Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

National Disease Research Interchange (NDRI, Philadelphia, PA) supplied consented post-mortem healthy and diabetic human eyes and corneas. NDRI's human tissue collection protocol is approved by the managerial committee and subject to National Institutes of Health oversight. This research has been conducted under the approved Cedars-Sinai Medical Center Institutional Review Board (IRB) exempt protocol EX-1055. Collaborating corneal surgeons, Drs. E. Maguen and Y. Rabinowitz, supplied discard corneoscleral rims for isolation of stem cell-enriched corneal epithelial cultures. This research has been conducted under the approved IRB protocol Pro00019393.

1. Human Corneal Organ Culture

Note: Normal and diabetic corneas or whole eyes are received in chilled corneal storage medium (e.g., Optisol GS) within 48 hr after death from the national supplier NDRI.

1. Remove the corneas from storage container with forceps and wash them twice in the antibiotic-antimycotic (ABAM) mixture. In case of whole eyes, cut the corneas out from the globes with curved scissors. Leave at least a 5 mm conjunctival rim and also wash the corneas in ABAM.
2. Prepare the mixture to be placed in the corneal concavity. It consists of serum-free minimum essential medium (MEM) containing ABAM, 1 mg/ml calf skin collagen (made from a stock solution of 10 mg/ml in 0.1 N acetic acid), 1% agar, and insulin-transferrin-selenite supplement⁷.
3. Microwave this mixture to boiling for sterilization and agar dissolution. It may take up to 1 min for everything to dissolve. Leave the tube cap partially open because the mixture overflows easily when it boils. For this reason, several boiling cycles may be needed, each for 10-15 sec. Cool down the mixture to 37-39 °C.
4. Place the corneas epithelial side down in sterile 60 mm dishes. Fill the corneal concavity with approximately 0.5 ml of the mixture described in 1.2. The mixture solidifies on the corneas within 2 min.
5. Place the corneas agar side down in sterile 60-mm dishes and add the full medium containing MEM with ABAM, and insulin-transferrin-selenite supplement⁷ (37 °C) to keep its level approximately at the limbus for air-liquid interface culture. The medium volume is 7-8 ml per dish.
6. Place the dishes with the corneas into a 5% CO₂ incubator humidified with a water pan (this way, humidity level is kept at or above 95%) at 35 °C (normal corneal temperature). Add 100 μl medium (two drops) daily on the epithelium to moisten the corneas.
7. Monitor the corneal morphology and the epithelial cell viability microscopically under transmitted light using a standard microscope with a 4X objective. Do so once a day.
8. Change the medium every three days. The corneas can be cultured for at least three weeks. For the gene therapy experiments, culture the corneas for 3-5 days, then transduce with genes of interest (4.1-4.4), leave for another 3-5 days depending on the transgene and subject to the wound healing protocol (2.1-2.4).

2. Epithelial Wound Healing

Note: In the diabetic corneas, the epithelial debridement by mechanical scraping is not feasible because the fragile epithelial basement membrane is detached in the process, which does not happen in the normal corneas. Thus, to keep the normal and diabetic corneas under similar conditions of wound healing, chemical removal of the epithelium with n-heptanol is used¹⁹. This procedure removes the epithelium but leaves behind an intact basement membrane in both the normal and diabetic corneas.

1. To perform central epithelial debridement²⁰, place a 5 mm filter paper disc soaked in n-heptanol on the central corneal anterior surface for 75 sec.
2. Remove the filter and wash the corneas in full medium. Fill the concavity with 0.5 ml agar-collagen mixture and culture as in 1.4. After debridement, the epithelial cells usually die and slough off leaving behind microscopically intact basement membrane⁷.
3. Use 8.5 mm disks to create larger epithelial wounds in the experiments with only limbal gene therapy. This will ensure the involvement of limbal stem cells in the healing process.
4. Monitor the corneal healing microscopically. Make photographs at 4X and 10X every day until the epithelial defect is completely healed. The healing process can take from 2 to 15 days depending on the state (normal or diabetic) and wound size.
   Note: During the healing process, black spider-like cells are observed at the top focal plane. These cells are apoptotic stromal keratocytes that died after the epithelial removal⁴. The healing is determined to be complete when these dead cells are overgrown by the healing epithelium and are no longer visible (Figure 2, inset).
5. After healing completion, cut the corneas in half, embed them in O.C.T. compound and process for indirect immunofluorescence on cryostat sections or for Western blots^16,21.

3. Isolation of Limbal Cells and Maintenance of Stem Cell-enriched Cultures

Note: Prepare the primary limbal epithelial stem cell (LESC)-enriched cultures from the corneoscleral rims. The rims coming from healthy donors and discarded after corneal transplantations are received from the collaborating surgeons in the standard corneal storage medium (e.g., Optisol). Otherwise, the LESC-enriched cultures can be obtained from the rims excised from normal and diabetic whole corneas or globes received from NDRI in the corneal storage medium.

