Gli autori desiderano ringraziare Gregg Sealy e Alain Conti per l'eccellente assistenza tecnica e il Prof. Zsuzsanna Izsvák del Max-Delbrück Center di Berlino per aver gentilmente fornito i plasmidi pSB100X e pT2-CAGGS-Venus. Questo lavoro è stato sostenuto dalla Fondazione nazionale svizzera per le scienze e dalla Commissione europea nell'ambito del Settimo programma quadro. Z.I è stato finanziato dal Consiglio europeo della ricerca, ERC Advanced [ERC-2011-ADG 294742].

<ol>
	<li>Zareba, M., Raciti, M. W., Henry, M. M., Sarna, T., Burke, J. M. <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+in+ARPE-19+cultures:+Do+melanosomes+confer+cytoprotection.">Oxidative stress in ARPE-19 cultures: Do melanosomes confer cytoprotection.</a> Free Radical Biology and Medicine. 40 (1), 87-100 (2006).</li><li>Gong, X., Draper, C. S., Allison, G. S., Marisiddaiah, R., Rubin, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+macular+carotenoid+lutein+in+human+retinal+pigment+epithelial+cells.">Effects of the macular carotenoid lutein in human retinal pigment epithelial cells.</a> Antioxidants. 6 (4), (2017).</li><li>Sacconi, R., Corbelli, E., Querques, L., Bandello, F., Querques, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Review+of+current+and+future+management+of+geographic+atrophy.">A Review of current and future management of geographic atrophy.</a> Ophthalmology and Therapy. 6, 69-77 (2017).</li><li>Al-Zamil, W. M., Yassin, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+developments+in+age-related+macular+degeneration:+a+review.">Recent developments in age-related macular degeneration: a review.</a> Clinical Interventions in Aging. 12, 1313-1330 (2017).</li><li>Kumar-Singh, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+complement+membrane+attack+complex+in+dry+and+wet+AMD+-+From+hypothesis+to+clinical+trials.">The role of complement membrane attack complex in dry and wet AMD - From hypothesis to clinical trials.</a> Experimental Eye Research. 184, 266-277 (2019).</li><li>Ung, L., Pattamatta, U., Carnt, N., Wilkinson-Berka, J. L., Liew, G., White, A. J. R. <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+reactive+oxygen+species.">Oxidative stress and reactive oxygen species.</a> Clinical Science. 131, 2865-2883 (2017).</li><li>Beatty, S., Koh, H. H., Phil, M., Henson, D., Boulton, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+oxidative+stress+in+the+pathogenesis+of+age-related+macular+degeneration.">The role of oxidative stress in the pathogenesis of age-related macular degeneration.</a> Survey of Ophthalmology. 45 (2), 115-134 (2000).</li><li>Alhasani, R. H., 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=Gypenosides+protect+retinal+pigment+epithelium+cells+from+oxidative+stress.">Gypenosides protect retinal pigment epithelium cells from oxidative stress.</a> Food and Chemical Toxicology. 112, 76-85 (2018).</li><li>. National Institute of Health Available from: <a target="_blank" href="https://nei.nih.gov/learn-about-eye-health/resources-for-health-educators/eye-health-data-and-statistics/age-related-macular-degeneration-amd-data-and-statistics">https://nei.nih.gov/learn-about-eye-health/resources-for-health-educators/eye-health-data-and-statistics/age-related-macular-degeneration-amd-data-and-statistics</a> (2020)</li><li>Mitchell, P., Liew, G., Gopinath, B., Wong, T. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+Degenerative+Diseases:+Mechanisms+and+Experimental+Therapies.">Retinal Degenerative Diseases: Mechanisms and Experimental Therapies.</a> Retinal Degenerative Diseases. , 699-706 (2016).</li><li>Schallenberg, M., Charalambous, P., Thanos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GM-CSF+regulates+the+ERK1/2+pathways+and+protects+injured+retinal+ganglion+cells+from+induced+death.">GM-CSF regulates the ERK1/2 pathways and protects injured retinal ganglion cells from induced death.</a> Experimental Eye Research. 89, 665-677 (2009).</li><li>Schallenberg, M., Charalambous, P., Thanos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GM-CSF+protects+rat+photoreceptors+from+death+by+activating+the+SRC-dependent+signalling+and+elevating+anti-apoptotic+factors+and+neurotrophins.">GM-CSF protects rat photoreceptors from death by activating the SRC-dependent signalling and elevating anti-apoptotic factors and neurotrophins.</a> Graefes Archives for Clinical and Experimental Ophthalmology. 250, 699-712 (2012).