High throughput gene expression analysis identifies reliable expression markers of human corneal endothelial cells

Abstract

Considerable interest has been generated for the development of suitable corneal endothelial graft alternatives through cell-tissue engineering, which can potentially alleviate the shortage of corneal transplant material. The advent of less invasive suture-less key-hole surgery options such as Descemet's Stripping Endothelial Keratoplasty (DSEK) and Descemet's Membrane Endothelial Keratoplasty (DMEK), which involve transplantation of solely the endothelial layer instead of full thickness cornea, provide further impetus for the development of alternative endothelial grafts for clinical applications. A major challenge for this endeavor is the lack of specific markers for this cell type. To identify genes that reliably mark corneal endothelial cells (CECs) in vivo and in vitro, we performed RNA-sequencing on freshly isolated human CECs (from both young and old donors), CEC cultures, and corneal stroma. Gene expression of these corneal cell types was also compared to that of other human tissue types. Based on high throughput comparative gene expression analysis, we identified a panel of markers that are: i) highly expressed in CECs from both young donors and old donors; ii) expressed in CECs in vivo and in vitro; and iii) not expressed in corneal stroma keratocytes and the activated corneal stroma fibroblasts. These were SLC4A11, COL8A2 and CYYR1. The use of this panel of genes in combination reliably ascertains the identity of the CEC cell type.

Figure 1. The human corneal tissue, the isolated endothelium and the cultured corneal endothelial cells.

A. Corneal stroma with an intact Descemet’s membrane (DM), arrowed (left) and a corneal stromal without the DM layer (right). B. Peeled CEC-DM complex in a typical DM-roll, with the endothelium on the outside. C. Confluent culture of human CECs at the second passage. Scale bars: 100 µm.

Figure 2. RNA-seq comparing gene expression of young and old CEC-DM, CEC culture and corneal stroma.

A. Sequencing depth (total uniquely mapping reads) of each sample. B. Hierarchical clustering shows that the young and old CECs cluster closer to each other, followed by CEC culture, and lastly the corneal stroma.

Figure 3. High-throughput QPCR analysis of genes expressed in both CEC-DM and CEC cultures.

BM: bone marrow; IVS: interventricular septum; SG: salivary gland; SM: skeletal muscle; SI: small intestine; SC: spinal cord; U/C: uterus/cervix; FB: fetal brain; FL: fetal liver; SF: stromal fibroblast; CC: CEC culture; CEC-DM-y: CEC-DM young; CEC-DM-o: CEC-DM-old.

Figure 4. High-throughput QPCR analysis of genes differentially expressed in CEC-DM and CEC cultures.

BM:bone marrow; IVS:interventricular septum; SG: salivary gland; SM: skeletal muscle; SI: small intestine; SC: spinal cord; U/C:uterus/cervix; FB:fetal brain; FL:fetal liver; SF:stromal fibroblast; CC:CEC culture; CEC-DM-y:CEC-DM young; CEC-DM-o:CEC-DM-old.

Figure 5. High-throughput QPCR analysis of genes lowly or not expressed in corneal stroma.

BM: bone marrow; IVS: interventricular septum; SG: salivary gland; SM: skeletal muscle; SI: small intestine; SC: spinal cord; U/C: uterus/cervix; FB: fetal brain; FL: fetal liver; SF: stromal fibroblast; CC: CEC culture; CEC-DM-y: CEC-DM young; CEC-DM-o: CEC-DM-old.

Figure 6. QPCR validation of gene expression in CEC-DM andcorneal stroma (keratocytes) from 3 individual donors, corneal stromal fibroblast cultures from 2 individual donors and CEC culture.

CEC: CEC-DM; stroma: corneal stroma (keratocytes); SF: stromal fibroblast. Student’s t-tests (two-tailed assuming non-equal variance): One asterisk indicates p<0.05; two asterisks indicate p<0.01.