Identification and isolation of human corneal endothelial cells (HCECs)
The present invention provides methods for the identification, isolation and/or enrichment of human corneal endothelial cells (HCECs). In some embodiments, the method comprises a positive selection process in which a cell population containing human corneal cells is contacted with a positive affinity reagent that selectively binds to HCECs relative to cells other than HCECs (e.g., corneal keratocytes, etc.) in the population and/or a negative selection process in which a cell population containing HCECs is contacted with a negative affinity reagent that selectively binds to cells other than HCECs in the population relative to HCECs. The present invention also provides reagents and kits for the identification, isolation and/or enrichment of HCECs as well as compositions that are enriched in HCECs.
1. A method of forming a composition enriched with human corneal endothelial cells comprising:
contacting a cell population containing human corneal endothelial cells expressing CD56 or CD166 with a first positive affinity reagent; and
selecting cells to which the first positive affinity reagent is bound,
wherein the first positive affinity reagent comprises an antibody that binds to CD56 surface protein expressed on the surface of human corneal endothelial cells, an antibody that binds to CD166 surface protein expressed on the surface of human corneal endothelial cells, an antibody that binds to coxsackievirus and adenovirus receptor (CAR) surface protein expressed on the surface of human corneal endothelial cells, or an antibody that binds to CD248 surface protein expressed on the surface of human corneal endothelial cells;
wherein the method comprises one or both of:
(i) the human corneal endothelial cells in the cell population express CD56, the first positive affinity reagent comprises an antibody or aptamer that binds to CD248 surface protein expressed on the surface of human corneal endothelial cells, an antibody or aptamer that binds to CAR surface protein expressed on the surface of human corneal endothelial cells, or an antibody or aptamer that binds to CD166 surface protein expressed on the surface of human corneal endothelial cells, and
(ii) further comprising:
contacting the cell population containing human corneal endothelial cells with a second positive affinity reagent; and
selecting cells to which the second positive affinity reagent is bound;
wherein the second positive affinity reagent differs from the first positive affinity reagent in regard to the identity of at least one surface protein that the first and second positive affinity reagents bind to and the second positive affinity reagent comprises an antibody or aptamer that binds to CD56 surface protein expressed on the surface of human corneal endothelial cells, an antibody or aptamer that binds to CD90 surface protein expressed on the surface of human corneal endothelial cells, an antibody or aptamer that binds to CD166 surface protein expressed on the surface of human corneal endothelial cells, an antibody or aptamer that binds to CD248 surface protein expressed on the surface of human corneal endothelial cells, or an antibody or aptamer that binds to CAR surface protein expressed on the surface of human corneal endothelial cells.
2. The method of claim 1 , wherein the human corneal endothelial cells in the cell population express CD56, the first positive affinity reagent comprises an antibody or aptamer that binds to CD248 surface protein expressed on the surface of human corneal endothelial cells, an antibody or aptamer that binds to CAR surface protein expressed on the surface of human corneal endothelial cells, or an antibody or aptamer that binds to CD166 surface protein expressed on the surface of human corneal endothelial cells.
3. The method of claim 2 , wherein said first positive affinity reagent comprises an antibody or aptamer that binds to CD166 surface protein expressed on the surface of human corneal endothelial cells.
4. The method of claim 1 , further comprising:
contacting the cell population containing human corneal endothelial cells with a second positive affinity reagent; and
selecting cells to which the second positive affinity reagent is bound;
wherein the second positive affinity reagent compositionally differs from the first positive affinity reagent and the second positive affinity reagent comprises an antibody or aptamer that binds to CD56 surface protein expressed on the surface of human corneal endothelial cells, an antibody or aptamer that binds to CD90 surface protein expressed on the surface of human corneal endothelial cells, an antibody or aptamer that binds to CD166 surface protein expressed on the surface of human corneal endothelial cells, an antibody or aptamer that binds to CD248 surface protein expressed on the surface of human corneal endothelial cells, or an antibody or aptamer that binds to CAR surface protein expressed on the surface of human corneal endothelial cells.
5. The method of claim 1 , further comprising:
contacting the cell population containing human corneal endothelial cells with a negative affinity reagent that selectively binds to human endothelial cells that have undergone a fibroblastic transformation relative to human corneal endothelial cells; and
removing cells that have undergone a fibroblastic transformation to which the negative affinity reagent is bound.
6. The method of claim 5 , wherein the negative affinity reagent comprises an antibody or aptamer that binds to CD109 surface protein expressed on the surface of human corneal endothelial cells.
7. The method of claim 1 , wherein the human corneal endothelial cells in the cell population express CD166, the first positive affinity reagent comprises an antibody or aptamer that binds to CD56 surface protein expressed on the surface of human corneal endothelial cells, an antibody or aptamer that binds to CD90 surface protein expressed on the surface of human corneal endothelial cells, an antibody or aptamer that binds to CD248 surface protein expressed on the surface of human corneal endothelial cells, or an antibody or aptamer that binds to CAR surface protein expressed on the surface of human corneal endothelial cells.
8. The method of claim 7 , wherein the first positive affinity reagent comprises an antibody or aptamer that binds to CD56 surface protein expressed on the surface of human corneal endothelial cells, an antibody or aptamer that binds to CD248 surface protein expressed on the surface of human corneal endothelial cells, or an antibody or aptamer that binds to CAR surface protein expressed on the surface of human corneal endothelial cells.