</li><li>Thumann, G., 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=Engineering+of+PEDF-expressing+primary+pigment+epithelial+cells+by+the+SB+transposon+system+delivered+by+pFAR4+plasmids.">Engineering of PEDF-expressing primary pigment epithelial cells by the SB transposon system delivered by pFAR4 plasmids.</a> Molecular Therapy - Nucleic Acids. 6, 302-314 (2017).</li><li>Garcia-Garcia, 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=Long-term+PEDF+release+in+rat+iris+and+retinal+epithelial+cells+after+Sleeping+Beauty+transposon-mediated+gene+delivery.">Long-term PEDF release in rat iris and retinal epithelial cells after Sleeping Beauty transposon-mediated gene delivery.</a> Molecular Therapy - Nucleic Acids. 9, 1-11 (2017).</li><li>Johnen, 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=Antiangiogenic+and+neurogenic+activities+of+Sleeping+Beauty-mediated+PEDF-transfected+RPE+cells+in+vitro+and+in+vivo.">Antiangiogenic and neurogenic activities of Sleeping Beauty-mediated PEDF-transfected RPE cells in vitro and in vivo.</a> BioMed Research International. 2015, (2015).</li><li>Weigel, A. L., Handa, J. T., Hjelmeland, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+analysis+of+H2O2-,+HNE-,+or+tBH-treated+ARPE-19+cells.">Microarray analysis of H2O2-, HNE-, or tBH-treated ARPE-19 cells.</a> Free Radical Biology &amp; Medicine. 33 (10), 1419-1432 (2002).</li><li>Allen, R. G., Tresini, M. <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+gene+regulation.">Oxidative stress and gene regulation.</a> Free Radical Biology and Medicine. 28 (3), 463-499 (2000).</li><li>Tate, D. J., Miceli, M. V., Newsome, D. 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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+direct+correlation+between+the+levels+of+ascorbic+acid+and+H2O2+in+aqueous+humor.">A direct correlation between the levels of ascorbic acid and H2O2 in aqueous humor.</a> Experimental Eye Research. 38, 87-93 (1984).</li><li>Geiger, R. C., Waters, C. M., Kamp, D. W., Glucksberg, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KGF+prevents+oxygen-mediated+damage+in+ARPE-19+cells.">KGF prevents oxygen-mediated damage in ARPE-19 cells.</a> Investigative Ophthalmology and Visual Science. 46, 3435-3442 (2005).</li><li>Campochiaro, P. 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=Lentiviral+vector+gene+transfer+of+endostatin/angiostatin+for+macular+degeneration+(GEM)+study.">Lentiviral vector gene transfer of endostatin/angiostatin for macular degeneration (GEM) study.</a> Human Gene Therapy. 28, 99-111 (2017).</li><li>Chen, X. -. D., Su, M. -. Y., Chen, T. -. T., Hong, H. -. Y., Han, A. -. D., Li, W. -. 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W., Van Houten, B., Conklin, C. A., Jin, G. F., Godley, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+peroxide+causes+significant+mitochondrial+DNA+damage+in+human+RPE+cells.">Hydrogen peroxide causes significant mitochondrial DNA damage in human RPE cells.</a> Experimental Eye Research. 68 (6), 765-772 (1999).</li><li>Ma, 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=Transgenic+overexpression+of+uncoupling+protein+2+attenuates+salt-induced+vascular+dysfunction+by+inhibition+of+oxidative+stress.">Transgenic overexpression of uncoupling protein 2 attenuates salt-induced vascular dysfunction by inhibition of oxidative stress.</a> American Journal of Hypertension. 27 (3), 345-354 (2014).</li><li>Johnen, 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=Sleeping+Beauty+transposon-mediated+transfection+of+retinal+and+iris+pigment+epithelial+cells.">Sleeping Beauty transposon-mediated transfection of retinal and iris pigment epithelial cells.</a> Investigative Ophthalmology and Visual Science. 53 (8), 4787-4796 (2012).</li><li>M&#225;t&#233;s, 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=Molecular+evolution+of+a+novel+hyperactive+Sleeping+Beauty+transposase+enables+robust+stable+gene+transfer+in+vertebrates.">Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates.</a> Nature Genetics. 41 (6), 753-761 (2009).</li><li>. 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Il protocollo qui presentato offre un approccio per analizzare la funzione antiossidante e protettiva di PEDF e GM-CSF prodotti da cellule trasfettate, che possono essere applicate a cellule trasfettate con qualsiasi gene benefico putativo. Nelle strategie terapeutiche geniche che hanno l'obiettivo di fornire proteine ai tessuti trapiantando cellule geneticamente modificate, è fondamentale ottenere informazioni sul livello di espressione proteica, sulla longevità dell'espressione e sull'efficacia della proteina espress...

