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 approved by the regional human ethics committee (Regionala Etikpr&#246;vningsn&#228;mnden i Stockholm).
1. Subretinal Injection of Sodium Iodate (NaIO3) into a Large-eyed Animal Model
<ol>
	<li>Anesthetize animals by intramuscular administration in the thigh with a mixture of&#160;35 mg/kg ketamine and 5 mg/kg xylazine in saline using a 30 G syringe, and dilate pupils with topical eyedrops using a mix of 0.75% cyclopentolate and 2.5% phenylephrine. Proper anesthetization is confirmed if the animal does not react to a hard pinch of its back leg.</li>
	<li>Place the rabbit under the surgical microscope with the head facing the surgeon (Figure 1A). Use a lid retractor to remove eyelids and nictitans membrane with a sterile cloth to minimize the risk of contamination. Use balanced salt solution (BSS) to prevent dryness while under anesthesia in both eyes.</li>
	<li>For microsurgery, use a 2-port (or optional 3-port) 25 G transvitreal pars plana technique with non-valved trocars for the insertion of microsurgical instruments (Figure 1B). The multifunction vitrectomy machine has ports to connect an infusion cannula, endoillumination, vitrector, and endolaser. For subretinal injections, only the endoillumination is mandatory, which makes a 2-port set-up sufficient. Insert the endoillumation through the upper left trocar and use the upper right trocar for the subretinal injection cannula.
	<ol>
		<li>For a 3-port set-up, use the lower temporal trocar for the BSS infusion cannula. If optional instruments (such as a vitrector) are used, insert them through the upper right trocar.</li>
		<li>Insert the upper 2 trocars 1-2 mm from the limbus using clawed forceps to grab and displace the conjunctiva overlying the insertion site.</li>
		<li>Make sure the trocars are inserted transsclerally at a 30-45&#176; limbus parallel angle, and proceed to the middle of the tip of the trocar. Then turn the trocar 90&#176; and advance into the eye, aiming at the posterior pole of the eye. This procedure will avoid post-surgical leakage from the sclerotomies, and also decrease the risk of endophthalmitis (i.e., bacterial infection in the eye). See also Figure 2B for trocar positions.</li>
		<li>Put a single use flat contact lens on the cornea to visualize the retina, with synthetic tears as contact gel between the eye and the contact lens (Figure 1A).</li>
		<li>Draw 500 &#181;L of NaIO3 into a 1 mL syringe connected to an extension tube (operated by the assistant) and a 38 G polytip cannula (operated by the surgeon).</li>
		<li>Insert the endoillumination probe through the upper left trocar and the injection cannula through the upper right trocar, and advance the cannula through the vitreous space towards the retina, aiming for the area just below the optic nerve head.</li>
		<li>Allow the tip of the cannula to slowly touch the retina until a focal whitening is visible. The injection itself will penetrate the retina, allowing for subretinal delivery. Do not let the cannula penetrate the retina, since this may cause hemorrhage.</li>
		<li>Inject 50 &#181;L of NaIO3 subretinally over a 5 s period. Since there is a natural cleavage plane between the retina and the underlying choroid, a clearly visible semitransparent bleb should gradually form during the injection.</li>
		<li>During the injection, slowly retract the needle, but make sure the tip is maintained within the bleb to minimize reflux.</li>
		<li>After removal of the endoillumation and injection cannula, remove the trocars using clawed forceps, and apply light pressure for 30 s to the self-sealing suture-less sclerotomies using the tip or the blunt end of the forceps.</li>
	</ol>
	</li>
	<li>Post-surgically give 10&#160;mL of saline subcutaneously to prevent dehydration.&#160;Do not give&#160;post-surgical&#160;topical steroids or antibiotics.&#160;For analgesics give 0.5 mL of buprenorphine 0.3 mg/ml subcutaneously after surgery, as well as the day after surgery.</li>
	<li>After usage, wash all instruments by immersing them for a couple of seconds firstly in 70% ethanol, secondly in 45% ethanol, and lastly in distilled water. Dry them properly with a paper towel.</li>
	<li>Attend animals until they regain sufficient consciousness and place them all single encaged. If required (e.g., for immunohistochemistry purposes), euthanize animals by intravenous injection of 100 mg/kg pentobarbital (see Table of Materials).</li>
	<li>Wait 7 days to proceed with the transplantation of hESC-RPE cells.</li>
</ol>
2. Subretinal Transplantation of hESC-RPE Cells in Treated Animals
<ol>
	<li>Administer 2 mg (100 &#181;L) of intravitreal triamcinolone in anesthetized animals using a 30 g injection needle inserted 1-2 mm from the limbus in the lower temporal quadrant 1 week before transplantation of hESC-RPE, and re-administer it every 3 months. Make sure to point the tip towards the posterior pole of the eye to avoid a lens touch.</li>
	<li>Culture a hESC-RPE monolayer as described previously1.</li>
