Functional Markers for Cultured Human Corneal Endothelial Cells

Abstract

Purpose : Corneal endothelial dysfunction is a leading cause of blindness and cell therapy is a potential approach to replace lamellar and penetrating keratoplasty. Protocols for culturing and expanding human corneal endothelial cells (HCECs) are being developed to bring cell therapy to the clinic, but it remains difficult to identify the HCECs with the best function. Here we describe a method to select the best HCECs for cell therapy

Methods : HCECs isolated from cadaveric donor corneas were cultured in proliferative media containing nerve growth factor, epidermal growth factor and bovine pituitary extract, and in stabilization media without growth factors. Expression of functional markers was assessed by flow cytometry and RT-PCR in HCECs with canonical, cobblestone morphology and in fibroblastic HCECs. Function was recorded by the trans-endothelial electrical resistance (TEER) assay.

Results : Canonical HCECs expressed high levels of CD56 and CD248. Expression levels decreased when HCECs exhibited fibroblastic morphology but in the presence of stabilization media, HCEC cultures maintained their canonical morphology and their function over a 6-week period.

Conclusions : CD56 and CD248 are novel HCEC surface markers that can help predict which cultures will have the highest barrier function. This is an invaluable resource for cell banking, and in the future we will use these identification and potency assays to explore protocols to derive corneal endothelial-like cells from pluripotent stem cells.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.