Name: subretinal transplantation of human embryonic stem cell derived-retinal pigment epithelial cells into a large-eyed model of geographic atrophy
Uploaded: 2018-01-22T14:30:00
Description: Watch this Scientific Journal Video about Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy at JoVE.com

Denne undersøgelse blev støttet af tilskud fra Karolinska Institut, Kronprinsesse Margareta fundament for de svagsynede, Edwin Jordan Foundation for oftalmologisk forskning, den svenske øjet Foundation, Kong Gustav V Foundation, ARMEC Lindeberg Foundation, og Cronqvist Foundation.

Følgende protokol følger retningslinjerne dyrs pleje af Karolinska Instituet. Alle dyreforsøg ved hjælp af New Zealand albino kaniner (Tabel af materialer) er blevet godkendt af det regionale Dyreetik udvalg (Stockholms Norra Djurförsöksetiska Nämnd) (tillade: dnr 56/15). Brugen af menneskelige stamceller (dnr 2011/745-31/3) og overførsel og manipulation af menneskelige stamceller-ÅV (dnr 2013/813-31/2) er også i overensstemmelse med svensk lovgivning og Karolinska Institutet forordninger, og e...

<ol>
	<li>Plaza Reyes, 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=Xeno-Free+and+Defined+Human+Embryonic+Stem+Cell-Derived+Retinal+Pigment+Epithelial+Cells+Functionally+Integrate+in+a+Large-Eyed+Preclinical+Model.">Xeno-Free and Defined Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells Functionally Integrate in a Large-Eyed Preclinical Model.</a> Stem Cell Rep. 6 (1), 9-17 (2016).</li><li>Bartuma, 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=In+Vivo+Imaging+of+Subretinal+Bleb-Induced+Outer+Retinal+Degeneration+in+the+Rabbit.">In Vivo Imaging of Subretinal Bleb-Induced Outer Retinal Degeneration in the Rabbit.</a> Invest Ophthalmol Vis Sci. 56 (4), 2423-2430 (2015).</li><li>Petrus-Reurer, 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=Integration+of+Subretinal+Suspension+Transplants+of+Human+Embryonic+Stem+Cell-Derived+Retinal+Pigment+Epithelial+Cells+in+a+Large-Eyed+Model+of+Geographic+Atrophy.">Integration of Subretinal Suspension Transplants of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells in a Large-Eyed Model of Geographic Atrophy.</a> Invest Ophthalmol Vis Sci. 58 (2), 1314-1322 (2017).</li><li>Carido, 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=Characterization+of+a+mouse+model+with+complete+RPE+loss+and+its+use+for+RPE+cell+transplantation.">Characterization of a mouse model with complete RPE loss and its use for RPE cell transplantation.</a> Invest Ophthalmol Vis Sci. 55 (8), 5431-5444 (2014).</li><li>Lund, R. 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=Human+embryonic+stem+cell-derived+cells+rescue+visual+function+in+dystrophic+RCS+rats.">Human embryonic stem cell-derived cells rescue visual function in dystrophic RCS rats.</a> Cloning Stem Cells. 8 (3), 189-199 (2006).</li><li>Vugler, 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=Elucidating+the+phenomenon+of+HESC-derived+RPE:+anatomy+of+cell+genesis,+expansion+and+retinal+transplantation.">Elucidating the phenomenon of HESC-derived RPE: anatomy of cell genesis, expansion and retinal transplantation.</a> Exp Neurol. 214 (2), 347-361 (2008).</li><li>Schwartz, S. 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=Embryonic+stem+cell+trials+for+macular+degeneration:+a+preliminary+report.">Embryonic stem cell trials for macular degeneration: a preliminary report.</a> Lancet. 379 (9817), 713-720 (2012).</li><li>Hughes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+schematic+eye+for+the+rabbit.">A schematic eye for the rabbit.</a> Vision Res. 12 (1), 123-138 (1972).</li><li>Blanch, R. J., Ahmed, Z., Berry, M., Scott, R. A., Logan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+retinal+injury.">Animal models of retinal injury.</a> Invest Ophthalmol Vis Sci. 53 (6), 2913-2920 (2012).</li><li>Nork, T. 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=Functional+and+anatomic+consequences+of+subretinal+dosing+in+the+cynomolgus+macaque.">Functional and anatomic consequences of subretinal dosing in the cynomolgus macaque.</a> Arch Ophthalmol. 130 (1), 65-75 (2012).</li></ol>

I denne protokol, er generation af en stor-eyed model GA og dens prækliniske brug for evaluering af menneskelige stamceller-ÅV integration i vivo beskrevet.
For oversættelse af regenerative behandlinger for GA og beslægtede sygdomme til klinikken7er det vigtigt at udvikle og optimere metoder, der trofast fange de kliniske metoder til transplantation og billedbehandling. Kaninen er i dette attraktive aspekt: det har et forholdsvis stort øje, der tillader in...