Il modello qui descritto, offre un approccio utile per valutare l'efficienza degli agenti biofarmaceutici per ridurre lo stress ossidativo nelle cellule. Abbiamo utilizzato il modello per studiare gli effetti protettivi di PEDF e GM-CSF sullo stress ossidativo mediato da H2O2sulle cellule epiteliali pigmentate retiniche, che sono esposte ad alti livelli di O2e luce visibile, e la fagocitosi delle membrane del segmento esterno dei fotorecettori, generando livelli significativi di specie reattive dell'ossigeno (ROS)1, 2. Sono considerati un importante contributo alla patogenesi della degenerazione maculare avascolare legata all'età (aAMD)3,4,5,6,7,8. Inoltre, vi è una diminuzione dei fattori neuroprotettivi sintetizzati RPE, in particolare il fattore derivato dall'epitelio pigmentato (PEDF), i fattori di crescita insulino-simili (IGF) e il fattore stimolante le colonie di macrofagi dei granulociti (GM-CSF) che porta alla disfunzione e alla perdita delle cellule RPE, seguito dalla morte dei fotorecettori e delle cellule gangliari retiniche (RGC)3,4,5 . L'AMD è una malattia complessa che deriva dall'interazione tra fattori metabolici, funzionali, genetici e ambientali4. La mancanza di trattamenti per l'aAMD è la principale causa di cecità nei pazienti di età superiore ai 60 anni nei paesiindustrializzati9,10. La ricostituzione dell'ambiente retinico neuroprotettivo e neurogeno mediante trapianto subretinico di cellule RPE geneticamente modificate sovraesprimendo PEDF e GM-CSF ha il potenziale per prevenire la degenerazione retinica mitigando gli effetti dello stress ossidativo, inibendo l'infiammazione e sostenendo la sopravvivenza cellulare11,12,13,14,15,16 . Anche se ci sono diverse metodologie per fornire geni alle cellule, abbiamo scelto il sistema di trasposoni iperattivi non virali della Bella Addormentata per fornire i geni PEDF e GM-CSF alle cellule RPE a causa del suo profilo di sicurezza, dell'integrazione dei geni nel genoma delle cellule ospiti e della sua propensione a integrare i geni consegnati in siti non trascrizionalmente attivi come abbiamo mostrato in precedenza17, 18,19.
Lo stress ossidativo cellulare può essere indotto in cellule coltivate in vitro da diversi agenti ossidativi, tra cui perossido di idrogeno (H2O2), 4-idronetonale (HNE), terzbutilidroperossido (tBH), alte tensioni di ossigeno e luce visibile (spettro completo o irradiazione UV)20,21. Le elevate tensioni di ossigeno e la luce richiedono attrezzature e condizioni speciali, che limitano la trasferibilità ad altri sistemi. Agenti come H2O2, HNE e tBH inducono cambiamenti molecolari e cellulari da stress ossidativo sovrapposti. Abbiamo scelto H2O2 per testare l'attività antiossidante di PEDF e GM-CSF perché è conveniente e biologicamente rilevante poiché è prodotto dalle cellule RPE come intermedio reattivo dell'ossigeno durante la fagocitosi del segmento esterno dei fotorecettori22 e si trova nei tessuti oculari in vivo23. Poiché l'ossidazione del glutatione può essere parzialmente responsabile della produzione di H2O2 nell'occhio, abbiamo analizzato i livelli di GSH / glutatione nei nostri studi, che sono legati allo stress ossidativo indotto da H2O2e alla capacità rigenerativa delle cellule21,22. L'analisi dei livelli di glutatione è particolarmente rilevante poiché partecipa ai meccanismi protettivi antiossidanti nell'occhio24. L'esposizione a H2O2 è usata frequentemente come modello per esaminare la suscettibilità allo stress ossidativo e l'attività antiossidante dellecellule RPE1,25,26, 27,28, 29,30e, inoltre, mostra somiglianze con il danno da stress ossidativo indotto dalla luce, una fonte "fisiologica" di stress ossidativo21.
Per valutare la funzionalità e l'efficacia dei fattori neuroprotettivi, abbiamo sviluppato un modello in vitro che consente l'analisi per quantificare l'effetto antiossidante dei fattori di crescita espressi da cellule geneticamente modificate per sovraesprimere PEDF e GM-CSF. Qui, mostriamo che le cellule RPE trasfettate con i geni per PEDF e GM-CSF sono più resistenti agli effetti dannosi di H2O2 rispetto alle cellule di controllo non trasfettate, come evidenziato dalla morfologia cellulare, densità, vitalità, livello intracellulare di glutatione ed espressione del gene UCP2, che codifica per la proteina di disaccoppiamento mitocondriale 2 che ha dimostrato di ridurre le specie reattive dell'ossigeno (ROS)31.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24-well plates</td>
			<td>Corning</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>Greiner</td>
			<td>7657160</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well culture plate white with clear flat bottom&#160;</td>
			<td>Costar</td>
			<td>3610</td>
			<td>Allows to check the cells before measuring the luminescence (GSH-Glo Assay)</td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>Corning&#160;</td>
			<td>353072</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylamid 40%</td>
			<td>Biorad</td>
			<td>161-0144</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>AMIMED</td>
			<td>4-05F00-H</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody anti-GMCSF</td>
			<td>ThermoFisher Scientific</td>
			<td>PA5-24184</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody anti-mouse IgG/IgA/IgM&#160;</td>
			<td>Agilent</td>
			<td>P0260</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody anti-PEDF&#160;</td>
			<td>Santa Cruz Biotechnology Inc</td>
			<td>sc-390172</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody anti-penta-His&#160;</td>
			<td>Qiagen</td>
			<td>34660</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody anti-phospho-Akt</td>
			<td>Cell Signaling Technology</td>
			<td>9271</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody anti-rabbit IgG H&#38;L-HRP</td>
			<td>Abcam</td>
			<td>ab6721</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody donkey anti-rabbit Alexa Fluor 594&#160;</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>A11034</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody goat anti-mouse Alexa 488</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11029</td>
