CD24 antigen; and
b) selecting for those cells that do not bind to the CD24 antigen, wherein the selected cells are enriched for CNS-SC,
the method further comprising the step of further enriching the population for CNS-SC by selecting from the remaining population of neural or neural-derived cells for cells that bind to monoclonal antibody AC133 or to monoclonal antibody 5E12.
18. The method according to claim 17 , wherein at least one of said antibodies is fluorochrome conjugated.
19. The method according to claim 18 , wherein said selecting with said fluorochrome conjugated antibodies is by flow cytometry.
20. The method according to claim 17 , wherein at least of one of said antibodies is conjugated to magnetic particles.
21. The method of claim 20 , wherein said selecting with said magnetic particle conjugated antibodies is by high gradient magnetic selection.
22. The method of claim 9 , wherein said elimination is accomplished by contacting the population of cells with monoclonal antibody 8G1 and removing those cells that bind to monoclonal antibody 8G1.
23. The method of claim 10 , wherein said elimination is accomplished by contacting the population of cells with monoclonal antibody 8G1 and removing those cells that bind to monoclonal antibody 8G1.