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: (a) contacting a cell population containing human corneal cells with a first positive affinity reagent that selectively binds to human corneal endothelial cells relative to human corneal endothelial cells that have undergone a fibroblastic transformation and selecting cells to which the first positive affinity reagent is bound, wherein said first positive affinity reagent comprises an antibody that binds to CD56 surface protein, an antibody that binds to CD166 surface protein, an antibody that binds to coxsackievirus and adenovirus receptor (CAR) surface protein, or an antibody that binds to CD248 surface protein, (b) contacting the cell population containing human corneal cells with a second positive affinity reagent that selectively binds to human corneal endothelial cells relative to human corneal endothelial cells that have undergone a fibroblastic transformation 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 and (c) optionally, contacting said cell population containing human corneal cells with a first negative affinity reagent that selectively binds to human corneal endothelial cells that have undergone a fibroblastic transformation relative to human corneal endothelial cells and removing the cells to which the first negative affinity reagent is bound.
2. The method of claim 1 , wherein said first positive affinity reagent comprises an antibody that binds to CD56 surface protein, an antibody that binds to CAR surface protein, or an antibody that binds to CD248 surface protein.
3. The method of claim 1 , wherein said second positive affinity reagent comprises an antibody that binds to CD56 surface protein, an antibody that binds to CAR surface protein, or an antibody that binds to CD248 surface protein.
4. The method of claim 1 , wherein said cell population containing human corneal cells is contacted with said first negative affinity reagent and wherein cells to which the first negative affinity reagent is bound are removed.
5. The method of claim 4 , wherein said first negative affinity reagent comprises an antibody that binds to CD109 surface protein.
6. The method of claim 1 , wherein the first positive affinity reagent is coupled to a first label, wherein the second positive affinity reagent is coupled to a second label, and wherein the optional first negative affinity reagent is coupled to a third label, wherein the first label, the second label and the third label may be the same or different.
7. A composition enriched with human corneal endothelial cells that is made by the method of claim 6 .
8. A kit comprising
two positive affinity reagents that selectively bind to human corneal endothelial cells relative to human corneal endothelial cells that have undergone a fibroblastic transformation, wherein said two positive affinity reagents comprise (i) a first positive affinity reagent comprising an antibody that binds to CD56 surface protein, an antibody that binds to CD166 surface protein, an antibody that binds to CAR surface protein, or an antibody that binds to CD248 surface protein, and (ii) a second positive affinity reagent that is different from the first positive affinity reagent and comprises an antibody that binds to CD56 surface protein, an antibody that binds to CD166 surface protein, an antibody that binds to CAR surface protein, or an antibody that binds to CD248 surface protein.
9. The kit of claim 8 , wherein said first positive affinity reagent comprises an antibody that binds to CD56 surface protein, an antibody that binds to CAR surface protein, or an antibody that binds to CD248 surface protein.
10. The kit of claim 8 , further comprising a negative affinity reagent that selectively binds to human corneal endothelial cells that have undergone a fibroblastic transformation relative to human corneal endothelial cells, wherein said negative affinity reagent comprises an antibody that binds a CD109 surface protein.
11. A composition comprising: (a) human corneal cells; (b) a first positive affinity reagent that selectively binds to human corneal endothelial cells relative to human corneal endothelial cells that have undergone a fibroblastic transformation, wherein said positive affinity reagent comprises an antibody that binds to CD56 surface protein, an antibody that binds to CD166 surface protein, an antibody that binds to CAR surface protein, or an antibody that binds to CD248 surface protein, and (c) a second positive affinity reagent that selectively binds to human corneal endothelial cells relative to human corneal endothelial cells that have undergone a fibroblastic transformation 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.
12. The composition of claim 11 , wherein said first positive affinity reagent comprises an antibody that binds to CD56 surface protein, an antibody that binds to CAR surface protein, or an antibody that binds to CD248 surface protein.
13. The composition claim 11 , wherein said second positive affinity reagent comprises an antibody that binds to CD56 surface protein, an antibody that binds to CAR surface protein, or an antibody that binds to CD248 surface protein.
14. The composition of claim 11 , wherein said composition further comprises a negative affinity reagent that selectively binds to human corneal endothelial cells that have undergone a fibroblastic transformation relative to human corneal endothelial cells, and wherein said negative affinity reagent comprises an antibody that binds to a CD 109 surface protein.