			<td></td>
		</tr>
		<tr>
			<td>ARPE-19 cell line</td>
			<td>ATCC</td>
			<td>CRL-2302</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A9418-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>chamber culture glass slides</td>
			<td>Corning</td>
			<td>354118</td>
			<td></td>
		</tr>
		<tr>
			<td>CytoTox-Glo Cytotoxicity Assay&#160;</td>
			<td>Promega</td>
			<td>G9291</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/Ham`s F12&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>D8062</td>
			<td></td>
		</tr>
		<tr>
			<td>Duo Set ELISA kit</td>
			<td>R&#38;D Systems&#160;</td>
			<td>DY215-05</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>ThermoFisher Scientific</td>
			<td>78440</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISAquant kit</td>
			<td>BioProducts MD</td>
			<td>PED613-10-Human</td>
			<td></td>
		</tr>
		<tr>
			<td>Eyes (human)</td>
			<td>Lions Gift of Sight Eye Bank (Saint Paul, MN)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FBS&#160;</td>
			<td>Brunschwig</td>
			<td>P40-37500</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount Aqueous Mounting Medium</td>
			<td>Sigma-Aldrich</td>
			<td>F4680-25ML</td>
			<td></td>
		</tr>
		<tr>
			<td>FLUOstar Omega plate reader&#160;</td>
			<td>BMG Labtech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism software (version 8.0)</td>
			<td>GraphPad Software, Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GSH-Glo Glutathione Assay</td>
			<td>Promega</td>
			<td>V6912</td>
			<td></td>
		</tr>
		<tr>
			<td>hydrogen peroxide (H2O2)</td>
			<td>Merck</td>
			<td>107209</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ software (image processing program)</td>
			<td>W.S. Rasband, NIH, Bethesda, MD, USA; https://imagej.nih.gov/ij/; 1997&#8211;2014</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazol&#160;</td>
			<td>Axonlab</td>
			<td>A1378.0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica DMI4000B microscope&#160;</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LightCycler 480 Instrument II&#160;</td>
			<td>Roche Molecular Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LightCycler 480 SW1.5.1 software</td>
			<td>Roche Molecular Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>71376-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Axonlab</td>
			<td>3468.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Neon Transfection System&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>MPK5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Neon Transfection System 10 &#181;L Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>MPK1096</td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer chamber</td>
			<td>Marienfeld-superior</td>
			<td>640010</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA superflow&#160;</td>
			<td>Qiagen</td>
			<td>30410</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose&#160;</td>
			<td>VWR</td>
			<td>732-3197</td>
			<td></td>
		</tr>
		<tr>
			<td>Omega Lum G Gel Imaging System</td>
			<td>Aplegen Life Science</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 1X</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P0781-100</td>
			<td></td>
		</tr>
		<tr>
			<td>PerfeCTa SYBR Green FastMix</td>
			<td>Quantabio</td>
			<td>95072-012</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>158127-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>23227</td>
			<td></td>
		</tr>
		<tr>
			<td>Primers&#160;</td>
			<td>Invitrogen&#160;</td>
			<td></td>
			<td>See Table 1 in Supplementary Materials</td>
		</tr>
		<tr>
			<td>pSB100X (250 ng/&#181;L)</td>
			<td></td>
			<td></td>
			<td>M&#225;t&#233;s et al., 2009. Provide by Prof. Zsuzsanna Izsvak</td>
		</tr>
		<tr>
			<td>pT2-CMV-GMCSF-His plasmid DNA (250 ng/&#181;L)</td>
			<td></td>
			<td></td>
			<td>Constructed using the existing pT2-CMV-PEDF-EGFP plasmid reported in Johnen, S. et al. (2012) IOVS, 53 (8), 4787-4796.</td>
		</tr>
		<tr>
			<td>pT2-CMV-PEDF-His plasmid DNA (250 ng/&#181;L)</td>
			<td></td>
			<td></td>
			<td>Constructed using the existing pT2-CMV-PEDF-EGFP plasmid reported in Johnen, S. et al. (2012) IOVS, 53 (8), 4787-4796.</td>
		</tr>
		<tr>
			<td>QIAamp DNA Mini Kit</td>
			<td>QIAGEN</td>
			<td>51304</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant hGM-CSF&#160;</td>
			<td>Peprotech</td>
			<td>100-11</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant hPEDF&#160;&#160;</td>
			<td>BioProductsMD</td>
			<td>004-096</td>
			<td></td>
		</tr>
		<tr>
			<td>ReliaPrep RNA Cell Miniprep System</td>
			<td>Promega</td>
			<td>Z6011</td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA buffer</td>
			<td>ThermoFisher Scientific</td>
			<td>89901</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase-free DNase Set</td>
			<td>QIAGEN</td>
			<td>79254</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Mini Kit</td>
			<td>QIAGEN</td>
			<td>74204</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Applichem</td>
			<td>A2572</td>
			<td></td>
		</tr>
		<tr>
			<td>Semi-dry transfer system for WB&#160;</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SuperMix qScript</td>
			<td>Quantabio</td>
			<td>95048-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-buffered saline (TBS)&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>15504020</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>AppliChem</td>
			<td>A4975</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>T4174</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween</td>
			<td>AppliChem&#160;</td>
			<td>A1390</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>ThermoFisher Scientific</td>
			<td>29700</td>
			<td></td>
		</tr>
		<tr>
			<td>WesternBright ECL HRP substrate</td>
			<td>Advansta</td>
			<td>K-12045-D50</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman nitrocellulose membrane</td>
			<td>Chemie Brunschwig</td>
			<td>MNSC04530301</td>
			<td></td>
		</tr>
	</tbody>
</table>