Denne protokol beskriver generation af en stor-eyed prækliniske model af geografisk atrofi (GA), der giver mulighed for evaluering af integration af transplanterede menneskelige stamceller-RPE i subretinal plads. Metoderne beskrevet i detaljer her har været brugt i 3 seneste publikationer, der viser produktionen af en beriget, rene og funktionelle befolkning af ÅV celler fra menneskelige stamceller1, samt oprettelsen af ydre nethinde beskadige og en GA-lignende fænotype induceret af subretinal injektion af fysiologisk saltopløsninger (dvs., BSS og PBS) eller NaIO3 i kanin øje2,3. Vi viste yderligere, at sub retinal suspension transplantationer af menneskelige stamceller-ÅV danner omfattende funktionelle encellelag med fotoreceptor rednings kapacitet2.
Flere fordele ledsage brugen af kanin øje for generation af en GA model af sygdom. For det første størrelsen af kaninøjne, der er 70% volumen af en voksen menneskelige øje, giver klinisk meningsfulde transplantation ved hjælp af en celle tæthed, der er meget lavere end rutinemæssigt anvendes i små gnavere øjne (1000 celler/mikroliter vs 50.000 celler/µL)4 , 5. for det andet, kirurgi i gnavere er normalt transscleral gennem årehinden, som bringer den retinale barriere og potentielt udløser en inflammatorisk reaktion og en mulig afvisning6. Begge faktorer kan sammen føre til multilayering og sammenklumpning af transplanterede celler, og en generelt dårlig integration af de transplanterede celler i en forstyrret indfødte retinale væv. Men, den store-eyed kanin model giver mulighed for en kirurgisk teknik med instrumentation identisk med en klinisk indstilling. For det tredje tillader en stor-eyed model også høj opløsning i vivo billeddannelse og overvågning af de transplanterede celler og den overliggende nethinden gennem tid1,2,3. Dermed, vi beskriver en klinisk relevante og omkostningseffektive prækliniske model, der bør være et attraktivt alternativ til gnavere for alle med interesse for forskning af den normale og syge nethinde og sub retinal rummet.

<table >
	<tbody>
		<tr >
			<td >NutriStem hESC XF differentiation medium &#8211;bFGF and &#8211;TGFb</td>
			<td >Biological Industries</td>
			<td >06-5100-01-1A</td>
			<td ></td>
		</tr>
		<tr >
			<td >TrypLE Select 1x</td>
			<td >Gibco, ThermoFisher Scientific Corp</td>
			<td >12563-011</td>
			<td ></td>
		</tr>
		<tr >
			<td >PBS without Ca2+ and Mg2+</td>
			<td >Gibco, ThermoFisher Scientific Corp</td>
			<td >14190-094</td>
			<td></td>
		</tr>
		<tr >
			<td >Cell strainer 40 &#956;m Nylon</td>
			<td >VWR</td>
			<td >732-2757</td>
			<td ></td>
		</tr>
		<tr >
			<td >Needle 30g 0.5&#8217;&#8217;; 0.3 x 13mm</td>
			<td >BD Microlance</td>
			<td >304827</td>
			<td ></td>
		</tr>
		<tr >
			<td >Acrodisc 25mm Syringe Filter</td>
			<td >Acrodisc</td>
			<td >PN4612</td>
			<td></td>
		</tr>
		<tr >
			<td >0.4 % trypan blue</td>
			<td >ThermoFisher Scientific Corp</td>
			<td >15250061</td>
			<td >Use at 0.2%</td>
		</tr>
		<tr >
			<td >NaIO3</td>
			<td >Sigma-Aldrich Corp</td>
			<td>S4007</td>
			<td ></td>
		</tr>
		<tr >
			<td >BSS</td>
			<td >Alcon Nordic A/S</td>
			<td >65079550</td>
			<td ></td>
		</tr>
		<tr >
			<td >70% Ethanol</td>
			<td >Solveco AB</td>
			<td>1047</td>
			<td></td>
		</tr>
		<tr >
			<td >Ketaminol, 100 mg/mL</td>
			<td >Intervet, Boxmeer</td>
			<td >511519</td>
			<td >Use 35 mg/kg ketamine</td>
		</tr>
		<tr >
			<td >Rompun vet, 20 mg/mL</td>
			<td >Bayer Animal Health</td>
			<td >22545</td>
			<td >Use 5 mg/kg xylazine</td>
		</tr>
		<tr >
			<td >Triescence, 40 mg/mL</td>
			<td >Alcon Nordic A/S</td>
			<td >412915</td>
			<td >2 mg intraviterial</td>
		</tr>
		<tr >
			<td >Cyklopentolat-phenylephrine, 0.75% + 2.5%</td>
			<td >APL</td>
			<td >321968</td>
			<td >Use 1 drop in each eye</td>
		</tr>
		<tr >
			<td >Viscotears</td>
			<td>Laboratoires Th&#233;a</td>
			<td >597562</td>
			<td ></td>
		</tr>
		<tr >
			<td >Topical saline</td>
			<td >Apotea AB</td>
			<td>7053249369080</td>
			<td></td>
		</tr>
		<tr >
			<td >Allfatal vet. 100 mg/mL</td>
			<td >Omnidea</td>
			<td>77168</td>
			<td >Use 100 mg/mL pentobarbital</td>
		</tr>
		<tr >
			<td >Extension tube (Hammer)</td>
			<td >MedOne Surgical Inc</td>
			<td >3223</td>
			<td ></td>
		</tr>
		<tr >
			<td >25G/38G polytip subretinal cannula</td>
			<td >MedOne Surgical Inc</td>
			<td >3219</td>
			<td >25G/38G</td>
		</tr>
		<tr >
			<td >Single Use Flat Lens</td>
			<td >Volk</td>
			<td >#VWFD10</td>
			<td ></td>
		</tr>
		<tr >
			<td >Barraquer Colibri lid retractor</td>
			<td >AgnTho&#39;s AB</td>
			<td >42-020-030</td>
			<td></td>
		</tr>
		<tr >
			<td >Non-valved trocars</td>
			<td >Alcon Nordic A/S</td>
			<td>8065751448</td>
			<td></td>
		</tr>
		<tr >
			<td >Clawed forceps</td>
			<td >Bausch &#38; Lomb Nordic AB</td>
			<td >ET1811</td>
			<td></td>
		</tr>
		<tr >
			<td >Alcon Accurus 400VS Vitrectomy machine</td>
			<td >Alcon Nordic A/S</td>
			<td >8065740238</td>
			<td ></td>
		</tr>
		<tr >
			<td >Accurus 25+ Gauge Vitrectomy TotalL Plus Pak</td>
			<td >Alcon Nordic A/S</td>
			<td >8065751493</td>
			<td ></td>
		</tr>
		<tr >
			<td >SD-OCT device</td>
			<td>Heidelberg Engineering</td>
			<td>Spectralis HRA+OCT</td>
			<td>Use Heidelberg Eye Explorer version 1.9.10.0</td>
		</tr>
		<tr >
			<td >24 well plates</td>
			<td >Sarstedt</td>
			<td >83.3922</td>
			<td></td>
		</tr>
		<tr >
			<td >Neubauer hemocytometer</td>
			<td >VWR</td>
			<td >631-0925</td>
			<td></td>
		</tr>
		<tr >
			<td >New Zealand albino rabbits</td>
			<td>Lidk&#246;pings Rabbit Farm, Sweden</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >hESC-RPE cells</td>
			<td ></td>
			<td ></td>
			<td>See reference number 1</td>
		</tr>
	</tbody>
</table>