	<li>Remove cell differentiation media (see Table of Materials) of a 24-well confluent hESC-RPE monolayer and wash each well with 500 &#181;L of PBS without Ca2+ and Mg2+. Repeat this action once more, for a total of 2 washes.</li>
	<li>Discard supernatant, add 500 &#181;L of trypsin per well, and incubate for 12 min at 37 &#176;C.</li>
	<li>Tilt the plate and carefully remove trypsin (cells should remain attached to the plate). Collect cells in 800 &#181;L of fresh 37 &#176;C prewarmed differentiation media by gentle pipetting, or even scraping if needed, to obtain a single cell suspension. 
	NOTE: Use a 40 &#181;m cell strainer if cell clumps are observed.</li>
	<li>Count cells in a hemocytometer chamber using 0.2% Trypan Blue, according to the manufacturer&#39;s instructions.</li>
	<li>Add 5 mL of differentiation media and centrifuge cells at room temperature, 300 x g for 5 min.</li>
	<li>Discard supernatant and resuspend pellet in freshly filter-sterilized PBS (passed through a 25 mm syringe filter) to a final concentration of 1,000 cells/&#181;L.</li>
	<li>Aliquot the previous cell suspension into 600 &#181;L aliquots and keep on ice until and during surgery.</li>
	<li>Anesthetize animals by intramuscular administration in the thigh with a mixture of&#160;35 mg/kg ketamine and 5 mg/kg xylazine in saline using a 30 G syringe. Dilate pupils with topical eyedrops using amix of 0.75% cyclopentolate and 2.5% phenylephrine. Proper anesthetization is confirmed if the animal does not react to a hard pinch in its back leg.</li>
	<li>Place the rabbit with the head facing the surgeon. Use a lid retractor to remove eyelids and nictitans membrane with a sterile cloth to minimize the risk of contamination. Use BSS to prevent dryness while under anesthesia in both eyes.</li>
	<li>For microsurgery, use a 2-port (or optional 3-port) 25 G transvitreal pars plana technique with trocars placed in the same positions as described in step 1.3 and Figure 2. If the previous sclerotomies are visible, perform trocar insertion just adjacent, but not through these, to minimize the risk of postoperative leakage.
	<ol>
		<li>Put a single use flat contact lens on the cornea to visualize the retina, with synthetic tears as the contact gel between the eye and the contact lens (Figure 1A).</li>
		<li>After proper tip positioning (see steps 1.3.5 and 1.3.6), inject 50 &#181;L of a gently-mixed hESC-RPE suspension (50,000 cells) subretinally. Aim for the center of the NaIO3 pretreated area distinguished by a characteristic &#34;metallic&#34; endo-illumination reflex. The neurosensory retina should separate easily creating a clearly visible bleb. Flush the needle with sterile H2O in between rabbits/after use to avoid needle clogging due to cell clumps. Change the needle when clogging is noticed.</li>
		<li>During the injection, slowly retract the needle but make sure the tip is maintained within the bleb to minimize reflux.</li>
		<li>After removal of the endoillumation and injection cannula, remove the trocars using clawed forceps, and apply light pressure for 30 s to the self-sealing suture-less sclerotomies using the tip or the blunt end of the forceps.</li>
	</ol>
	</li>
	<li>Post-surgically give 10&#160;mL of saline subcutaneously to prevent dehydration.&#160;Do not give&#160;post-surgical&#160;topical steroids or antibiotics.&#160;For analgesics give 0.5 mL of buprenorphine 0.3 mg/ml subcutaneously after surgery, as well as the day after surgery.</li>
	<li>After usage, wash all instruments by immersing them for a couple of seconds, firstly in 70% ethanol, secondly in 45% ethanol, and lastly in distilled water. Dry them properly with a paper towel.</li>
	<li>Attend animals until they regain sufficient consciousness and place them all single encaged. If required (e.g. for immunohistochemistry purposes), euthanize animals by intravenous injection of 100 mg/kg pentobarbital.</li>
</ol>
3. In Vivo Retinal and Subretinal Imaging
<ol>
	<li>Use a spectral domain optical coherence tomography (SD-OCT) device with the accompanying software (see Table of Materials) to obtain cross-sectional b-scans of the treated animals, according to manufacturer&#39;s instructions. To avoid image blurring, make sure to keep the cornea moist by flushing with topical saline every 30-60 s.
	<ol>
		<li>Place the anesthetized and pupil-dilated animals (see steps 1.1 and 2.10) in an adjustable mount to obtain an unobstructed path from the instrument light source to the rabbit retina.</li>
		<li>Obtain at least 3 OCT scans with simultaneous infrared-confocal scanning laser ophthalmoscopy (IR-cSLO) reflectance reference images representing the upper, central, and lower portion of the injected area.</li>
		<li>Obtain en-face fundus images with cSLO blue, green, infrared, and multicolor laser reflectance (i.e., multiple simultaneous laser colors), respectively.</li>
		<li>Capture blue light autofluorescence (BAF) images using the blue-light laser capability of the SD-OCT device.</li>
	</ol>
	</li>
</ol>

