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

Summary

An example of adenoviral gene therapy in the human diabetic organ-cultured corneas is presented towards the normalization of delayed wound healing and markedly reduced epithelial stem cell marker expression in these corneas. It also describes the optimization of this process in stem cell-enriched limbal epithelial cultures.

Abstract

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

Introduction

The diabetic corneal disease mainly results in degenerative epithelial (keratopathy) and nerve (neuropathy) changes. It is often manifested by the abnormalities of epithelial wound healing and corneal nerve reduction^1-4. An estimated 60-70% diabetics have various corneal problems^1,3. Our studies have identified several marker proteins with altered expression in human diabetic corneas including the downregulation of c-met proto-oncogene (hepatocyte growth factor receptor) and the upregulation of matrix metalloproteinase-10 (MMP-10) and cathepsin F^{5, 6}. We have also documented significantly decreased expression of several putative epithelial stem cell markers in the human diabetic corneas.

In the previous studies we have developed an adenoviral-based gene therapy to normalize the levels of diabetes-altered markers using human diabetic corneal organ culture system, which shows slow wound healing, diabetic marker changes, and stem cell marker expression reduction similar to the ex vivo corneas^7,8. This persistence of changes appears to be due to the existence of epigenetic metabolic memory⁹. This culture system was further used for gene therapy. The targets for this therapy were chosen from markers with either reduced expression in diabetic corneas (c-met proto-oncogene), or increased expression (MMP-10 and cathepsin F).

The adenoviral (AV) therapy was used in the whole organ-cultured corneas or the corneoscleral peripheral limbal compartment only. This compartment harbors epithelial stem cells that renew the corneal epithelium and actively participate in the wound healing^4,10-15. Here, protocols are provided for normal and diabetic human corneal organ culture, epithelial wound healing, isolation and characterization of stem cell-enriched limbal cell cultures, and adenoviral cell and corneal transduction. Our results show the feasibility of this therapy for normalizing marker expression and wound healing in diabetic corneas for possible future transplantation. They also suggest that the combination therapy is the most efficacious way to restore normal marker pattern and epithelial healing in the diabetic cornea^16-18.

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Protocol

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).

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Results

We have shown previously that in the corneal organ cultures, the differences in the expression of diabetic markers (e.g., basement membrane proteins and integrin α3β1) and wound healing between the normal and diabetic corneas are preserved. This culture system was subjected to the gene therapy aimed at normalizing the levels of diabetes-altered markers, c-met, MMP-10, and cathepsin F.

When the whole corneal ep...

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Discussion

The cornea appears to be an ideal tissue for gene therapy due to its surface location where the gene delivery, as well as the evaluation of efficacy and side effects, are easy. However, a clinical translation of this powerful approach is still slow due to scarce information on genetic causes of the corneal diseases and the gene therapy targets²⁴. Diabetic complications including corneal alterations may be largely epigenetic in nature, which translates into metabolic memory⁹. For this reason, the dia...

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Materials

Name	Company	Catalog Number	Comments
minimum essential medium	Thermo Fisher Scientific	11095-080
Optisol-GS	Bausch & Lomb	50006-OPT
ABAM antibiotic-antimycotic mixture	Thermo Fisher Scientific	15240062
calf skin collagen	Sigma-Aldrich	C9791
agar, tissue culture grade	Sigma-Aldrich	A1296
n-heptanol	Sigma-Aldrich	72954-5ML-F
O.C.T. compound	VWR International	25608-930
Dispase II	Roche Applied Science	4942078001
keratinocyte serum-free medium (KSFM)	Thermo Fisher Scientific	17005042
EpiLife medium with calcium	Thermo Fisher Scientific	MEPI500CA
N2 medium supplement, 100x	Thermo Fisher Scientific	17502-048
B27 medium supplement, 50x	Thermo Fisher Scientific	17504-044
human keratinocyte growth supplement, 100x	Thermo Fisher Scientific	S-001-5
trypsin 0.25% - EDTA 0.02% with phenol red	Thermo Fisher Scientific	25200056
trypsin 0.25% with phenol red	Thermo Fisher Scientific	15050065
soybean trypsin inhibitor	Sigma-Aldrich	T6414
fetal bovine serum	Thermo Fisher Scientific	26140079
insulin-transferrin-selenite supplement (ITS)	Sigma-Aldrich	I3146-5ML
antibody to keratin 14	Santa Cruz Biotechnology	sc-53253
antibody to keratin 15	Santa Cruz Biotechnology	sc-47697
antibody to keratin 17	Santa Cruz Biotechnology	SC-58726
antibody to ΔNp63α	Santa Cruz Biotechnology	sc-8609
antibody to PAX6	BioLegend	PRB-278P-100
antibody to nidogen-1	R&D Systems	MAB2570
antibody to integrin α3β1	EMD Millipore	MAB1992
human fibronectin	BD Biosciences	354008
human laminin	Sigma-Aldrich	L4445
human type IV collagen	Sigma-Aldrich	C6745-1ML
adenovirus expressing MMP-10 shRNA	Capital BioSciences	custom made
adenovirus expressing cathepsin F shRNA	Capital BioSciences	custom made
adenovirus expressing scrambled shRNA and GFP	Capital BioSciences	custom made
adenovirus expressing c-met	OriGene (plasmid)	SC323278
adenovirus expressing GFP	KeraFAST	FVQ002
sildenafil citrate, 25 mg	Pfizer	from pharmacy
epidermal growth factor	Thermo Fisher Scientific	PHG0311
poly-L-lysine	Sigma-Aldrich	P4707
polybrene	Sigma-Aldrich	107689-10G
ViraDuctin	Cell Biolabs	AD-200
ibiBoost	ibidi, Germany	50301
phosphate buffered saline (PBS)	Thermo Fisher Scientific	10010049
Corning round end spatula	Dow Corning	3005
60 mm Petri dishes	Thermo Fisher Scientific	174888
Nunc Lab-Tek II multiwell chamber slides	Sigma-Aldrich	C6807
200 μl pipet tips	Bioexpress	P-1233-200	other suppliers available
inverted microscope	Nikon	Diaphot	other suppliers/models available
humidified CO2 incubator	Thermo Fisher Scientific	370 (Steri-Cycle)	other suppliers/models available
fluorescent microscope	Olympus, Japan	BX-40	other suppliers/models available
dissecting stereo microscope	Leica, Germany	S4 E	other suppliers/models available