subretinal transplantation of human embryonic stem cell derived-retinal pigment epithelial cells into a large-eyed model of geographic atrophy

Geografisk atrofi (GA), den sene fase af tørre aldersrelateret makuladegeneration er karakteriseret ved tab af retinale pigment epitel (ÅV) lag, hvilket fører til efterfølgende degeneration af vitale retinal strukturer (fx, fotoreceptorer) forårsager alvorlig synsnedsættelse. Ligeledes er ÅV-tab og nedgang i synsstyrke set i langsigtede følger op af patienter med avanceret våd aldersrelateret makuladegeneration (AMD) intravitreal anti-vaskulær endotel vækstfaktor (VEGF) behandling. Derfor, på den ene side det er grundlæggende at effektivt udlede ÅV celler fra en ubegrænset kilde, der kunne tjene som substitutionsterapi. På den anden side er det vigtigt at vurdere adfærd og integration af de afledte celler i en model af sygdom indebærer kirurgisk og Billeddannende metoder som tæt som muligt på dem, der anvendes i mennesker. Her, vi fremlægge en detaljeret protokol baseret på vores tidligere publikationer, der beskriver generation af prækliniske model af GA albino kaninøjne for evaluering af den menneskelige embryonale stamceller afledt retinale pigment epitel celler (menneskelige stamceller-RPE) i en klinisk relevante indstilling. Differentierede menneskelige stamceller-ÅV kan transplanteres ind i naive øjne eller øjne med NaIO3-induceret GA-lignende retinal degeneration ved hjælp af en 25 G transvitreal pars plana teknik. Evaluering af degenererede og transplanterede områder udføres af multimodale høj opløsning non-invasiv real-time imaging.

Repræsentant i vivo billeder af BAF, IR-cSLO og SD-okt af en normal albino kanin nethinden er vist i figur 2. Bemærk de forskellige retinal lag med deres karakteristiske niveauer af lysrefleksion fanget af SD-okt-instrument.
I tallene 1A og figur 1B, setup hen til oprette sub retinale blebs er illustreret: en låget retraktoren e...

Watch this Scientific Journal Video about Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy at JoVE.com

Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy

Subretinal Transplantation af humane embryonale stamceller afledt retinal Pigment epitel celler ind i en stor-eyed Model af geografisk atrofi

Retinale pigment epitel celler kunne fungere som en celle-substitutionsterapi for avancerede form af tørre aldersrelateret makuladegeneration. Denne protokol beskriver generation af en stor-eyed model af geografisk atrofi og subretinal transplantation af humane embryonale stamceller-afledt retinale pigment epitel celler ind i denne model af sygdom.

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, ...

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