<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>

None of the authors have competing interests or conflicting interests.

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 &amp;ndash;bFGF and &amp;ndash;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 Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>Gibco, ThermoFisher Scientific Corp</td><td>14190-094</td><td></td></tr><tr><td>Cell strainer 40 &amp;mu;m Nylon</td><td>VWR</td><td>732-2757</td><td></td></tr><tr><td>Needle 30 G 0.5&amp;rsquo;&amp;rsquo;; 0.3 mm x 13 mm</td><td>BD Microlance</td><td>304827</td><td></td></tr><tr><td>Acrodisc 25 mm 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>NaIO&lt;sub&gt;3&lt;/sub&gt;</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&amp;eacute;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>25 G/38 G polytip subretinal cannula</td><td>MedOne Surgical Inc</td><td>3219</td><td>25 G/38 G</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&#039;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 &amp;amp; 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&amp;ouml;pings Rabbit Farm, Sweden</td><td></td><td></td></tr><tr><td>hESC-RPE cells</td><td></td><td></td><td>See reference number &lt;sup class=&quot;xref&quot;&gt;1&lt;/sup&gt;</td></tr><tr><td>Buprenodale Vet, 0.3 mg/ml</td><td>Dechra</td><td>660500</td><td>Use 0.5 mL buprenorphine subcutaneously</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, ...

subretinal-transplantation-human-embryonic-stem-cell-derived-retinal

Research

JoVE Journal

Biology

10.0K Views.  Karolinska Institutet. Retinale pigment epitheliale cellen kunnen dienen als een cel-vervangende therapie voor de geavanceerde vorm van droge leeftijdsgebonden Macula degeneratie. Dit protocol beschrijft de generatie van een grote-eyed model van geografische atrofie en subretinal transplantatie van menselijke embryonale stamcellen cel afkomstige retinale pigment epitheliale cellen in dit model van ziekte.

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

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

No cure has been discovered for age-related macular degeneration (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex multifactorial disease with an unknown etiology, although it is largely thought to occur due to death or dysfunction of the retinal pigment epithelium (RPE), a monolayer of cells that underlies the retina and provides critical support for photoreceptors. RPE cell replacement strategies may hold great promise for providing therapeutic relief for a large subset of AMD patients, and RPE cells that strongly resemble primary human cells (hRPE) have been generated in multiple independent labs, including our own. In addition, the uses for iPS-RPE are not limited to cell-based therapies, but also have been used to model RPE diseases. These types of studies may not only elucidate the molecular bases of the diseases, but also serve as invaluable tools for developing and testing novel drugs. We present here an optimized protocol for directed differentiation of RPE from stem cells. Adding nicotinamide and either Activin A or IDE-1, a small molecule that mimics its effects, at specific time points, greatly enhances the yield of RPE cells. Using this technique we can derive large numbers of low passage RPE in as early as three months.

