Name: adenoviral gene therapy for diabetic keratopathy: effects on wound healing and stem cell marker expression in human organ-cultured corneas and limbal epithelial cells
Uploaded: 2016-04-07T15:00:00
Description: Watch this Scientific Journal Video about Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epitheli

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.

National Disease Research Interchange (NDRI, Philadelphia, PA) supplied consented post-mortem healthy and diabetic human eyes and corneas. NDRI&#39;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...

<ol>
	<li>Bikbova, G., Oshitari, T., Tawada, A., Yamamoto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+changes+in+diabetes+mellitus.">Corneal changes in diabetes mellitus.</a> Curr Diabetes Rev. 8 (4), 294-302 (2012).</li><li>Calvo-Maroto, A. M., Perez-Cambrod&#237;, R. J., Albar&#225;n-Diego, C., Pons, A., Cervi&#241;o, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+quality+of+the+diabetic+eye:+a+review.">Optical quality of the diabetic eye: a review.</a> Eye (Lond). 28 (11), 1271-1280 (2014).</li><li>Tripathy, K., Chawla, R., Sharma, Y. R., Venkatesh, P., Vohra, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+changes+in+diabetes+mellitus.">Corneal changes in diabetes mellitus.</a> DOS Times. 20 (5), 55-58 (2015).</li><li>Ljubimov, A. V., Saghizadeh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+corneal+wound+healing.">Progress in corneal wound healing.</a> Prog Retin Eye Res. 49, 17-45 (2015).</li><li>Saghizadeh, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+matrix+metalloproteinase-10+and+matrix+metalloproteinase-3+in+human+diabetic+corneas:+a+possible+mechanism+of+basement+membrane+and+integrin+alterations.">Overexpression of matrix metalloproteinase-10 and matrix metalloproteinase-3 in human diabetic corneas: a possible mechanism of basement membrane and integrin alterations.</a> Am J Pathol. 158 (2), 723-734 (2001).</li><li>Saghizadeh, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteinase+and+growth+factor+alterations+revealed+by+gene+microarray+analysis+of+human+diabetic+corneas.">Proteinase and growth factor alterations revealed by gene microarray analysis of human diabetic corneas.</a> Invest Ophthalmol Vis Sci. 46 (10), 3604-3615 (2005).</li><li>Kabosova, A., Kramerov, A. A., Aoki, A. M., Murphy, G., Zieske, J. D., Ljubimov, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+diabetic+corneas+preserve+wound+healing,+basement+membrane,+integrin+and+MMP-10+differences+from+normal+corneas+in+organ+culture.">Human diabetic corneas preserve wound healing, basement membrane, integrin and MMP-10 differences from normal corneas in organ culture.</a> Exp Eye Res. 77 (2), 211-217 (2003).</li><li>Saghizadeh, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+of+epithelial+stem+cell+marker+patterns+in+human+diabetic+corneas+and+effects+of+c-met+gene+therapy.">Alterations of epithelial stem cell marker patterns in human diabetic corneas and effects of c-met gene therapy.</a> Mol Vis. 17, 2177-2190 (2011).</li><li>Kowluru, R. A., Kowluru, A., Mishra, M., Kumar, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+and+epigenetic+modifications+in+the+pathogenesis+of+diabetic+retinopathy.">Oxidative stress and epigenetic modifications in the pathogenesis of diabetic retinopathy.</a> Prog Retin Eye Res. 48 (Sep), 40-61 (2015).</li><li>Lehrer, M. S., Sun, T. T., Lavker, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+of+epithelial+repair:+modulation+of+stem+cell+and+transit+amplifying+cell+proliferation.">Strategies of epithelial repair: modulation of stem cell and transit amplifying cell proliferation.</a> J Cell Sci. 111 (Pt 19), 2867-2875 (1998).</li><li>Lu, L., Reinach, P., Kao, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+epithelial+wound+healing.">Corneal epithelial wound healing.</a> Exp Biol Med. 226 (7), 653-664 (2001).