Discovery of molecular markers to discriminate corneal endothelial cells in the human body

Abstract

The corneal endothelium is a monolayer of hexagonal corneal endothelial cells (CECs) on the inner surface of the cornea. CECs are critical in maintaining corneal transparency through their barrier and pump functions. CECs in vivo have a limited capacity in proliferation, and loss of a significant number of CECs results in corneal edema called bullous keratopathy which can lead to severe visual loss. Corneal transplantation is the most effective method to treat corneal endothelial dysfunction, where it suffers from donor shortage. Therefore, regeneration of CECs from other cell types attracts increasing interests, and specific markers of CECs are crucial to identify actual CECs. However, the currently used markers are far from satisfactory because of their non-specific expression in other cell types. Here, we explored molecular markers to discriminate CECs from other cell types in the human body by integrating the published RNA-seq data of CECs and the FANTOM5 atlas representing diverse range of cell types based on expression patterns. We identified five genes, CLRN1, MRGPRX3, HTR1D, GRIP1 and ZP4 as novel markers of CECs, and the specificities of these genes were successfully confirmed by independent experiments at both the RNA and protein levels. Notably none of them have been documented in the context of CEC function. These markers could be useful for the purification of actual CECs, and also available for the evaluation of the products derived from other cell types. Our results demonstrate an effective approach to identify molecular markers for CECs and open the door for the regeneration of CECs in vitro.

Fig 1. Bioinformatics analysis flow chart

FPKM: Fragments Per Kilobase of exon per Million mapped fragments.

Fig 2. qRT-PCR analysis of 6 corneal endothelial cell marker candidate genes within ocular tissues.

CECs: corneal endothelial cells, cCECs: cultured corneal endothelial cells, C.stroma: corneal stroma, C.epi.: corneal epithelial cells, iris pig. epi.: iris pigment epithelial cells, TM: trabecular meshwork, CB: ciliary body, RPE: Retinal pigment epithelial cells, ON: optic nerve. Y axis indicates % ACTB, and error bars represent standard deviation of four replicates.

Fig 3. Immunofluorescence staining of human donor corneal tissue sections.

(A) Green signals represent the expression of each protein detected by the specific antibody. (B) Negative control by using isotype specific rabbit IgG (green signal) and goat IgG (red signal) as primary antibodies. Hoechst 33342-stained nuclei are shown in blue. A white arrow indicates the corneal endothelium. All scale bars indicate 50 μm.