induction and analysis of oxidative stress in sleeping beauty transposon-transfected human retinal pigment epithelial cells

Lo stress ossidativo svolge un ruolo fondamentale in diverse malattie degenerative, tra cui la degenerazione maculare legata all'età (AMD), una patologia che colpisce circa 30 milioni di pazienti in tutto il mondo. Porta ad una diminuzione dei fattori neuroprotettivi sintetizzati dall'epitelio pigmentato retinico (RPE), ad esempio il fattore derivato dall'epitelio pigmentato (PEDF) e il fattore stimolante le colonie di granulociti-macrofagi (GM-CSF), seguiti dalla perdita di cellule RPE e infine dalla morte dei fotorecettori e delle cellule gangliari retiniche (RGC). Ipotizziamo che la ricostituzione dell'ambiente retinico neuroprotettivo e neurogenico mediante trapianto subretinico di cellule RPE trasfettate sovraesprimendo PEDF e GM-CSF abbia il potenziale per prevenire la degenerazione retinica mitigando gli effetti dello stress ossidativo, inibendo l'infiammazione e sostenendo la sopravvivenza cellulare. Utilizzando il sistema di trasposone della Bella Addormentata nel Bosco(SB100X)le cellule RPE umane sono state trasfettate con i geni PEDF e GM-CSF e hanno mostrato un'integrazione genica stabile, un'espressione genica a lungo termine e una secrezione proteica utilizzando qPCR, western blot, ELISA e immunofluorescenza. Per confermare la funzionalità e la potenza del PEDF e del GM-CSF secreti dalle cellule RPE trasfettate, abbiamo sviluppato un test in vitro per quantificare la riduzione dello stress ossidativo indotto da H2O2sulle cellule RPE in coltura. La protezione cellulare è stata valutata analizzando la morfologia cellulare, la densità, il livello intracellulare di glutatione, l'espressione genica UCP2 e la vitalità cellulare. Sia le cellule RPE trasfettate che sovraesprimono PEDF e/o GM-CSF che le cellule non trasfettate ma pretrattate con PEDF e/o GM-CSF (disponibili in commercio o purificate da cellule trasfettate) hanno mostrato una significativa protezione delle cellule antiossidanti rispetto ai controlli non trattati. L'attuale modello H2O2è un approccio semplice ed efficace per valutare l'effetto antiossidante di fattori che possono essere efficaci per il trattamento di AMD o malattie neurodegenerative simili.

Induzione dello stress ossidativo nelle cellule epiteliali pigmentate retiniche umane ARPE-19 e cellule hRPE primarie sono state trattate con concentrazioni variabili di H2O2 per 24 ore ed è stato quantificato il livello intracellulare del glutatione antiossidante (Figura 2A,B). H2O2 a 50 μM e 100 μM non ha influenzato la produzione di glutatione, mentre a 350 μM c'è stata u...

