Identification and isolation of human corneal endothelial cells (hcecs)

Abstract

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.

What is claimed is: 
     
         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 cells other than human corneal endothelial cells and (b) selecting cells to which the first positive affinity reagent is bound. 
     
     
         2 . The method of  claim 1 , wherein the first positive affinity reagent selectively binds to human corneal endothelial cells relative to corneal keratocytes, human corneal endothelial cells of lower utility, or both. 
     
     
         3 . The method of  claim 1 , wherein said first positive affinity reagent comprises an antibody or aptamer that binds to a corneal protein selected from protein products of genes X1 through X26 of Table 2. 
     
     
         4 . The method of  claim 1 , wherein said first positive affinity reagent comprises an antibody or aptamer that binds to a protein product of gene X5 of Table 2, an antibody or aptamer that binds to a protein product of gene X15 of Table 2, or an antibody or aptamer that binds to a protein product of X25 of Table 2. 
     
     
         5 . The method of  claim 1 , wherein said first positive affinity reagent comprises (a) an antibody or aptamer that binds to SEQ ID NO (8), (b) an antibody or aptamer that binds to one or more of SEQ ID NO (27), SEQ ID NO (28), SEQ ID NO (29), SEQ ID NO (30) or SEQ ID NO (31), or (c) an antibody or aptamer that binds to one or more of SEQ ID NO (53), SEQ ID NO (54), SEQ ID NO (55), SEQ ID NO (56) or SEQ ID NO (57). 
     
     
         6 . The method of any of  claims 1 - 5 , wherein the first positive affinity reagent comprises an antibody or aptamer that is coupled to a solid matrix. 
     
     
         7 . The method of any of  claims 1 - 6 , wherein the first positive affinity reagent comprises an antibody or aptamer that is coupled to a label. 
     
     
         8 . The method of  claim 7 , wherein the label is selected from a magnetic label, a hapten (e.g., biotin) and a fluorescent label. 
     
     
         9 . The method of any of  claims 1 - 8 , further comprising (a) contacting said cell population containing human corneal cells with a second positive affinity reagent that selectively binds to human corneal endothelial cells relative to cells other than human corneal endothelial cells and (b) selecting cells to which the second positive affinity reagent is bound, wherein the second positive affinity reagent differs from the first positive affinity reagent. 
     
     
         10 . The method of  claim 9 , wherein said second positive affinity reagent comprises an antibody or aptamer that binds to a corneal protein selected from protein products of genes X1 through X26 of Table 2. 
     
     
         11 . The method of  claim 9 , wherein said second positive affinity reagent comprises an antibody or aptamer that binds to a protein product of gene X5 of Table 2, an antibody or aptamer that binds to a protein product of gene X15 of Table 2, or an antibody or aptamer that binds to a protein product of X25 of Table 2. 
     
     
         12 . The method of  claim 9 , wherein said second positive affinity reagent comprises (a) an antibody or aptamer that binds to SEQ ID NO (8), (b) an antibody or aptamer that binds to one or more of SEQ ID NO (27), SEQ ID NO (28), SEQ ID NO (29), SEQ ID NO (30) or SEQ ID NO (31), or (c) an antibody or aptamer that binds to one or more of SEQ ID NO (53), SEQ ID NO (54), SEQ ID NO (55), SEQ ID NO (56) or SEQ ID NO (57). 
     
     
         13 . The method of any of  claims 9 - 12 , wherein the second positive affinity reagent comprises an antibody or aptamer that is coupled to a solid matrix or a label. 
     
     
         14 . The method of any of  claims 1 - 13 , further comprising (a) contacting said cell population containing human corneal cells with a first negative affinity reagent that selectively binds to cells other than human corneal endothelial cells relative to human corneal endothelial cells and (b) removing the cells to which the first negative affinity reagent is bound. 
     
     
         15 . The method of  claim 14 , wherein the first negative affinity reagent selectively binds to corneal keratocytes, human corneal endothelial cells of lower utility, or both, relative to human corneal endothelial cells. 
     
     
         16 . The method of  claim 14 , wherein said first negative affinity reagent comprises an antibody or aptamer that binds to a corneal protein selected from protein products of genes Y1 through Y23 of Table 2 and protein products of genes Z1 through Z8 of Table 2. 
     
