Abstract
Purpose :
The most common genetic cause of FECD is an intronic CTG repeat expansion in TCF4. Primary CEnC cultures derived from FECD specimens are limited due to their reduced mitotic capacity and loss of characteristics with passaging. The purpose of this study was to generate immortalized human CEnC lines derived from normal donor and FECD patients to provide better models to study FECD.
Methods :
Descemet membrane and endothelium were collected from donor corneas (n=2: 67 year old female and 68 year old female), and FECD patients undergoing endothelial keratoplasty (n=3: 54 year old female, 74 year old female, and 61 year old male). Blood samples were collected for genetic analysis. Primary CEnCs were cultured and immortalized using the simian virus 40 (SV40) T antigen, followed by puromycin selection. FECD-associated markers were assessed by Western blot and quantitative real-time PCR analysis. Morphological features were assessed by immunocytochemistry. Cell migration was detected by scratch assay with live imaging.
Results :
Two normal cell lines (HCEnC-SV-67F-17, HCEnC-SV-68F-15) and 3 FECD cells lines were generated (FECD-SV-54F-73, FECD-SV-74F-22, and FECD-SV-61M-80) with SV-40 immortalization at passage 0. Genetic analysis with triplet-repeat primed PCR from blood samples identified an expansion of the CTG repeat sequence in the TCF4 gene in 2 out 3 samples (FECD-SV-54F-73 with 73 repeats, and FECD-SV-61M-80 with 80 repeats). FECD cell lines retained the TCF4 repeat expansion until at least passage 7. FECD-SV-54F-73 and FECD-SV-61M-80 had increased cell doubling time compared to FECD-SV-74F-22 and normal cell lines. Cells lines were successfully passaged until at least passage 13. There was variability in expression of FECD-associated markers fibronectin, Snail1, ZEB1 and TGF-β1 between FECD cell lines. FECD cell lines expressed altered levels of ZO-1, N-cadherin, and Na+/K+ ATPase and displayed a fibroblastic morphology as detected by filamentous-actin staining.
Conclusions :
We generated 2 normal and 3 FECD cell lines, 2 of which had a TCF4 repeat expansion. TCF4 repeat expansion was retained during immortalization and was consistent with patient blood analysis. There was variability in morphological and functional phenotypes between the 3 FECD cell lines suggesting heterogeneity between FECD patients.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.