Watch this Scientific Journal Video about Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells at JoVE.com

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Presentiamo un protocollo per lo sviluppo e l'uso di un modello di stress ossidativo trattando le cellule epiteliali pigmentate retiniche con H2O2, analizzando la morfologia cellulare, la vitalità, la densità, il glutatione e il livello di UCP-2. È un modello utile per studiare l'effetto antiossidante delle proteine secrete dalle cellule trasfettate con trasposone per trattare la degenerazione neuroretinica.

Induzione e analisi dello stress ossidativo nelle cellule epiteliali del pigmento retinico umano trasforato dalla bella addormentata nel bosco

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 ...

induction-analysis-oxidative-stress-sleeping-beauty-transposon

Research

JoVE Journal

Medicine

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

2.5K Views.  University of Geneva. Presentiamo un protocollo per lo sviluppo e l'uso di un modello di stress ossidativo trattando le cellule epiteliali pigmentate retiniche con H2O2, analizzando la morfologia cellulare, la vitalità, la densità, il glutatione e il livello di UCP-2. È un modello utile per studiare l'effetto antiossidante delle proteine secrete dalle cellule trasfettate con trasposone per trattare la degenerazione neuroretinica.

Video: Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome compared to matched normal tissue. Based on these analyses it is evident that there are many undiscovered genetic drivers of cancer1. Unfortunately these drivers are hidden within a much larger number of passenger anomalies in the genome that do not directly contribute to tumor formation. Another aspect of the cancer genome is that there is considerable genetic heterogeneity within similar tumor types. Each tumor can harbor different mutations that provide a selective advantage for tumor formation2. Performing an unbiased forward genetic screen in mice provides the tools to generate tumors and analyze their genetic composition, while reducing the background of passenger mutations. The Sleeping Beauty (SB) transposon system is one such method3. The SB system utilizes mobile vectors (transposons) that can be inserted throughout the genome by the transposase enzyme. Mutations are limited to a specific cell type through the use of a conditional transposase allele that is activated by Cre Recombinase. Many mouse lines exist that express Cre Recombinase in specific tissues. By crossing one of these lines to the conditional transposase allele (e.g. Lox-stop-Lox-SB11), the SB system is activated only in cells that express Cre Recombinase. The Cre Recombinase will excise a stop cassette that blocks expression of the transposase allele, thereby activating transposon mutagenesis within the designated cell type. An SB screen is initiated by breeding three strains of transgenic mice so that the experimental mice carry a conditional transposase allele, a concatamer of transposons, and a tissue-specific Cre Recombinase allele. These mice are allowed to age until tumors form and they become moribund. The mice are then necropsied and genomic DNA is isolated from the tumors. Next, the genomic DNA is subjected to linker-mediated-PCR (LM-PCR) that results in amplification of genomic loci containing an SB transposon. LM-PCR performed on a single tumor will result in hundreds of distinct amplicons representing the hundreds of genomic loci containing transposon insertions in a single tumor4. The transposon insertions in all tumors are analyzed and common insertion sites (CISs) are identified using an appropriate statistical method5. Genes within the CIS are highly likely to be oncogenes or tumor suppressor genes, and are considered candidate cancer genes. The advantages of using the SB system to identify candidate cancer genes are: 1) the transposon can easily be located in the genome because its sequence is known, 2) transposition can be directed to almost any cell type and 3) the transposon is capable of introducing both gain- and loss-of-function mutations6. The following protocol describes how to devise and execute a forward genetic screen using the SB transposon system to identify candidate cancer genes (Figure 1).

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

A method of identifying unknown drivers of carcinogenesis using an unbiased approach is described. The method uses the Sleeping Beauty transposon as a random mutagen directed to specific tissues. Genomic mapping of transposon insertions that drive tumor formation identifies novel oncogenes and tumor suppressor genes

Identificazione dei<em&gt; Bella Addormentata</em&gt; Inserimenti trasposone nei tumori solidi utilizzando Linker-mediata PCR

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome ...

Ricerca

Medicina

Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl

Retinal detachment (RD) describes a separation of the neurosensory retina from the retinal pigmented epithelium (RPE). The RPE is essential for normal function of the light sensitive neurons, the photoreceptors. Detachment of the retina from the RPE creates a physical gap that is filled with extracellular fluid. RD initiates cellular and molecular adverse events that affect both the neurosensory retina and the RPE since the physiological exchange of ions and metabolites is severely perturbed. The consequence for vision is related to the duration of the detachment since a rapid reapposition of the two tissues results in the restoration of vision 1. The treatment of RD is exclusively surgical. Removal of vitreous gel (vitrectomy) is followed by the removal non essential part of the retina around the detached area to favor retinal detachment. The removed retinal specimens are res nullius (nothing) and consequently normally discarded. To recover RNA from these surgical specimens, we developed the procedure jouRNAl that allows RNA conservation during the transfer from the surgical block to the laboratory. We also standardized a protocol to purify RNA by cesium chloride ultracentrifugation to assure that the purified RNAs are suitable for global gene expression analysis. The quality of the RNA was validated both by RT-PCR and microarray analysis. Analysis of the data shows a simultaneous involvement of inflammation and photoreceptor degeneration during RD.