     
         17 . The method of  claim 14 , wherein said first negative affinity reagent comprises an antibody or aptamer that binds to a protein product of gene Y6 of Table 2. 
     
     
         18 . The method of  claim 14 , wherein said first negative affinity reagent comprises an antibody or aptamer that binds to one or more of SEQ ID NO (66), SEQ ID NO (67) or SEQ ID NO (68). 
     
     
         19 . The method of any of  claims 14 - 18 , wherein the first negative affinity reagent comprises an antibody or aptamer that is coupled to a solid matrix or a label. 
     
     
         20 . A composition enriched with human corneal endothelial cells that is made by the method of any of  claims 1 - 19 . 
     
     
         21 . A kit comprising (a) a positive affinity reagent that selectively binds to human corneal endothelial cells relative to cells other than human corneal endothelial cells and (b) a negative affinity reagent that selectively binds to cells other than human corneal endothelial cells relative to human corneal endothelial cells. 
     
     
         22 . The kit of  claim 21 , wherein said positive affinity reagent comprises an antibody or aptamer that binds to a corneal protein selected from protein products of genes X1 through X26 of Table 2. 
     
     
         23 . The kit of  claim 21 , wherein said first positive affinity reagent comprises an antibody or aptamer that binds to a protein product of gene X5 of Table 2, an antibody or aptamer that binds to a protein product of gene X15 of Table 2, or an antibody or aptamer that binds to a protein product of X25 of Table 2. 
     
     
         24 . The kit of  claim 21 , wherein said first positive affinity reagent comprises (a) an antibody or aptamer that binds to SEQ ID NO (8), (b) an antibody or aptamer that binds to one or more of SEQ ID NO (27), SEQ ID NO (28), SEQ ID NO (29), SEQ ID NO (30) or SEQ ID NO (31), or (c) an antibody or aptamer that binds to one or more of SEQ ID NO (53), SEQ ID NO (54), SEQ ID NO (55), SEQ ID NO (56) or SEQ ID NO (57). 
     
     
         25 . The kit of any of  claims 21 - 24 , wherein the positive affinity reagent comprises an antibody or aptamer that is coupled to a solid matrix or a label. 
     
     
         26 . The kit of any of  claims 21 - 25 , wherein said negative affinity reagent comprises an antibody or aptamer that binds to a corneal protein selected from protein products of genes Y1 through Y23 and protein products of genes Z1 through Z8 of Table 2. 
     
     
         27 . The kit of any of  claims 21 - 25 , wherein said negative affinity reagent comprises an antibody or aptamer that binds to a protein product of gene Y6 of Table 2. 
     
     
         28 . The kit of any of  claims 21 - 25 , wherein said first negative affinity reagent comprises an antibody or aptamer that binds to one or more of SEQ ID NO (66), SEQ ID NO (67) or SEQ ID NO (68). 
     
     
         29 . The kit of any of  claims 21 - 28 , wherein the negative affinity reagent comprises an antibody or aptamer that is coupled to a solid matrix or a label. 
     
     
         30 . An affinity reagent selected from (a) a solid matrix or a label coupled to an antibody or aptamer that binds to a corneal protein selected from protein products of genes X1 through X26 of Table 2 and (b) a solid matrix or a label coupled to an antibody or aptamer that binds to a corneal protein selected from protein products of genes Y1 through Y23 and protein products of genes Z1 through Z8 of Table 2. 
     
     
         31 . A composition enriched with human corneal endothelial cells comprising: (a) human corneal cells and (b) a first positive affinity reagent that selectively binds to human corneal endothelial cells relative to cells other than human corneal endothelial cells. 
     
     
         32 . The composition of  claim 31 , comprising human corneal endothelial cells to which the first positive affinity reagent is bound. 
     
     
         33 . The composition of any of  claims 31 - 32 , wherein the first positive affinity reagent selectively binds to human corneal endothelial cells relative to corneal keratocytes, human corneal endothelial cells of lower utility, or both. 
     
     
         34 . The composition of any of  claims 31 - 33 , wherein said first positive affinity reagent comprises an antibody or aptamer that binds to a corneal protein selected from protein products of genes X1 through X26 of Table 2. 
     
