Identification of novel molecular markers through transcriptomic analysis in human fetal and adult corneal endothelial cells

Abstract

The corneal endothelium is composed of a monolayer of corneal endothelial cells (CECs), which is essential for maintaining corneal transparency. To better characterize CECs in different developmental stages, we profiled mRNA transcriptomes in human fetal and adult corneal endothelium with the goal to identify novel molecular markers in these cells. By comparing CECs with 12 other tissue types, we identified 245 and 284 signature genes that are highly expressed in fetal and adult CECs, respectively. Functionally, these genes are enriched in pathways characteristic of CECs, including inorganic anion transmembrane transporter, extracellular matrix structural constituent and cyclin-dependent protein kinase inhibitor activity. Importantly, several of these genes are disease target genes in hereditary corneal dystrophies, consistent with their functional significance in CEC physiology. We also identified stage-specific markers associated with CEC development, such as specific members in the transforming growth factor beta and Wnt signaling pathways only expressed in fetal, but not in adult CECs. Lastly, by the immunohistochemistry of ocular tissues, we demonstrated the unique protein localization for Wnt5a, S100A4, S100A6 and IER3, the four novel markers for fetal and adult CECs. The identification of a new panel of stage-specific markers for CECs would be very useful for characterizing CECs derived from stem cells or ex vivo expansion for cell replacement therapy.

Figure 1.

Adult and fetal CECs exhibit distinct global mRNA expression patterns. (A) Unbiased hierarchical clustering shows fetal and adult CEC relationship with representative samples from other tissues and cell types. (B) Three-dimensional scatterplot of the first three principal components (PCA1, PCA2 and PCA3) demonstrate that fetal samples cluster to hESCs, NPCs and HUVECs, and adult CEC cluster closer to brain samples. Ninety-seven samples from 12 different tissue and cell types were downloaded from the GEO database. Solid and open arrows point out adult and fetal CECs.

Figure 2.

Heatmap analysis of the up-regulated gene in adult and fetal CECs. Heatmap of relative expression levels for genes that are highly expressed in (A) adult or (B) fetal CECs.

Figure 3.

GO analysis of CEC tissue-specific signature genes. Bar graphs showing the significance of enrichment terms for a set of uniquely expressed genes in (A) adult CECs and (B) fetal CECs compared with 12 tissue and cell types. P-values <0.05.

Figure 4.

GO analysis of CEC stage-specific signature genes. Bar graphs showing the significance of enrichment terms for a set of higher expressed genes in (A) adult over fetal CECs and (B) fetal over adult CECs. P-values <0.05.

Figure 5.

Immunostaining of novel marker proteins expressed in adult and fetal CECs. The bottom monolayer of the samples depicts corneal endothelium. A red signal shows the expression of each antigen as detected by a specific antibody. The blue signal is Hoesch dye. Left panels show adult CEC samples and right panels display fetal CEC samples. (A) and (B) are immunofluorescent labeling of Wnt5a in adult and fetal CECs; (C) and (D) detect S100A4 expression in adult and fetal CECs; (E) and (F) for S100A6; (G) and (H) showing IER3 protein localization in adult and fetal CECs. Scale bar, 20 μm.