We used retinal samples from retinectomy for a transcriptomic analysis of retinal detachment. We developed a procedure that allows RNA conservation between the surgical blocks and the laboratory. We standardized a protocol to purify RNA by cesium chloride ultracentrifugation to assure that the purified RNAs are suitable for microarray analysis.

Analisi trascrittomica di campioni chirurgici retiniche umane Utilizzo ufficiale

Retinal detachment (RD) describes a separation of the neurosensory retina  from the retinal pigmented epithelium (RPE). The RPE is essential for normal  ...

Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment epithelium (RPE) cells. RPE provide critical support for photoreceptors and death or dysfunction of RPE cells is characteristic of age-related macular degeneration (AMD), the leading cause of permanent vision loss in people age 55 and older. While no cure for AMD has been identified, implantation of healthy RPE in diseased eyes may prove to be an effective treatment, and large numbers of RPE cells can be readily generated from pluripotent stem cells. Several interesting questions regarding the safety and efficacy of RPE cell delivery can still be examined in animal models, and well-accepted protocols used to inject RPE have been developed. The technique described here has been used by multiple groups in various studies and involves first creating a hole in the eye with a sharp needle. Then a syringe with a blunt needle loaded with cells is inserted through the hole and passed through the vitreous until it gently touches the RPE. Using this injection method, which is relatively simple and requires minimal equipment, we achieve consistent and efficient integration of stem cell-derived RPE cells in between the host RPE that prevents significant amount of photoreceptor degeneration in animal models. While not part of the actual protocol, we also describe how to determine the extent of the trauma induced by the injection, and how to verify that the cells were injected into the subretinal space using in vivo imaging modalities. Finally, the use of this protocol is not limited to RPE cells; it may be used to inject any compound or cell into the subretinal space.

Here we present a community accepted protocol in multimedia format for subretinally injecting a bolus of RPE cells in rats and mice. This approach can be used for determining rescue potentials, safety profiles, and survival capacities of grafted RPE cells upon implantation in animal models of retinal degeneration.

Esecuzione sottoretinico Iniezioni in roditori di agire cellule retiniche epitelio pigmentato in sospensione

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Studiare cancro del pancreas staminali caratteristiche cellulari per lo sviluppo di nuove strategie di trattamento

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.
Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.
The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

Un passo per passo protocollo per la chirurgia sottoretinico in Conigli

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

L&#39;esaurimento delle cellule di topo da xenotrapianti tumorali umani migliora in modo significativo Analisi A valle delle cellule bersaglio

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Electroporation-Based Genetic Modification of Primary Human Pigment Epithelial Cells Using the Sleeping Beauty Transposon System

Our increasingly aging society leads to a growing incidence of neurodegenerative diseases. So far, the pathological mechanisms are inadequately understood, thus impeding the establishment of defined treatments. Cell-based additive gene therapies for the increased expression of a protective factor are considered as a promising option to medicate neurodegenerative diseases, such as age-related macular degeneration (AMD). We have developed a method for the stable expression of the gene encoding pigment epithelium-derived factor (PEDF), which is characterized as a neuroprotective and anti-angiogenic protein in the nervous system, into the genome of primary human pigment epithelial (PE) cells using the Sleeping Beauty (SB) transposon system. Primary PE cells were isolated from human donor eyes and maintained in culture. After reaching confluence, 1 x 104 cells were suspended in 11 &#181;L of resuspension buffer and combined with 2 &#181;L of a purified solution containing 30 ng of hyperactive SB (SB100X) transposase plasmid and 470 ng of PEDF transposon plasmid. Genetic modification was carried out with a capillary electroporation system using the following parameters: two pulses with a voltage of 1,100 V and a width of 20 ms. Transfected cells were transferred into culture plates containing medium supplemented with fetal bovine serum; antibiotics and antimycotics were added with the first medium exchange. Successful transfection was demonstrated in independently performed experiments. Quantitative polymerase chain reaction (qPCR) showed the increased expression of the PEDF transgene. PEDF secretion was significantly elevated and remained stable, as evaluated by immunoblotting, and quantified by enzyme-linked immunosorbent assay (ELISA). SB100X-mediated transfer allowed for a stable PEDF gene integration into the genome of PE cells and ensured the continuous secretion of PEDF, which is critical for the development of a cell-based gene addition therapy to treat AMD or other retinal degenerative diseases. Moreover, analysis of the integration profile of the PEDF transposon into human PE cells indicated an almost random genomic distribution.

We have developed a protocol to transfect primary human pigment epithelial cells by electroporation with the gene encoding pigment epithelium-derived factor (PEDF) using the Sleeping Beauty (SB) transposon system. Successful transfection was demonstrated by quantitative polymerase chain reaction (qPCR), immunoblotting, and enzyme-linked immunosorbent assay (ELISA).