</li><li>Rama, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limbal+stem-cell+therapy+and+long-term+corneal+regeneration.">Limbal stem-cell therapy and long-term corneal regeneration.</a> N Engl J Med. 363 (2), 147-155 (2010).</li><li>Di Girolamo, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracing+the+fate+of+limbal+epithelial+progenitor+cells+in+the+murine+cornea.">Tracing the fate of limbal epithelial progenitor cells in the murine cornea.</a> Stem Cells. 48 (1), 203-225 (2014).</li><li>Amitai-Lange, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage+tracing+of+stem+and+progenitor+cells+of+the+murine+corneal+epithelium.">Lineage tracing of stem and progenitor cells of the murine corneal epithelium.</a> Stem Cells. 33 (1), 230-239 (2015).</li><li>Di Girolamo, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moving+epithelia:+Tracking+the+fate+of+mammalian+limbal+epithelial+stem+cells.">Moving epithelia: Tracking the fate of mammalian limbal epithelial stem cells.</a> Prog Retin Eye Res. 48 (Sep), 203-225 (2015).</li><li>Saghizadeh, M., Kramerov, A. A., Yu, F. S., Castro, M. G., Ljubimov, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normalization+of+wound+healing+and+diabetic+markers+in+organ+cultured+human+diabetic+corneas+by+adenoviral+delivery+of+c-met+gene.">Normalization of wound healing and diabetic markers in organ cultured human diabetic corneas by adenoviral delivery of c-met gene.</a> Invest Ophthalmol Vis Sci. 51 (4), 1970-1980 (2010).</li><li>Saghizadeh, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+wound+healing,+kinase+and+stem+cell+marker+expression+in+diabetic+organ-cultured+human+corneas+upon+MMP-10+and+cathepsin+F+gene+silencing.">Enhanced wound healing, kinase and stem cell marker expression in diabetic organ-cultured human corneas upon MMP-10 and cathepsin F gene silencing.</a> Invest Ophthalmol Vis Sci. 54 (13), 8172-8180 (2013).</li><li>Saghizadeh, M., Dib, C. M., Brunken, W. J., Ljubimov, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normalization+of+wound+healing+and+stem+cell+marker+patterns+in+organ-cultured+human+diabetic+corneas+by+gene+therapy+of+limbal+cells.">Normalization of wound healing and stem cell marker patterns in organ-cultured human diabetic corneas by gene therapy of limbal cells.</a> Exp Eye Res. 129 (Dec), 66-73 (2014).</li><li>Hatchell, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Damage+to+the+epithelial+basement+membrane+in+the+corneas+of+diabetic+rabbits.">Damage to the epithelial basement membrane in the corneas of diabetic rabbits.</a> Arch Ophthalmol. 101 (3), 469-471 (1983).</li><li>Chung, J. H., Kim, W. K., Lee, J. S., Pae, Y. S., Kim, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+topical+Na-hyaluronan+on+hemidesmosome+formation+in+n-heptanol-induced+corneal+injury.">Effect of topical Na-hyaluronan on hemidesmosome formation in n-heptanol-induced corneal injury.</a> Ophthalmic Res. 30 (2), 96-100 (1998).</li><li>Saghizadeh, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-driven+overexpression+of+proteinases+in+organ-cultured+normal+human+corneas+leads+to+diabetic-like+changes.">Adenovirus-driven overexpression of proteinases in organ-cultured normal human corneas leads to diabetic-like changes.</a> Brain Res Bull. 81 (2-3), 262-272 (2010).</li><li>Sareen, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+human+limbal-derived+induced+pluripotent+stem+cells+into+limbal-like+epithelium.">Differentiation of human limbal-derived induced pluripotent stem cells into limbal-like epithelium.</a> Stem Cells Transl Med. 3 (9), 1002-1012 (2014).</li><li>Funari, V. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentially+expressed+wound+healing-related+microRNAs+in+the+human+diabetic+cornea.">Differentially expressed wound healing-related microRNAs in the human diabetic cornea.</a> PLoS One. 8 (12), e84425 (2013).