Matériel et méthodes

This study was conducted in accordance with our institution’s guidelines (CHU de Québec-Université Laval, Québec, Canada; Hôpital Maisonneuve-Rosemont (HMR), Montréal, QC, Canada and Faculté de médecine; Centre hospitalier de l'Université de Montréal, Université de Montréal, Montréal, QC, Canada) and the Declaration of Helsinki.

2.5.1 FECD and healthy human corneal specimens

Specimens from 24 late-stage FECD patients were collected following graft surgery as described previously (Zaniolo et al. 2012). These specimens were randomly assigned and used for gene profiling and qPCR analysis (n=6, age range: 58 to 82 years, mean ± SD: 69 ± 10 years), for transendothelial electrical resistance (TEER) and 10 kDA FITC-Dextran permeability analysis (n=4; age range: 57 to 71 years, mean ± SD: 66 ± 5 years), to engineer FECD endothelia (n=7, age range: 58 to 77 years, mean ± SD: 67 ± 8 years) and for immunostaining analysis (native ex vivo FECD specimens; n=7, age range: 57 to 82 years, mean ± SD: 70 ± 6 years).

Donor human corneas, without endothelial disease (“Healthy”) and unsuitable for transplantation, were obtained from the Centre universitaire d’ophtalmologie (CUO) eye bank (Québec, Québec, Canada). Six were used for gene expression profiling and qPCR (age range: 58 to 82 years, mean ± SD: 71 ± 10 years), four for TEER and permeability analysis (age range: 56 to 72 years, mean ± SD: 66 ± 7 years), two to engineer healthy endothelia (aged 47 and 68 years), nine as carriers for the engineered corneal endothelia (age range: 54 to 84 years, mean ± SD: 69 ± 10 years) and eight for immunostaining experiments (native ex vivo healthy tissue; range: 68 to 84 years, mean ± SD: 72 ± 12).

2.5.2 Isolation and culture of corneal endothelial cells from healthy and FECD

corneas

Isolation and culture of healthy corneal endothelial cells (CEC) were performed as described previously (C. Zhu and Joyce 2004). Briefly, Descemet’s membranes were peeled off from healthy donor corneas and incubated overnight in culture medium at 37°C. Descemet’s membranes were then incubated in ethylenediaminetetraacetic acid (EDTA) 0.02% buffered solution (Sigma-Aldrich, St. Louis, MO, USA) and incubated for 1 hour. Physical force (up and down through a flamed-polished pipet) was used to detach cells from the Descemet membrane. After centrifugation, cells were seeded in culture dishes covered with FNC coating mix (Athena Enzyme Systems, Baltimore, MD), in culture medium made

of OptiMem-I (Invitrogen, Burlington, ON, Canada), 8 % fetal bovine serum (HyClone, Logan, UT), 5 ng/mL human epidermal growth factor (Austral Biologicals, San Ramon, CA), 20 ng/mL nerve growth factor (Biomedical Technologies, Stoughton, MA), 100 μg/mL bovine pituitary extract (Biomedical Technologies), 20 μg/mL ascorbic acid (Sigma-Aldrich), 0.08 % chondroitin sulfate (Sigma), 25 μg/mL gentamicin sulfate (Schering Pointe Claire, Qc, Canada) and 100 IU/mL penicillin G (Sigma-Aldrich). The same procedure was used for the isolation and culture of FECD endothelial cells, with the exception that the peeling was performed by corneal surgeon during the DSAEK procedure (Zaniolo et al. 2012). Cells were used at passage 2 or 3 for all experiments. Cultures were maintained at confluency for at least two weeks before being used for the experiments.

2.5.3 Gene expression profiling

Cells were maintained at confluency for 25-40 days. Hydrocortisone [0.4 µg/mL] (Calbiochem, La Jolla, CA, USA) was added to the growth medium in the last 7 days. Total RNA was isolated using RNeasy Mini Kit (Qiagen, Toronto, ON, Canada) or TRIzol (Ambion; Life Technologies, Grand Island, NY, USA). All microarray analyses were conducted by the gene profiling service of the molecular genetic platform (Genetiquemoleculaire.com) from the CUO-Recherche (Québec, QC, Canada). RNA was labeled with cyanine 3-CTP using Agilent One-Color Microarray-Based Gene Expression Analysis kit (Agilent Technologies Canada Inc., Mississauga, ON, Canada). Obtained cRNA were incubated on a G4851A SurePrint G3 Human GE 8x60K array slide (60,000 probes, Agilent Technologies). After being washed and stained, the slides were digitized with an Agilent SureScan Scanner. The results were analyzed by RMA (Robust Multiarray Analysis) using the ArrayStar V12 (DNASTAR, Madison, WI, USA) software as described previously (Duval et al. 2015; Landreville et al. 2011; Molloy-Simard et al. 2012). The color scale used to display the log2 expression level values was determined by the hierarchical clustering algorithm of the Euclidian metric distance between genes. Genes indicated in dark blue correspond to those whose expression is very low whereas highly expressed genes are shown in orange/red. The microarray data presented in this study complied with the Minimum Information About a Microarray Experiment (MIAME) requirements (Brazma et al. 2001) and have been deposited in the NCBI’s Gene Expression Omnibus (GEO) repository (Series record GSE112039).

