Corneal Endothelial Cells Over the Past Decade: Are We Missing the Mark(er)?

Abstract

Corneal endothelial dysfunction is one of the leading causes of corneal edema and visual impairment, requiring corneal endothelial transplantation. The treatments are limited, however, by both logistics and a global donor shortage. As a result, corneal researchers are striving to develop tissue-engineered constructs as an alternative. Recently, the clinical results of the first patients treated using a novel corneal endothelial cell therapy were reported, and it is likely many more will follow shortly. As we move from lab to clinic, it is crucial that we establish accurate and robust methods of proving the cellular identity of these products, both in genotype and phenotype.In this review, we summarized all of the markers and techniques that have been reported during the development of corneal endothelial cell therapies over the past decade. The results show the most frequently used markers were very general, namely Na+/K+ ATPase and zonula occludens-1 (ZO-1). While these markers are expressed in nearly every epithelial cell, it is the hexagonal morphology that points to cells being corneal endothelium in nature. Only 11% of articles aimed at discovering novel markers, while 30% were already developing cell therapies. Finally, we discuss the potential of functional testing of cell products to demonstrate potency in parallel with identity markers.With this review, we would like to highlight that, while this is an exciting era in corneal endothelial cell therapies, there is still no accepted consensus on a unique endothelial marker panel. We must ask the question of whether or not we are getting ahead of ourselves and whether we need to refocus on basic science rather than enter clinics prematurely.

Transcriptome dataset of human corneal endothelium based on ribosomal RNA-depleted RNA-Seq data

“…However, this high expression of PTGDS is consistent with a report indicating that PTGDS is expressed in both the stroma and the endothelium 38 . By contrast, expression of genes often used as endothelial markers 36 was generally high. The expression of Tight Junction Protein 1 (TJP1, also known as ZO-1) was relatively low when compared with other endothelial markers, but this lower expression level was consistent with a previous description of lower expression of TJP1 mRNA compared to TJP1 protein 26 .…”

“…The correlation among seven samples was tested by the Spearman's rank correlation coefficient test, and the resulting correlation matrix was drawn with the 'ggcorrplot' library of R. In the heatmap analysis, the values were normalized with 'zFPKM' and heatmap analysis was performed with 'pheatmap' (both the libraries were obtained from Bioconductor). Marker genes reported to show specific expression in different corneal tissues, such as the epithelium, stroma, and endothelium, were selected as follows: PAX6 and WNT7 were referred from a representative study that performed a functional analysis of the corneal epithelium 31 ; ALDH3A1, CHST6, KERA, and PTGDS were extracted from expression markers of the corneal stroma or keratocytes commonly reported in four articles [32][33][34][35] ; and ATP1A1, TJP1, COL8A1, and SLC4A11 were selected from highly ranked investigated genes related to the corneal endothelium in a comprehensive review article 36 . The expression data of each gene used in the heatmap were evaluated for the existence of outlier samples by the Smirnov-Grubbs test with the 'outliers' library of R.…”

Transplantation of human induced pluripotent stem cell-derived neural crest cells for corneal endothelial regeneration

“…Previous studies have reported the in vitro induction of corneal endothelial-like cells derived from hESCs and hiPSCs [ 14 , 40 , 41 ], but the in vivo recovery outcome was unsatisfactory when transplantation in animal models of corneal endothelial dysfunction [ 41 ]. The most difficult is the lack of definitive specific markers to identify and enrich the differentiated CECs [ 42 , 43 ]. Moreover, the differentiated cells may experience an incomplete maturation process through the in vitro induction, although they exhibited the positive staining of several corneal endothelial cell markers.…”

A single cell transcriptome atlas reveals the heterogeneity of the healthy human cornea and identifies novel markers of the corneal limbus and stroma

“…Data of the 19,472 sequenced cells were embedded in a uniform manifold approximation and projection (UMAP) and unbiased low resolution clustering revealed five major cell clusters (Figure 1b, left). The expression of specific corneal layer marker (keratin 5 (KRT5) for epithelium 4 , keratocan (KERA) for stromal cells 5 , and transporter-like protein 11 (SLC4A11) for endothelium 6 ) identified the three main layers within the five identified clusters (Figure 1c). Differential gene expression profiling of the clusters further confirmed the identification of a corneal epithelial cell cluster comprising 5,964, a corneal stromal cell cluster comprising 12,344 cells, a corneal endothelial cell cluster comprising 842 cell, and non-corneal clusters of blood/lymphatic vessel endothelial cells comprising 36 cells, and immune cells comprising 216 cells (Figure 2).…”