</li><li>Mohan, R. R., Rodier, J. T., Sharma, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+gene+therapy:+basic+science+and+translational+perspective.">Corneal gene therapy: basic science and translational perspective.</a> Ocul Surf. 11 (3), 150-164 (2013).</li><li>Liu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+tropism+of+adenoviruses+and+adeno-associated+viruses+to+corneal+cells:+implications+for+corneal+gene+therapy.">Different tropism of adenoviruses and adeno-associated viruses to corneal cells: implications for corneal gene therapy.</a> Mol Vis. 14, 2087-2096 (2008).</li><li>Thomas, C. E., Ehrhardt, A., Kay, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+problems+with+the+use+of+viral+vectors+for+gene+therapy.">Progress and problems with the use of viral vectors for gene therapy.</a> Nat Rev Genet. 4 (5), 346-358 (2003).</li><li>Sharma, G. D., He, J., Bazan, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P38+and+ERK1/2+coordinate+cellular+migration+and+proliferation+in+epithelial+wound+healing:+evidence+of+cross-talk+activation+between+MAP+kinase+cascades.">P38 and ERK1/2 coordinate cellular migration and proliferation in epithelial wound healing: evidence of cross-talk activation between MAP kinase cascades.</a> J Biol Chem. 278 (24), 21989-21997 (2003).</li><li>Saika, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+p38+MAP+kinase+in+regulation+of+cell+migration+and+proliferation+in+healing+corneal+epithelium.">Role of p38 MAP kinase in regulation of cell migration and proliferation in healing corneal epithelium.</a> Invest Ophthalmol Vis Sci. 45 (1), 100-109 (2004).</li><li>Xu, K. P., Li, Y., Ljubimov, A. V., Yu, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+glucose+suppresses+epidermal+growth+factor+receptor/phosphatidylinositol+3-kinase/Akt+signaling+pathway+and+attenuates+corneal+epithelial+wound+healing.">High glucose suppresses epidermal growth factor receptor/phosphatidylinositol 3-kinase/Akt signaling pathway and attenuates corneal epithelial wound healing.</a> Diabetes. 58 (5), 1077-1085 (2009).</li><li>Xu, K., Yu, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+epithelial+wound+healing+and+EGFR+signaling+pathways+in+the+corneas+of+diabetic+rats.">Impaired epithelial wound healing and EGFR signaling pathways in the corneas of diabetic rats.</a> Invest Ophthalmol Vis Sci. 52 (6), 3301-3308 (2011).</li><li>Takamura, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aldose+reductase+inhibitor+counteracts+the+enhanced+expression+of+matrix+metalloproteinase-10+and+improves+corneal+wound+healing+in+galactose-fed+rats.">Aldose reductase inhibitor counteracts the enhanced expression of matrix metalloproteinase-10 and improves corneal wound healing in galactose-fed rats.</a> Mol Vis. 19, 2477-2486 (2013).</li><li>Byun, Y. S., Kang, B., Yoo, Y. S., Joo, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(ADP-ribose)+polymerase+inhibition+improves+corneal+epithelial+innervation+and+wound+healing+in+diabetic+rats.">Poly(ADP-ribose) polymerase inhibition improves corneal epithelial innervation and wound healing in diabetic rats.</a> Invest Ophthalmol Vis Sci. 56 (3), 1948-1955 (2015).</li><li>Deng, S. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+limbal+stem+cell+deficiency+by+in+vivo+laser+scanning+confocal+microscopy:+a+microstructural+approach.">Characterization of limbal stem cell deficiency by in vivo laser scanning confocal microscopy: a microstructural approach.</a> Arch Ophthalmol. 130 (4), 440-445 (2012).</li><li>Lagali, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+morphology+of+the+limbal+palisades+of+Vogt+correlates+with+progressive+stem+cell+deficiency+in+aniridia-related+keratopathy.">In vivo morphology of the limbal palisades of Vogt correlates with progressive stem cell deficiency in aniridia-related keratopathy.</a> Invest Ophthalmol Vis Sci. 54 (8), 5333-5342 (2013).</li></ol>

