Transcriptional profiles along cell programming into corneal epithelial differentiation

Using the rabbit corneal epithelial cell line RCE1(5T5) as a model, we analyzed three differentiation stages, distinguished on basis to the growth state of cultured cells and after studying the expression of transcription factors such as Oct4, Pax6 and ΔNp63α, selected differentiation markers, and signaling or epigenetic markers such as Notch receptors and Prdm3. Namely, proliferative non-differentiated cells, committed cells, and cells that constitute a stratified epithelium with a limbal epithelial-like structure. RNAseq based transcriptome analysis showed that 4891 genes were differentially expressed among these stages displaying distinctive gene signatures: proliferative cells had 1278 genes as gene signature, and seem to be early epithelial progenitors with an Oct4⁺, KLF4⁺, Myc⁺, ΔNp63α⁺, ABCG2⁺, Vimentin⁺, Zeb1⁺, VANGL1⁺, Krt3^-, Krt12^- phenotype. Committed cells had a gene signature with 417 genes and displayed markers indicative of the beginning of corneal differentiation, and genes characteristic of proliferative cells; we found the possible participation of Six3 and Six4 transcription factors along this stage. The third stage matches with a stratified corneal epithelium (gene signature comprising 979 genes) and is typified by an increase in the expression of WNT10A and NOTCH 2 and 3 signaling and Cux1 transcription factor, besides Pax6, KLF4 or Sox9. The differentiated cells express about 50% of the genes that belong to the Epidermal Differentiation Complex (EDC). Analysis of the differences between corneal epithelium and epidermis could be crucial to understand the regulatory mechanisms that lead to the expression of the differentiated phenotype.