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

This study was supported by grants from the Karolinska Institute, the Crown Princess Margareta&#39;s Foundation for the Visually Impaired, the Edwin Jordan Foundation for Ophthalmological Research, the Swedish Eye Foundation, the King Gustav V Foundation, the ARMEC Lindeberg Foundation, and the Cronqvist Foundation.

The following protocol follows the animal care guidelines of Karolinska Instituet. All animal experiments using New Zealand albino rabbits (Table of Materials) have been approved by the regional animal ethics committee (Stockholms Norra Djurf&#246;rs&#246;ksetiska N&#228;mnd) (permit: dnr 56/15). The use of hESC (dnr 2011/745-31/3) and the transfer and manipulation of hESC-RPE (dnr 2013/813-31/2) is also in accordance with the Swedish legislation and Karolinska Institutet regulations, and has been approv...

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	<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>

In this protocol, the generation of a large-eyed model of GA and its preclinical use for evaluating hESC-RPE integration in vivo is described.
For translation of regenerative therapies for GA and related diseases into the clinic7, it is important to develop and optimize methods that faithfully capture the clinical methods for transplantation and imaging. The rabbit is in this aspect attractive: it has a relatively large eye that permits intraocular surgery and ...

This protocol describes the generation of a large-eyed preclinical model of geographic atrophy (GA) that allows the evaluation of integration of transplanted hESC-RPE in the subretinal space. The methods described in detail here have been used in 3 recent publications that demonstrate the production of an enriched, pure, and functional population of RPE cells from hESC1, as well as the creation of outer retinal damage and a GA-like phenotype induced by the subretinal injection of physiologic salt solutions (i.e., BSS and PBS) or NaIO3 in the rabbit eye2,3. We further demonstrated that sub-retinal suspension transplants of hESC-RPE form extensive functional monolayers with photoreceptor rescue capacity2.
Several advantages accompany the use of the rabbit eye for the generation of a GA model of disease. Firstly, the size of the rabbit eye, which is 70% the volume of an adult human eye, allows clinically meaningful transplantation using a cell density that is much lower than routinely used in small rodent eyes (1,000 cells/&#181;L vs. 50,000 cells/&#181;L)4,5. Secondly, surgery in rodents is usually transscleral through the choroid, which compromises the retinal barrier and potentially triggers an inflammatory response and a possible rejection6. Both factors together may lead to multilayering and clumping of transplanted cells, and an overall poor integration of the transplanted cells in a disrupted native retinal tissue. However, the large-eyed rabbit model allows performing a surgical technique with instrumentation identical to a clinical setting. Thirdly, a large-eyed model also permits high-resolution in vivo imaging and monitoring of the transplanted cells and the overlying retina through time1,2,3. Thus, we describe a clinically relevant and cost-efficient preclinical model that should be an attractive alternative to rodents for anyone with an interest in research of the normal and diseased retina and the sub-retinal space.

<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

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, which leads to subsequent degeneration of vital retinal structures (e.g., photoreceptors) causing severe vision impairment. Similarly, RPE-loss and decrease in visual acuity is seen in long-term follow up of patients with advanced wet age-related macular degeneration (AMD) receiving intravitreal anti-vascular endothelial growth factor (VEGF) treatment. Therefore, on the one hand, it is fundamental to efficiently derive RPE cells from an unlimited source that could serve as replacement therapy. On the other hand, it is important to assess the behavior and integration of the derived cells in a model of the disease entailing surgical and imaging methods as close as possible to those applied in humans. Here, we provide a detailed protocol based on our previous publications that describes the generation of a preclinical model of GA using the albino rabbit eye, for evaluation of the human embryonic stem cell derived retinal pigment epithelial cells (hESC-RPE) in a clinically relevant setting. Differentiated hESC-RPE are transplanted into naive eyes or eyes with NaIO3-induced GA-like retinal degeneration using a 25 G transvitreal pars plana technique. Evaluation of degenerated and transplanted areas is performed by multimodal high-resolution non-invasive real-time imaging.

Representative in vivo images of BAF, IR-cSLO, and SD-OCT of a normal albino rabbit retina are shown in Figure 2. Note the different retinal layers with their distinctive levels of light reflection captured by the SD-OCT instrument.
In Figures 1A and Figure 1B, the setup to create sub-retinal blebs is illustrated: a lid retractor i...

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

Retinal pigment epithelial cells could serve as a cell-replacement therapy for the advanced form of dry age-related macular degeneration. This protocol describes the generation of a large-eyed model of geographic atrophy and the subretinal transplantation of human embryonic stem cell-derived retinal pigment epithelial cells into this model of disease.

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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