1. If the whole corneas or globes are used, first isolate the limbal areas. To do this, place the cornea on a sterile plastic dish with epithelial side up and remove the central area with a 9-mm trephine (circular corneal knife). Discard this central part. Then excise the limbal zone using a 13 mm trephine and discard the outer conjunctival part. To isolate cells use the bagel-like limbal rim. Before cell isolation, remove the endothelial cells and adhering remnants of the iris from the inner side of the cornea opposite to the surface epithelium with a sterile cotton swab.
2. Isolate limbal cell sheets using dispase treatment. Incubate each corneoscleral rim with 2.4 U/ml dispase II in 1.5 ml keratinocyte serum-free medium (KSFM) supplemented with 10% fetal bovine serum (FBS) at 37 °C for 2 hr²².
3. Gently ease the limbal epithelial cell sheet off the rim under a dissecting stereo microscope with a forceps and dissociate the cells in 1 ml of 0.25% trypsin – 0.02% EDTA solution for 30 min at room temperature.
4. Wash the cells in 10 ml of medium (e.g., Epilife) and pellet them at 300 x g in a table-top centrifuge for 5 min at room temperature.
5. Resuspend the cells in culture medium: medium with N2, B27 and human keratinocyte growth supplements, and 10 ng/ml epidermal growth factor (EGF).
6. Seed the cells at 3,000-5,000 cells/ml in flasks or 60 mm dishes coated with a mixture of human basement membrane proteins including fibronectin, type IV collagen, and laminin (FCL), at 0.5-1 μg/cm², in the KSFM medium.
7. Culture the cells in a humidified (95% or higher humidity) CO₂ incubator at 37 °C until they form fully confluent monolayers. This may take 1-2 weeks.
   Note: Regularly confirm the cell identity by positive immunocytochemical staining for putative LESC markers including PAX6, keratin (K) 14, K15, K17, and ΔNp63α. The staining details including the primary antibodies have been published^8,16-18,22.
8. To passage the confluent cells, treat them with 1 ml 0.05% trypsin solution (1:5 dilution of the 0.25% solution) for 10 min at 37 °C. Add 5 ml soybean trypsin inhibitor solution (10 mg/ml) diluted 1:4 in medium, and centrifuge the cells as in 3.5.
9. Wash the cell pellet in 3 ml of diluted soybean trypsin inhibitor solution. Plate the cells onto FCL-coated (see 3.7) dishes or glass chamber slides at 2 x 10⁴ cells/ml.

4. Adenoviral Transduction of Organ-cultured Corneas

Note: The recombinant adenoviruses (AV) include AV-vector (no gene inserted), AV-cmet (with the c-met gene open reading frame), AV-shM10 (with shRNA to MMP-10), and AV-shCF (with shRNA to cathepsin F). They are E1/E3-deleted type 5 AV expressing genes under the control of the major immediate early cytomegalovirus promoter. The AV-cmet viruses are generated using AV vector pAd/CMV/V5-DEST¹⁶. The AV-shRNA viruses are custom generated by subcloning of shRNA sequences along with hH1 promoter and GFP tag sequence from iLenti-EGFP vector into a replication-incompetent (-E1/-E3) human AV type 5 genome using Adeno-4 expression system¹⁷.

1. Transduce one organ-cultured cornea of each pair with AV-vector and another cornea, with a gene expression-modulating AV. Use the AV-cmet at 0.8 to 1.25 x 10⁸ plaque-forming units (pfu) per cornea in culture medium for 48 hr at 37 °C keeping the corneas under the medium surface each in a well of a 24-well dish. Use the AV-shRNA viruses at 1-2 x 10⁸ pfu per cornea.
2. Use the viruses for transduction in full medium supplemented with 75 μg/ml sildenafil citrate. This reagent activates the caveolin transport facilitating viral uptake by the epithelial cells²¹. Due to short half-life of sildenafil in aqueous solutions, re-add the same amount to the medium 4-5 hr later. Standard treatment is for 48 hr.
3. Transfer corneas to new dishes with a round end sterile spatula and culture in the medium without AV keeping the medium level at the limbus. After the additional 4-8 days at the liquid-air interface, process the AV-treated corneas for various analyses or test for epithelial wound healing (see above). Routinely moisten the corneas during culture by adding 100 μl medium (two drops) on top of the cornea.
4. For the transduction of the limbal cells only, incubate the viruses with the corneas at the air-liquid interface with medium level at the limbus, to avoid transduction of the central epithelium.

5. Adenoviral Transduction of Cultured Limbal Cells

1. Maintain the LESC-enriched cultures in the medium with 10 ng/ml EGF (see 3.5) on plastic dishes coated with the FCL mixture at 0.5 μg/cm².
2. Transduce cultured cells that have reached 70-80% confluency with the AV harboring the green fluorescent protein gene (AV-GFP) or the AV-scrambled shRNA-GFP in a range of multiplicity of infection (MOI: 1-300 pfu/cell), in the medium with 2 ng/ml EGF. Perform the transduction for 4 hr at 37 °C in a minimal volume (0.2 ml) followed by 20 hr in 0.5 ml medium. The caveolin transport activator sildenafil is not needed for the cell culture transduction.
3. Use one of the following reagents that facilitate the AV binding to the cell surface to enhance the AV transduction efficiency: either polycations (1 μg/ml poly-L-lysine or 5 μg/ml polybrene), or 6 μl/ml transduction reagent (e.g., ViraDuctin), or 20 μl/ml transduction enhancer (e.g., ibiBoost).
4. After replacing the medium for the one without the AV, incubate cultured cells for 4 days, in the humidified CO₂ incubator and evaluate GFP expression level using an inverted fluorescent microscope.
5. Use the scratch wound healing assay to evaluate cell migration in the AV-transduced cultures. Grow the cells in the multiwell chamber slides. Make the wounds in a monolayer by scratching cells in a straight linear motion with a 200 μl pipette tip. After wounding, change the medium for a fresh one to remove the detached cells.
6. Photograph the wounds every day with a digital camera attached to an inverted microscope at 4X magnification. Record the time when the healing is complete (the wound edges come into contact along the entire wound).