Purpose: In vitro expansion of primary corneal endothelial cells (pCEnC) has emerged as a potential solution to the global shortage of donor tissue, and thus it is important to comprehensively assess the methods for growing pCEnC in vitro. Transcriptomic (RNA-seq) analysis of pCEnC after growth using two expansion protocols for pCEnC identified differentially expressed genes involved in either solute transport or cell barrier function (adhesion and glycocalyx). The purpose of this study was to validate the RNA-seq results for these genes using qPCR.
Methods: Primary cell cultures (P0) were established from donor corneas (5 pairs) using two expansion protocols (Tryp/LN/F99 and CollA/COLIV/M4M5). After achieving 100% confluence, cells were lysed in Tri-Reagent and total RNA was isolated. First-strand synthesis of poly-A RNA was performed using oligo-dT primers. The resulting products were used as template for the qPCR reactions that were performed using KAPA SYBR Fast qPCR mix. qPCR results were analyzed using the comparative CT (2-∆∆Ct) method using RAB7 as the reference gene.
Results: All of the selected transporter genes (e.g., SLC4A4, SLC9A7, SLC16A12, SLC4A11) expressed in ex vivo CEnC (evCEnC) showed higher expression in pCEnC cultured in M4M5 compared with cells in F99. Although pCEnC cultured in M4M5 demonstrated greater barrier function compared with cells cultured in F99, cells in M4M5 showed lower expression of a majority of selected cell adhesion genes (e.g., CDH11, CDH3, CADM3), but a minority of these genes were not expressed in evCEnC. A majority of selected glycocalyx-associated genes (e.g. DCN, GPC4, LUM) expressed in evCEnC showed higher expression in pCEnC cultured in M4M5 compared with cells in F99.
Conclusion: Taken together, these results demonstrate that M4M5 is better than F99 at establishing pCEnC with gene expression profiles characteristic of evCEnC. Subsequent investigation using in vivo models of CEnC dysfunction will establish the use of M4M5 as a viable option in cell-replacement therapy for the management of corneal endothelial cell disease.