Cell-surface signatures for isolating neurons from cell cultures derived from pluripotent stem cells
The inventors disclose methods and systems that provide for the isolation and purification of neurons directly from heterogeneous cell cultures. The expression of various cell adhesion molecules was examined in pluripotent stem cells. Changes in the expression of one or more of these molecules correlates with the progression of cells from non-lineage committed to neural cells. Using one or more antibodies for these molecules in combination with antibodies specific for CD200 will enable one to identify and isolate neurons from a population cells.
1. A method of distinguishing neurons from other cells in a cell culture, the method comprising:
contacting the cell culture with a first antibody that specifically binds to CD200 and at least a second antibody that specifically binds to a protein selected from the group consisting of HLA-A, HLA-B, HLA-C, CD49f, CD151 and CD340; and
distinguishing cells that are CD200 + cells and also exhibit low to negative expression of one or more of HLA-A, HLA-B, HLA-C, CD49f, CD151 and CD340 from other cells in the cell culture wherein cells that are CD200 + and also exhibit one or more of HLA-A − , HLA-B − , HLA-C −1 , CD49f − , CD151 − or CD340 − are neurons.
2. The method of claim 1 wherein the cell culture comprises human induced pluripotent stem cells (hiPSC), human embryonic stem cells (hEPSC), neural crest cells, fibroblast cells, neural stem cells or any combination thereof.
3. The method of claim 1 wherein the cell culture is derived from a heterogeneous population of cells.
4. The method of claim 1 wherein the cell culture comprises neural rosettes.
5. The method of claim 1 wherein the cell culture comprises neural epithelium.
6. The method of claim 1 where the CD200 + cells are CD200 med and CD200 high .
7. The method of claim 1 wherein the cell culture has not undergone a neural stem cell enriching step.
8. The method of claim 1 wherein the step of distinguishing cells is carried out by means of a flow cytometer.
9. The method of claim 1 wherein the first and second antibodies are detectably and discriminably labeled by direct conjugation with a fluorophore.
10. The method according to claim 9 , wherein the method further comprises identifying cells that are CD200 + cells and also exhibit low to negative expression of one or more of HLA-A, HLA-B, HLA-C, CD49f, CD151 and CD340 as neurons.
11. The method according to claim 10 , wherein the cells identified as neurons are HLA-A − /HLA-B − /HLA-C − /CD 151 − /CD49f − /CD340 − /CD200 + .
12. The method of claim 1 further comprising enriching the population of cells that are CD200 ++ cells and also exhibit one or more of HLA-A − , HLA-B − , HLA-C − , CD49f − , CD151 − or CD340 − .
13. A system for the isolation or enrichment of neurons from a cell culture comprising:
a first antibody that specifically binds to CD200;
a second antibody that specifically binds to a protein selected from the group consisting of HLA-A, HLA-B, HLA-C, CD49f, CD151 and CD340;
a cell culture comprising neurons; and
a flow cytometer configured to sort cells that are CD200 + and also exhibit one or more of HLA-A − , HLA-B − , HLA-C − , CD49f − , CD151 − or CD340 − .
14. The system of claim 13 wherein the first and second antibodies are fluorescently labeled antibodies.