Reconstruction of a Corneal Endothelium Using Cells From Patients With Fuchs Endothelial Corneal Dystrophy

Abstract

Purpose: : To characterize a reconstructed corneal endothelium bioengineered by seeding cultured Fuchs endothelial corneal endothelium (FECD) cells on the denuded Descemet’s membrane of a human cornea.

Methods: : Descemet’s membranes from patients undergoing Descemet stripping automated endothelial keratoplasty (n=6) were used to isolate and culture FECD endothelial cells. Second or third-passaged FECD endothelial cells were seeded on top of a previously decellularized human cornea. After 2 weeks in culture, in order to allow for attachment and spreading of seeded cells, the reconstructed corneas were stained with alizarin red and fixed for histology and transmission electron microscopy (TEM). Immunofluorescence labeling of various proteins was also performed.

Results: : Amplification of the cultured FECD cells generated between 88000 and 1.1 million cells. FECD cell morphology on Descemet’s membrane was polygonal. Histology and TEM revealed a monolayer of tightly packed cells well adhered to Descemet’s membrane. The reconstructed FECD corneal endothelium expressed K8/18 and did not express a-SMA, confirming the absence of endothelial-mesenchymal transition. Clusterin expression was faint and uniform in all constructs. FECD cells also expressed the function-related proteins Na+-K+/ATPase and Na+/HCO3-. Collagen type IV expression was visible as a thin and uniform line on the endothelial side of Descemet’s membrane.

Conclusions: : This study demonstrates the feasibility of reconstructing a corneal endothelium using cells from patients with FECD. This represents an additional step towards a better understanding of this endothelial dystrophy. Potential applications are numerous, including in vitro pathophysiological and pharmacological studies.

Keywords: cornea: endothelium • degenerations/dystrophies