Novel monoclonal antibodies for enrichment and characterization of human corneal endothelial cells

Abstract

Purpose: Corneal transplantation is the primary treatment for patients with corneal endothelial damage. Worldwide shortage of donor corneas has provoked much interest in cultured human corneal endothelial cells (hCENC) isolated from cadaveric corneas. Together with the use of tissue-engineered constructs, this may serve as an alternative option for transplant grade corneas. However, tools for the characterization of cultured hCENC and enrichment of hCENC from are lacking. The discovery of novel markers against hCENC will be valuable for both research and clinical applications.

Methods: In this study, we describe the discovery of novel monoclonal antibodies specific for hCENC using cadaveric expanded hCENCs as an immunogen in a mouse cell based immunization model. Antibodies recognizing antigen targets on the cell surface were selected using flow cytometry and further validated by immunostaining of frozen corneal tissue sections. Antibody specificity was determined by secondary screening with multiple human cell lines. The magnetic-activated sorting (MACS) system was used as a proof of concept study for enrichment of hCENCs from cell mixtures using hCENC-specific antibody.

Results: Two antibodies TAG-1A3 and TAG-2A12 were found to be specific to the corneal endothelial monolayer via immunostaining of frozen tissue sections. Both antibodies were found to be able to characterize cultured hCENC from multiple donors. Importantly, TAG-2A12 was found to be binding specifically only to cultured hCENC and not to other cell types such as human corneal stromal fibroblasts (hCSF) and human pluripotent stem cells (hPSC).

Conclusions: Both antibodies have shown promising applications to quantitatively assess hCENC quality. It is also noteworthy that TAG-2A12 could be used as a tool for enrichment of hCENC from cell mixtures of hCSF and hPSC.

Keywords: 481 cornea: endothelium • 423 antigen presentation/processing