2.5.4 Quantitative PCR

The RNA was reverse-transcribed to cDNA with SuperScript II Reverse Transcriptase (Invitrogen) following the manufacturer’s instructions. Primers used for amplification were for ATP1A1: 5’-TGTCCAGAATTGCAGGTCTTTG-3’ and 5’- TGCCCGCTTAAGAATAGGTAGGT-3’ (Murphy et al. 2004), for ATP1B1: 5’- TGATCCCAAGAGCTATGAGGC-3’ and 5’-GCACATCGCCACAATCTTCAAAA-3’, for SLC4A4: 5’-CGGCATCAAACCAAGAAATCC-3’ and 5’-CATGGCTGG GCTACCATTAT-3’, for SLC16A1: 5’-TTGGAGTCATTGGAGGTCTTG-3’ and 5’- TGGTCGCCTCTTGTAGAAATAC-3’, and for SLC16A3: 5’-CCATGCTCTACGGGACAGG- 3’ and 5’-GCTTGCTGAAGTAGCGGTT-3’. B2M was used as housekeeping gene and primers were 5′-GTGCTCGCGCTACTCTCTC-3′ and 5′-GTCAACTTCAATGTCGGAT-3′ (Boudjadi et al. 2013). Quantitative PCR was performed using a Rotor Gene Q real-time PCR Cycler (Qiagen). Synthesis of double-stranded DNA during PCR was monitored using Brilliant III UltraFast SYBR Green (Agilent Technologies). A standard calibration curve was prepared for each gene using serial dilutions of the calibrator sample. Crossing point values were plotted to the log of the relative concentration of each dilution. This standard curve was used to correct for differences in the efficiency of the PCR reactions. The relative gene expression was then calculated by the ΔΔCT method and normalized to B2M expression (Pfaffl 2001).

2.5.5 Trans-endothelial resistance and 10 kDa FiTC-Dextran permeability analysis

Cells were seeded in quadruplet on FNC-coated 12 mm 0.4 µm polycarbonate isopore membrane filters (Millipore PIHP01250) inserted in a 24-well plate. TEER was measured after each medium change (n=5 measures per insert). For the permeability analysis, 10 kDa Dextran-FiTC (40 µg/ml) was added into the insert. The plates were then incubated for 2.5 hours before collecting the medium from the bottom of the well. Fluorescence was measured using the CytoFluor serie 4000 PerSeptive Biosystems. Inserts without cells were used as a treshold control for both tests. Fibroblasts, which do not create polarized cell barrier, were used as a negative control.

2.5.6 Tissue engineering of the corneal endothelium

Corneas were devitalized as described previously, using three freeze-thaw cycles and stored at -20°C until use (Proulx et al. 2009). Before cell seeding, they were rinsed to remove dead cells. Corneas with detached Descemet’s membrane were discarded. Upon seeding, devitalized corneas were laid down, posterior side facing up, in a well of a six-well plate.

Cultured cells were seeded in 200 μL of culture medium on the Descemet’s membrane and allowed to adhere for four hours. The corneas were then immersed in culture medium and further cultured for two weeks.

2.5.7 Tissue fixation and immunofluorescence analysis

Corneas were embedded in Optimal Cutting Temperature compound (Somagen, Edmonton, Alberta, Canada) and frozen in liquid nitrogen. They were then stored at -80°C. Immunofluorescence analysis was performed as previously described (Proulx et al. 2009). Briefly, 10 μm cryosections were fixed for 10 minutes at -20°C in acetone (100%, EMD, Mississauga, Ontario, Canada) and immunostained with a mouse anti-Na+_/K+ _{ATPase α1} (Millipore, Billerica, MA, USA), a mouse anti-Na+_/K+ _{ATPase β1 (Thermo Scientific,} Waltham, MA, USA), a rabbit anti-Na+/HCO3- cotransporter (Chemicon, Temecula, CA, USA), a mouse anti-aquaporin 1 (Novus Biologicals, Oakville, ON, Canada), a mouse anti- monocarboxylate cotransporter 1 (Santa Cruz, Dallas, TX, USA) and a rabbit anti- monocarboxylate cotransporter 4 (Santa Cruz, Dallas, TX, USA) as primary antibodies. Goat anti-mouse (Invitrogen, Waltham, MA, USA) and chicken anti-rabbit (Invitrogen, Waltham, MA, USA) antibodies conjugated with Alexa 594 were used as secondary antibodies. The antibodies were diluted in phosphate buffer saline with 1% bovine serum albumin (Sigma- Aldrich, St. Louis, MO, USA) at room temperature for 1 hour with primary antibodies and 45 minutes with secondary antibodies. Nuclei were stained with Hoechst reagent 33258 (Sigma-Aldrich, St. Louis, MO, USA). The fluorescence was observed using a Zeiss Axio Image.Z2 microscope (Carl Zeiss, Toronto, ON, Canada). Immunofluorescence analysis omitting the primary antibody was used as negative control to distinguish background signal from primary antibody signal.

2.5.8 Statistical analyses

Statistical analyses consisted in Student's t-distribution test for differences in means. Mean values were reported with their standard deviation (SD). A p value of less than 0.05 was considered to be statistically significant. All statistical tests were two-sided. The analyses were conducted using the Microsoft Excel software.