     
         35 . The composition of any of  claims 31 - 33 , wherein said first positive affinity reagent comprises an antibody or aptamer that binds to a protein product of gene X5 of Table 2, an antibody or aptamer that binds to a protein product of gene X15 of Table 2, or an antibody or aptamer that binds to a protein product of X25 of Table 2. 
     
     
         36 . The composition of any of  claims 31 - 33 , wherein said first positive affinity reagent comprises (a) an antibody or aptamer that binds to SEQ ID NO (8), (b) an antibody or aptamer that binds to one or more of SEQ ID NO (27), SEQ ID NO (28), SEQ ID NO (29), SEQ ID NO (30) or SEQ ID NO (31), or (c) an antibody or aptamer that binds to one or more of SEQ ID NO (53), SEQ ID NO (54), SEQ ID NO (55), SEQ ID NO (56) or SEQ ID NO (57). 
     
     
         37 . The composition of any of  claims 31 - 36 , wherein the first positive affinity reagent comprises an antibody or aptamer that is coupled to a label. 
     
     
         38 . The composition of  claim 37 , wherein the label is selected from a magnetic label, a hapten (e.g., biotin) and a fluorescent label. 
     
     
         39 . The composition of any of  claims 31 - 38 , further comprising a second positive affinity reagent that selectively binds to human corneal endothelial cells relative to cells other than human corneal endothelial cells, wherein the second positive affinity reagent differs from the first positive affinity reagent. 
     
     
         40 . The composition of  claim 39 , comprising human corneal endothelial cells to which the second positive affinity reagent is bound. 
     
     
         41 . The composition of any of  claims 39 - 40 , wherein said second positive affinity reagent comprises an antibody or aptamer that binds to a corneal protein selected from protein products of genes X1 through X26 of Table 2. 
     
     
         42 . The composition of any of  claims 39 - 40 , wherein said second positive affinity reagent comprises an antibody or aptamer that binds to a protein product of gene X5 of Table 2, an antibody or aptamer that binds to a protein product of gene X15 of Table 2, or an antibody or aptamer that binds to a protein product of X25 of Table 2. 
     
     
         43 . The composition of any of  claims 39 - 40 , wherein said second positive affinity reagent comprises (a) an antibody or aptamer that binds to SEQ ID NO (8), (b) an antibody or aptamer that binds to one or more of SEQ ID NO (27), SEQ ID NO (28), SEQ ID NO (29), SEQ ID NO (30) or SEQ ID NO (31), or (c) an antibody or aptamer that binds to one or more of SEQ ID NO (53), SEQ ID NO (54), SEQ ID NO (55), SEQ ID NO (56) or SEQ ID NO (57). 
     
     
         44 . The composition of any of  claims 39 - 43 , wherein the second positive affinity reagent comprises a label. 
     
     
         45 . The composition of any of  claims 31 - 44 , comprising a measurable amount of one or more negative affinity reagents that selectively binds to cells other than human corneal endothelial cells relative to human corneal endothelial cells. 
     
     
         46 . The composition of  claim 45 , wherein the one or more negative affinity reagents selectively binds to corneal keratocytes, human corneal endothelial cells of lower utility, or both, relative to human corneal endothelial cells. 
     
     
         47 . The composition of any of  claim 45 - 46 , wherein said one or more negative affinity reagents comprise one or more antibodies that bind to one or more corneal proteins selected from protein products of genes Y1 through Y23 of Table 2 and protein products of genes Z1 through Z8 of Table 2. 
     
     
         48 . The composition of any of  claims 45 - 46 , wherein said one or more negative affinity reagents comprise an antibody or aptamer that binds to a protein product of gene Y6 of Table 2. 
     
     
         49 . The composition of any of  claims 45 - 46 , wherein said one or more negative affinity reagents comprise an antibody or aptamer that binds to one or more of SEQ ID NO (66), SEQ ID NO (67) or SEQ ID NO (68). 
     
     
         50 . The composition of any of  claims 45 - 49 , wherein the one or more negative affinity reagents comprise an antibody or aptamer that is coupled to a label.