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 differentiating between corneal endothelial cells with a non-fibroblastic phenotype and corneal endothelial cells with a fibroblastic phenotype in a sample comprising corneal endothelial cells with a non-fibroblastic phenotype and corneal endothelial cells with a fibroblastic phenotype, comprising at least one of:
detecting corneal endothelial cells with a non-fibroblastic phenotype in the sample by detecting at least one 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, wherein the detecting comprises contacting the sample with a detection agent that binds the marker and detecting any binding between the detection agent and the marker, and wherein the cell-surface marker present on the corneal endothelial cell with a non-fibroblastic phenotype is expressed at a higher level compared to the expression level of the same cell-surface marker on a corneal endothelial cell with a fibroblastic phenotype, and
detecting corneal endothelial cells with a fibroblastic phenotype in the sample by detecting at least one 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, wherein the detecting comprises contacting the sample with a detection agent that binds the marker and detecting any binding between the detection agent and the marker, and wherein the cell-surface marker present on the corneal endothelial cell with a fibroblastic phenotype is expressed at a higher level compared to the expression level of the same cell-surface marker on a corneal endothelial cell with a non-fibroblastic phenotype.
2. The method of claim 1 , wherein the method comprises detecting at least one cell-surface marker present on corneal endothelial cells with a fibroblastic phenotype and detecting at least one cell-surface marker present on corneal endothelial cells with a non-fibroblastic phenotype.
3. The method of claim 1 , wherein the method comprises detecting at least one cell-surface marker present on corneal endothelial cells with a non-fibroblastic phenotype selected from the group consisting of CD98, CD166, and CD340.
4. The method of claim 1 , wherein the method comprises detecting at least one cell-surface marker present on corneal endothelial cells with a fibroblastic phenotype selected from the group consisting of CD9, CD49e, CD44, and CD73.
5. The method of claim 1 , wherein the method comprises detecting at least one cell-surface marker present on corneal endothelial cells with a non-fibroblastic phenotype selected from the group consisting of CD98, CD166, and CD340, and detecting at least one cell-surface marker present on corneal endothelial cells with a fibroblastic phenotype selected from the group consisting of CD9, CD49e, CD44, and CD73.
6. The method of claim 1 , wherein the method comprises detecting at least one of CD166 and CD73.
7. The method according to claim 1 , wherein the detection agent for detecting corneal endothelial cells with a non-fibroblastic phenotype, and the detection agent for detecting corneal endothelial cells with a fibroblastic phenotype, if used, comprise(s) a staining agent.
8. The method of claim 1 , further comprising determining the purity of the sample based on the relative amount of binding detected of a cell-surface marker present on corneal endothelial cells with a non-fibroblastic phenotype as compared to the binding detected of a cell-surface marker present on corneal endothelial cells with a fibroblastic phenotype.
9. The method of claim 1 , further comprising, prior to the detecting, preparing the sample of corneal endothelial cells by inducing corneal endothelial cells from undifferentiated cells.
10. The method of claim 9 , wherein the undifferentiated cells are induced pluripotent stem (iPS) cells or embryonic stem (ES) cells.