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...

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 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 corneas7,8. 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 healing4,10-15. 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 cornea16-18.

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		<tr height="20">
			<td height="20" style="height:20px;width:384px;">minimum essential medium</td>
			<td style="width:277px;">Thermo Fisher Scientific</td>
			<td style="width:169px;">11095-080</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Optisol-GS&#160;</td>
			<td>Bausch &#38; Lomb</td>
			<td>50006-OPT</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ABAM antibiotic-antimycotic mixture</td>
			<td>Thermo Fisher Scientific</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">calf skin collagen&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>C9791</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">agar, tissue culture grade</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>A1296</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">n-heptanol</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>72954-5ML-F</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">O.C.T. compound&#160;</td>
			<td>VWR International</td>
			<td>25608-930</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dispase II&#160;</td>
			<td>Roche Applied Science</td>
			<td>4942078001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">keratinocyte serum-free medium (KSFM)&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>17005042</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EpiLife medium with calcium</td>
			<td>Thermo Fisher Scientific</td>
			<td>MEPI500CA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N2 medium supplement, 100x</td>
			<td>Thermo Fisher Scientific</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B27 medium supplement, 50x</td>
			<td>Thermo Fisher Scientific</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">human keratinocyte growth supplement, 100x</td>
			<td>Thermo Fisher Scientific</td>
			<td>S-001-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">trypsin 0.25% - EDTA 0.02% with phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">trypsin 0.25% with phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>15050065</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">soybean trypsin inhibitor&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>T6414</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">fetal bovine serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">insulin-transferrin-selenite supplement (ITS)</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>I3146-5ML</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">antibody to keratin 14</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-53253</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">antibody to keratin 15</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-47697</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">antibody to keratin 17</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-58726</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">antibody to &#916;Np63&#945;</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-8609</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">antibody to PAX6</td>
			<td>BioLegend</td>
			<td>PRB-278P-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">antibody to nidogen-1</td>
			<td>R&#38;D Systems</td>
			<td>MAB2570</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">antibody to integrin &#945;3&#946;1</td>
			<td>EMD Millipore</td>
			<td>MAB1992</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">human fibronectin</td>
			<td>BD Biosciences</td>
			<td>354008</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">human laminin</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>L4445</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">human type IV collagen</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>C6745-1ML</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">adenovirus expressing MMP-10 shRNA</td>
			<td>Capital BioSciences</td>
			<td>custom made</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">adenovirus expressing cathepsin F shRNA</td>
			<td>Capital BioSciences</td>
			<td>custom made</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">adenovirus expressing scrambled shRNA and GFP</td>
			<td>Capital BioSciences</td>
			<td>custom made</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">adenovirus expressing c-met</td>
			<td>OriGene (plasmid)</td>
			<td>SC323278</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">adenovirus expressing GFP</td>
			<td>KeraFAST</td>
			<td>FVQ002</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">sildenafil citrate, 25 mg</td>
			<td>Pfizer</td>
			<td>from pharmacy</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">epidermal growth factor&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>PHG0311</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">poly-L-lysine</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>P4707</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">polybrene</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>107689-10G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ViraDuctin</td>
			<td>Cell Biolabs</td>
			<td>AD-200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ibiBoost</td>
			<td>ibidi, Germany</td>
			<td>50301</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">phosphate buffered saline (PBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010049</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Corning round end spatula&#160;</td>
			<td>Dow Corning</td>
			<td>3005</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">60-mm petri dishes</td>
			<td>Thermo Fisher Scientific</td>
			<td>174888</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nunc Lab-Tek II multiwell chamber slides&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C6807</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">200 microliter pipet tips</td>
			<td>Bioexpress</td>
			<td>P-1233-200</td>
			<td>other suppliers available</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">inverted microscope&#160;</td>
			<td>Nikon</td>
			<td>Diaphot</td>
			<td>other suppliers/models available</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">humidified CO2 incubator&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>370 (Steri-Cycle)</td>
			<td>other suppliers/models available</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">fluorescent microscope</td>
			<td>Olympus, Japan</td>
			<td>BX-40</td>
			<td>other suppliers/models available</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">dissecting stereo microscope</td>
			<td>Leica, Germany</td>
			<td>S4 E</td>
			<td>other suppliers/models available</td>
		</tr>
	</tbody>
</table>

adenoviral gene therapy for diabetic keratopathy: effects on wound healing and stem cell marker expression in human organ-cultured corneas and limbal epithelial cells

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.

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 &#945;3&#946;1) 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...

Watch this Scientific Journal Video about Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epitheli

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

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 ...

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