9. The method of claim 7 , wherein the first positive affinity reagent comprises an antibody or aptamer that binds to CD56 surface protein expressed on the surface of human corneal endothelial cells.
10. A composition enriched with human corneal endothelial cells comprising:
human corneal endothelial cells expressing at least one marker selected from the group consisting of: CD56, CD90, CD166, and coxsackie virus and adenovirus receptor (CAR); and
a first positive affinity reagent, wherein the first positive affinity reagent is an antibody against a surface protein expressed on the surface of human corneal endothelial cells, the surface protein selected from the group consisting of: CD56, CD90, CD166, CD248, and CAR,
wherein the marker expressed on the human corneal endothelial cells and the surface protein detected by the first positive affinity reagent are compositionally different;
wherein at least one of:
(i) the human corneal endothelial cell expresses CD56 and the first positive affinity reagent comprises an antibody or aptamer that binds to CD248 surface protein expressed on the surface of human corneal endothelial cells, an antibody or aptamer that binds to CAR surface proteins expressed on the surface of human corneal endothelial cells, an antibody or aptamer that binds to CD166 surface protein expressed on the surface of human corneal endothelial cells, or an antibody or aptamer that binds to CD90 surface protein expressed on the surface of human corneal endothelial cells and
(ii) the human corneal endothelial cell expresses CD56 and the first positive affinity reagent comprises an antibody or aptamer that binds to CD248 surface protein expressed on the surface of human corneal endothelial cells, an antibody or aptamer that binds to CAR surface protein expressed on the surface of human corneal endothelial cells, or an antibody or aptamer that binds to CD166 surface protein expressed on the surface of human corneal endothelial cells.
11. The composition of claim 10 , wherein the human corneal endothelial cell expresses CD56 and the first positive affinity reagent comprises an antibody or aptamer that binds to CD248 surface protein expressed on the surface of human corneal endothelial cells, an antibody or aptamer that binds to CAR surface proteins expressed on the surface of human corneal endothelial cells, an antibody or aptamer that binds to CD166 surface protein expressed on the surface of human corneal endothelial cells, or an antibody or aptamer that binds to CD90 surface protein expressed on the surface of human corneal endothelial cells.
12. The composition of claim 11 , wherein the first positive affinity reagent is an antibody or aptamer that binds to CD248 surface protein expressed on the surface of human corneal endothelial cells, an antibody or aptamer that binds to CAR surface protein expressed on the surface of human corneal endothelial cells, or an antibody or aptamer that binds to CD166 surface protein expressed on the surface of human corneal endothelial cells.
13. The composition of claim 12 , wherein the first positive affinity reagent is an antibody or aptamer that binds to CD166 surface protein expressed on the surface of human corneal endothelial cells.
14. The composition of claim 10 , wherein the composition further comprises a second positive affinity reagent that is compositionally different from the first positive affinity reagent, and the second positive affinity reagent is an antibody or aptamer that binds to CD90 surface protein expressed on the surface of human corneal endothelial cells, an antibody or aptamer that binds to CD166 surface protein expressed on the surface of human corneal endothelial cells, an antibody or aptamer that binds to CD248 surface protein expressed on the surface of human corneal endothelial cells, or an antibody or aptamer that binds to CAR surface protein expressed on the surface of human corneal endothelial cells.
15. The composition of claim 10 , wherein the human corneal endothelial cells express CD166 and the first positive affinity reagent is an antibody or aptamer that binds to CD56 surface protein expressed on the surface of human corneal endothelial cells, an antibody or aptamer that binds to CD90 surface protein expressed on the surface of human corneal endothelial cells, an antibody or aptamer that binds to CD248 surface protein expressed on the surface of human corneal endothelial cells, or an antibody or aptamer that binds to CAR surface protein expressed on the surface of human corneal endothelial cells.
16. The composition of claim 15 , wherein the first positive affinity reagent is an antibody or aptamer that binds to CD56 surface protein expressed on the surface of human corneal endothelial cells, an antibody or aptamer that binds to CD248 surface protein expressed on the surface of human corneal endothelial cells, or an antibody or aptamer that binds to CAR surface protein expressed on the surface of human corneal endothelial cells.
17. The composition of claim 15 , wherein the first positive affinity reagent is an antibody or aptamer that binds to CD56 surface protein expressed on the surface of human corneal endothelial cells.
18. The composition of claim 10 , wherein the composition further comprises a second positive affinity reagent that is compositionally different from the first positive affinity reagent, and the second positive affinity reagent is an antibody or aptamer that binds to CD56 surface protein expressed on the surface of human corneal endothelial cells, an antibody or aptamer that binds to CD90 surface protein expressed on the surface of human corneal endothelial cells, an antibody or aptamer that binds to CD248 surface protein expressed on the surface of human corneal endothelial cells, or an antibody or aptamer that binds to CAR surface protein expressed on the surface of human corneal endothelial cells.
19. The composition of claim 18 , wherein the positive affinity reagents are coupled to a label.
20. The composition of claim 19 , wherein the positive affinity reagents are coupled to different labels.