Modificazione genetica basata sull'elettroporazione delle cellule epiteliali pigmentate umane primarie utilizzando il sistema di trasposone della bella addormentata nel bosco

Our increasingly aging society leads to a growing incidence of neurodegenerative diseases. So far, the pathological mechanisms are inadequately ...

Directed Induction of Retinal Organoids from Human Pluripotent Stem Cells

Retinal cell transplantation is a promising therapeutic approach, which could restore the retinal architecture and stabilize or even improve the visual capabilities to the degenerated retina. Nevertheless, progress in cell replacement therapy presently faces the challenges of requiring an off-the-shelf source of high quality and standardized human retinas. Therefore, an easy and stable protocol is needed for the experiments. Here, we develop an optimized protocol, based on a self-organizing method with the use of exogenous molecules and reagent A as well as manual excision to generate the three-dimensional human retina organoids (RO). The human Pluripotent Stem Cells (PSCs)-derived RO expresses specific markers for photoreceptors. With the addition of COCO, a multifunctional antagonist, the differentiation efficiency of photoreceptor precursors and cones is significantly increased. The efficient use of this system, which has the benefits of cell lines and primary cells, and without the sourcing issues associated with the latter, could produce confluent retinal cells, especially photoreceptors. Thus, the differentiation of PSCs to RO provides an optimal and biorelevant platform for disease modelling, drug screening and cell transplantation.

Using a self-organizing method, we develop a protocol with the addition of COCO that could significantly increase the generation of photoreceptors.

Induzione diretta di organoidi retinici da cellule staminali pluripotenti umane

Retinal cell transplantation is a promising therapeutic approach, which could restore the retinal architecture and stabilize or even improve the visual ...

Retinal Organoid Induction System for Derivation of 3D Retinal Tissues from Human Pluripotent Stem Cells

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to differentiate into all types of retinal cells, even mini-retinal tissues, hold huge promises for patients with these diseases and many opportunities in disease modeling and drug screening. However, the induction process from hPSCs to retinal cells is complicated and time-consuming. Here, we describe an optimized retinal induction protocol to generate retinal tissues with high reproducibility and efficiency, suitable for various human pluripotent stem cells. This protocol is performed without the addition of retinoic acid, which benefits the enrichment of cone photoreceptors. The advantage of this protocol is the quantification of EB size and plating density to significantly enhance the efficiency and repeatability of retinal induction. With this method, all major retinal cells sequentially appear and recapitulate the main steps of retinal development. It will facilitate downstream applications, such as disease modeling and cell therapy.

Here we describe an optimized retinal organoid induction system, which is suitable for various human pluripotent stem cell lines to generate retinal tissues with high reproducibility and efficiency.

Sistema di induzione organoide retinica per la derivazione di tessuti retinici 3D da cellule staminali pluripotenti umane

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to ...

Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These conditions include retinitis pigmentosa (RP) and advanced forms of age-related macular degeneration (AMD), such as geographic atrophy (GA). Together, these disorders represent a significant proportion of currently untreatable blindness globally. These unmet medical needs have generated heightened academic interest in developing methods of RPE replacement. Among the animal models commonly utilized for preclinical testing of therapeutics, the non-human primate (NHP) is the only animal model that has a macula. As it shares this anatomical similarity with the human eye, the NHP eye is an important and appropriate preclinical animal model for the development of advanced therapy medicinal products (ATMPs) such as RPE cell therapy.
This manuscript describes a method for the submacular transplantation of an RPE monolayer, cultured on a polyethylene terephthalate (PET) cell carrier, underneath the macula onto a surgically created RPE wound in immunosuppressed NHPs. The fovea-the central avascular portion of the macula-is the site of the greatest mechanical weakness during the transplantation. Foveal trauma will occur if the initial subretinal fluid injection generates an excessive force on the retina. Hence, slow injection under perfluorocarbon liquid (PFCL) vitreous tamponade is recommended with a dual-bore subretinal injection cannula at low intraocular pressure (IOP) settings to create a retinal bleb. 
Pretreatment with an intravitreal plasminogen injection to release parafoveal RPE-photoreceptor adhesions is also advised. These combined strategies can reduce the likelihood of foveal tears when compared to conventional techniques. The NHP is a key animal model in the preclinical phase of RPE cell therapy development. This protocol addresses the technical challenges associated with the delivery of RPE cellular therapy in the NHP eye.

The non-human primate (NHP) is an ideal model for studying human retinal cellular therapeutics due to the anatomical and genetic similarities. This manuscript describes a method for submacular transplantation of retinal pigment epithelial cells in the NHP eye and strategies to prevent intraoperative complications associated with macular manipulation.

Trapianto di epitelio pigmentato retinico in un modello di primati non umani per malattie degenerative della retina

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These ...