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Developmental Biology

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

Published: April 7th, 2016

DOI:

10.3791/54058

1Eye Program, Board of Governors Regenerative Medicine Institute, Departments of Biomedical Sciences and Neurosurgery, Cedars-Sinai Medical Center, 2David Geffen School of Medicine, University of California, Los Angeles

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.

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.

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 reduction1-4. An estimated 60-70% diabetics have various corneal problems1,3. 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 F5, 6. We have also documented significantly decreased expression of several putative epithe....

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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-e.......

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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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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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We gratefully acknowledge financial support by NIH/NEI R01 EY13431 (AVL), CTSI grant UL 1RR033176 (AVL), and grants from the Regenerative Medicine Institute, Cedars-Cedars Medical Center.

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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 microliter 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

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