Corneal endothelial cell marker
The purpose of the present invention is to provide a method of purification and preparation of cultured corneal endothelial cells, and in particular, to provide cell surface markers for use in corneal endothelial cells not including transformed cells. Provided are cell markers for distinguishing normal cells and transformed cells, in particular normal and transformed corneal endothelium cells. These cell markers relate to specific cell surface markers, for example, to a normal corneal endothelial surface marker such as CD166, and a transformed cell surface marker such as CD73. By using the transformed cell surface marker such as CD73 to remove transformed cells by sorting, it becomes possible to improve purity of a normal cultured corneal endothelium. By using normal corneal endothelial surface marker such as CD166, or by combined use with the transformed cell surface marker, it becomes possible to provide a means for verifying the purity of a prepared corneal endothelium.
1. A method for determining the fibroblastic status of a corneal endothelial cell, comprising:
(a) contacting a sample comprising the corneal endothelial cell with one or more detection agents selected from:
(i) one or more non-fibroblastic detection agents for endothelial cells with a non-fibroblastic phenotype, wherein each non-fibroblastic detection agent binds a cell-surface marker present on corneal endothelial cells with a non-fibroblastic phenotype selected from the group consisting of CD166, HLA-A2, CD66a, CD66c, CD66d, CD66e, CD98, CD59, CD54, CD340, CD47, EGF-R, CD29, CD74, CD165, CD221, CD49a, SSEA-4, CD130, and CD49f, and
(ii) one or more fibroblastic detection agents for endothelial cells with a fibroblastic phenotype, wherein each fibroblastic detection agent binds a cell-surface marker present on corneal endothelial cells with a fibroblastic phenotype selected from the group consisting of CD26, CD9, CD49b, CD49e, CD13, CD99, CD105, CD63, CD58, CD201, CD56, CD44, CD55, CD71, CD73, CD91, HLA-DQ, CD164, CD49d, CD49c, CD90, MICA/B, CD46, CD140b, CD146, CD147, CD81, CD151, CD200, and CD10;
(b) detecting any binding between the detection agent(s) and the marker(s) expressed on the cell; and
(c) determining the cell has a non-fibroblastic phenotype when a cell-surface marker bound by one or more of the non-fibroblastic detection agents is expressed on the cell at a higher level as compared to its expression level on a corneal endothelial cell with a fibroblastic phenotype, or
determining the cell has a fibroblastic phenotype when a cell-surface marker bound by one or more of the fibroblastic detection agents is expressed on the cell at a higher level as compared to its expression level on a corneal endothelial cell with a non-fibroblastic phenotype.
2. The method of claim 1 , wherein the corneal endothelial cell is present in an organism.
3. The method of claim 1 , wherein the corneal endothelial cell is of an organism in need of diagnosis of a corneal endothelial disease.
4. The method of claim 3 , wherein the corneal endothelial disease is a disease associated with a fibroblast.
5. The method of claim 4 , wherein the corneal endothelial disease is selected from the group consisting of bullous keratopathy, corneal endothelial disorders due to trauma or intraocular surgery, and corneal dystrophy including Fuchs corneal endothelial dystrophy and posterior polymorphous corneal endothelial dystrophy.
6. The method according to claim 3 , further comprising determining that the organism is suffering from irreversible dystrophy when a cell-surface marker bound by one or more of the fibroblastic detection agents is expressed on the cell at a higher level as compared to its expression level on a corneal endothelial cell with a non-fibroblastic phenotype.
7. A method of preparing a corneal endothelial cell sample with an elevated ratio of corneal endothelial cells with a non-fibroblastic phenotype to corneal endothelial cells with a fibroblastic phenotype, comprising:
depleting a sample comprising corneal endothelial cells with a non-fibroblastic phenotype and corneal endothelial cells with a fibroblastic phenotype of corneal endothelial cells expressing a cell-surface marker selected from the group consisting of CD26, CD9, CD49b, CD49e, CD13, CD99, CD105, CD63, CD58, CD201, CD56, CD44, CD55, CD71, CD73, CD91, HLA-DQ, CD164, CD49d, CD49c, CD90, MICA/B, CD46, CD140b, CD146, CD147, CD81, CD151, CD200, and CD10 at a higher level as compared to its expression level on a corneal endothelial cell with a non-fibroblastic phenotype, thereby elevating the ratio in the sample of corneal endothelial cells with a non-fibroblastic phenotype to corneal endothelial cells with a fibroblastic phenotype.
8. The method of claim 7 , further comprising, after the depleting, administering the corneal endothelial cell sample to a subject in need of prevention or treatment of a corneal endothelial disease.
9. The method of claim 1 , wherein the method comprises contacting the sample comprising the corneal endothelial cell with one or more non-fibroblastic detection agent(s) that bind a cell-surface marker selected from CD166, CD98, and CD340.
10. The method of claim 1 , wherein the method comprises contacting the sample comprising the corneal endothelial cell with one or more fibroblastic detection agent(s) that bind a cell-surface marker selected from CD9, CD49e, CD44, and CD73.
11. The method of claim 1 , wherein the method comprises contacting the sample comprising the corneal endothelial cell with a non-fibroblastic detection agent that binds cell-surface marker CD166.
12. The method of claim 1 , wherein the method comprises contacting the sample comprising the corneal endothelial cell with a fibroblastic detection agent that binds cell-surface marker CD73.
13. The method of claim 1 , wherein the method comprises contacting the sample comprising the corneal endothelial cell with a non-fibroblastic detection agent that binds cell-surface marker CD166 and with a fibroblastic detection agent that binds cell-surface marker CD73.
14. The method of claim 7 , wherein the method comprises depleting the sample of corneal endothelial cells expressing a cell-surface marker selected from CD9, CD49e, CD44, and CD73 at a higher level as compared to its expression level on a corneal endothelial cell with a non-fibroblastic phenotype.
15. The method of claim 7 , wherein the method comprises depleting the sample of corneal endothelial cells expressing cell-surface marker CD73 at a higher level as compared to its expression level on a corneal endothelial cell with a non-fibroblastic phenotype.