Stem cell-derived retinal pigment epithelium (RPE) cells may be used for multiple applications including cell-based therapies for retinal degeneration, disease modeling, and drug studies. Here we present a simple protocol for reproducibly deriving RPE from stem cells.

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

Developmental Biology

Subretinal Transplantation of MACS Purified Photoreceptor Precursor Cells into the Adult Mouse Retina

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability in industrialized countries. Replacement of degenerated photoreceptors by cell transplantation represents a possible treatment option in future clinical applications. Indeed, recent preclinical studies demonstrated that immature photoreceptors, isolated from the neonatal mouse retina at postnatal day 4, have the potential to integrate into the adult mouse retina following subretinal transplantation. Donor cells generated a mature photoreceptor morphology including inner and outer segments, a round cell body located at the outer nuclear layer, and synaptic terminals in close proximity to endogenous bipolar cells. Indeed, recent reports demonstrated that donor photoreceptors functionally integrate into the neural circuitry of host mice. For a future clinical application of such cell replacement approach, purified suspensions of the cells of choice have to be generated and placed at the correct position for proper integration into the eye. For the enrichment of photoreceptor precursors, sorting should be based on specific cell surface antigens to avoid genetic reporter modification of donor cells. Here we show magnetic-associated cell sorting (MACS) - enrichment of transplantable rod photoreceptor precursors isolated from the neonatal retina of photoreceptor-specific reporter mice based on the cell surface marker CD73. Incubation with anti-CD73 antibodies followed by micro-bead conjugated secondary antibodies allowed the enrichment of rod photoreceptor precursors by MACS to approximately 90%. In comparison to flow cytometry, MACS has the advantage that it can be easier applied to GMP standards and that high amounts of cells can be sorted in relative short time periods. Injection of enriched cell suspensions into the subretinal space of adult wild-type mice resulted in a 3-fold higher integration rate compared to unsorted cell suspensions.

Cell transplantation represents a strategy for the treatment of retinal degeneration characterized by photoreceptor loss. Here we describe a method for enrichment of transplantable photoreceptors and their subretinal grafting into adult mice.

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability ...

Medicine

Engineering Transplantation-suitable Retinal Pigment Epithelium Tissue Derived from Human Embryonic Stem Cells

Several pathological conditions of the eye affect the functionality and/or the survival of the retinal pigment epithelium (RPE). These include some forms of retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Cell therapy is one of the most promising therapeutic strategies proposed to cure these diseases, with already encouraging preliminary results in humans. However, the method of preparation of the graft has a significant impact on its functional outcomes in vivo. Indeed, RPE cells grafted as a cell suspension are less functional than the same cells transplanted as a retinal tissue. Herein, we describe a simple and reproducible method to engineer RPE tissue and its preparation for an in vivo implantation. RPE cells derived from human pluripotent stem cells are seeded on a biological support, the human amniotic membrane (hAM). Compared to artificial scaffolds, this support has the advantage of having a basement membrane that is close to the Bruch&#39;s membrane where endogenous RPE cells are attached. However, its manipulation is not easy, and we developed several strategies for its proper culturing and preparation for grafting in vivo.

We describe a method to engineer a retinal tissue composed of retinal pigment epithelial cells derived from human pluripotent stem cells cultured on top of human amniotic membranes and its preparation for grafting in animal models.

Several pathological conditions of the eye affect the functionality and/or the survival of the retinal pigment epithelium (RPE). These include some forms ...

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.

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

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.

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

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

Neuroscience

Subretinal Implantation of RPE on a Carrier in Minipigs: Guidelines for Preoperative Preparations, Surgical Techniques, and Postoperative Care

Degenerative disorders of the retina (including age-related macular degeneration), which originate primarily at or within the retinal pigmented epithelial (RPE) layer, lead to a progressive disorganization of the retinal anatomy and the deterioration of visual function. The substitution of damaged RPE cells (RPEs) with in vitro cultured RPE cells using a subretinal cell carrier has shown potential for re-establishing the anatomical structure of the outer retinal layers and is, therefore, being further studied. Here, we present the principles of a surgical technique that allows for the effective subretinal transplantation of a cell carrier with cultivated RPEs into minipigs. The surgeries were performed under general anesthesia and included a standard lens-sparing three-port pars plana vitrectomy (PPV), subretinal application of a balanced salt solution (BSS), a 2.7 mm retinotomy, implantation of a nanofibrous cell carrier into the subretinal space through an additional 3.0 mm sclerotomy, fluid-air exchange (FAX), silicone oil tamponade, and closure of all the sclerotomies. This surgical approach was used in 29 surgeries (18 animals) over the past 8 years with a success rate of 93.1%. Anatomic verification of the surgical placement was carried out using in vivo fundus imaging (fundus photography and optical coherence tomography). The recommended surgical steps for the subretinal implantation of RPEs on a carrier in minipig eyes can be used in future preclinical studies using large-eye animal models.

