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.
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Method Article
Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells
In This Article
Summary
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 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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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 ....
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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 epithelium w.......
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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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Disclosures
The authors declare no financial interests.
Acknowledgements
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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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 |
References
- Bikbova, G., Oshitari, T., Tawada, A., Yamamoto, S. Corneal changes in diabetes mellitus. Curr Diabetes Rev. 8 (4), 294-302 (2012).
- Calvo-Maroto, A. M., Perez-Cambrodí, R. J., Albarán-Diego, C., Pons, A., Cerviño, A. Optical quality of the diabetic eye: a review. Eye (Lond). 28 (11), 1271-1280 (2....
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