The subretinal implantation of retinal pigmented epithelium (RPE) is one of the most promising approaches for the treatment of degenerative retinal diseases. However, the performance of preclinical studies on large-eye animal models remains challenging. This report presents guidelines for the subretinal transplantation of RPE on a cell carrier into minipigs.

Degenerative disorders of the retina (including age-related macular degeneration), which originate primarily at or within the retinal pigmented epithelial ...

Bioengineering

Subretinal Transplantation of Human Embryonic Stem Cell-Derived Retinal Tissue in a Feline Large Animal Model

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and Leber Congenital Amaurosis (LCA) cause progressive and debilitating vision loss. There is an unmet need for therapies that can restore vision once photoreceptors have been lost. Transplantation of human pluripotent stem cell (hPSC)-derived retinal tissue (organoids) into the subretinal space of an eye with advanced RD brings retinal tissue sheets with thousands of healthy mutation-free photoreceptors and has a potential to treat most/all blinding diseases associated with photoreceptor degeneration with one approved protocol. Transplantation of fetal retinal tissue into the subretinal space of animal models and people with advanced RD has been developed successfully but cannot be used as a routine therapy due to ethical concerns and limited tissue supply. Large eye inherited retinal degeneration (IRD) animal models are valuable for developing vision restoration therapies utilizing advanced surgical approaches to transplant retinal cells/tissue into the subretinal space. The similarities in globe size, and photoreceptor distribution (e.g., presence of macula-like region area centralis) and availability of IRD models closely recapitulating human IRD would facilitate rapid translation of a promising therapy to the clinic. Presented here is a surgical technique of transplanting hPSC-derived retinal tissue into the subretinal space of a large animal model allowing assessment of this promising approach in animal models.

Presented here is a surgical technique for transplanting human pluripotent stem cell (hPSC)-derived retinal tissue into the subretinal space of a large animal model.

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and ...

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.

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

Transpupillary-Guided Trans-Scleral Transplantation of Subretinal Grafts in a Retinal Degeneration Mouse Model

Transplantation of photoreceptor cells and retinal pigment&#160;epithelial (RPE) cells provide a potential therapy for retinal degeneration diseases. Subretinal transplantation of therapeutic donor cells into mouse recipients is challenging due to the limited surgical space allowed by the small volume of the mouse eye. We developed a trans-scleral surgical transplantation platform with direct transpupillary vision guidance to facilitate the subretinal delivery of exogenous cells in mouse recipients. The platform was tested using retinal cell suspensions and three-dimensional retinal sheets collected from rod-rich Rho::EGFP mice and cone-rich OPN1LW-EGFP;NRL-/- mice, respectively. Live/dead assay showed low cell mortality for both forms of donor cells. Retinal grafts were successfully delivered into the subretinal space of a mouse model of retinal degeneration, Rd1/NS, with minimum surgical complications as detected by multimodal confocal scanning laser ophthalmoscope (cSLO) imaging. Two months post-transplantation, histological staining demonstrated evidence of advanced maturation of the retinal grafts into &#39;adult&#39; rods and cones (by robust Rho::EGFP, S-opsin, and OPN1LW:EGFP expression, respectively) in the subretinal space. Here, we provide a surgical platform that can enable highly accurate subretinal delivery with a low rate of complications in mouse recipients. This technique offers precision and relative ease of skill acquisition. Furthermore, the technique could be used not only for studies of subretinal cell transplantation but also for other intraocular therapeutic studies including gene therapies.

This protocol presents a transpupillary vision-guided trans-scleral approach to safely and precisely deliver subretinal cellular grafts, with a low rate of surgical complications, in mouse recipients with or without retinal degeneration.

Transplantation of photoreceptor cells and retinal pigment&#160;epithelial (RPE) cells provide a potential therapy for retinal degeneration diseases. ...

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

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