这项研究得到了卡研究所、皇冠公主玛加丽塔·瓦尔斯特伦的视力受损基金会、埃德温∙乔丹基金会眼科研究、瑞典眼基金会、国王古斯塔夫五世基金会、ARMECLindeberg 基金会和 Cronqvist 基金会

下面的协议遵循卡 Instituet 的动物保育指南。所有使用新西兰白化兔的动物实验 (材料表) 已得到区域动物伦理委员会 (召开 Norra Djurförsöksetiska Nämnd) 的批准 (许可证: dnr 56/15)。使用干细胞 (dnr 2011/745-31/3) 和干细胞-RPE (dnr 2013/813-31/2) 的转让和操纵也符合瑞典立法和卡学院条例, 并已得到区域人类伦理委员会的批准 (Regionala Etikprövningsnämnden 我斯德哥尔摩)。
1. 视网膜注射碘酸钠 (NaIO3) 成大眼动物模型
<ol><li>麻醉动物通过肌肉注射在大腿与35毫克/千克氯胺酮和5毫克/千克嗪在盐水中使用30克注射器, 并扩大学生的局部眼药液使用混合0.75% 喷和2.5% 肾上腺素。如果动物对其后腿的硬捏没有反应, 适当的麻醉被证实。</li>
	<li>将兔子置于外科显微镜下, 头部朝向外科医生 (图 1A)。使用盖子牵引器去除眼睑和 nictitans 膜与无菌布, 以尽量减少污染的风险。使用平衡盐溶液 (BSS), 以防止在两眼麻醉时干燥。</li>
	<li>
对于显微外科, 使用2端口 (或可选3端口) 25 克 transvitreal 部扁平技术与非瓣套管插入显微外科仪器 (图 1B)。多功能玻璃体切割机有端口连接输液套管, endoillumination, vitrector, 和 endolaser。对于视网膜注射, 只有 endoillumination 是强制性的, 这使得一个2端口设置足够。插入 endoillumation 通过左上方的套管针, 并使用右上套管针视网膜注射套管。
	<ol>
<li>对于一个3端口设置, 使用较低的时间套管针的 BSS 输液套管。如果使用可选仪器 (如 vitrector), 请将其插入右上角的套管针。</li>
		<li>用爪钳将上部2套管 1-2 mm 从角膜缘上插入, 并将结膜覆盖在插入部位上。</li>
		<li>确保套管入 transsclerally 在一个 30 - 45°的平行角 , 并进行到套管针的尖端中间。然后转动套管针90°和前进到眼睛, 瞄准后极的眼睛。这一做法将避免手术后的 sclerotomies 渗漏, 也减少了眼内炎 (即,细菌感染的眼睛) 的风险。另请参阅图 2B以了解套管针位置。</li>
		<li>在角膜上放置一个单一的使用平板隐形眼镜来可视化视网膜, 用合成的泪水作为眼睛和隐形眼镜之间的接触凝胶 (图 1A)。</li>
		<li>绘制500µL 的 NaIO3到1毫升注射器连接到延伸管 (由助手操作) 和 38 G polytip 套管 (由外科医生操作)。</li>
		<li>插入 endoillumination 探针通过左上角套管针和注射套管通过右上方的套管针, 并推进套管通过玻璃空间向视网膜, 瞄准的区域略低于视神经头。</li>
		<li>允许导管的尖端慢慢地接触视网膜, 直到焦点美白是可见的。注射本身将穿透视网膜, 允许视网膜分娩。不要让套管穿透视网膜, 因为这可能导致出血。</li>
		<li>在5秒内注入50µL 的 NaIO3下。由于视网膜和底层脉络膜之间有一个天然的劈裂平面, 所以在注射过程中, 明显可见的半透明疱疹应该逐渐形成。</li>
		<li>在注射过程中, 慢慢缩回针头, 但要确保在疱疹内保持尖端, 以减少回流。</li>
		<li>去除 endoillumation 和注射套管后, 取出套管使用爪钳, 并适用于三十年代的光压力自密封缝合-少 sclerotomies 使用尖端或钝端的镊子。</li>
	</ol>
</li>
	<li>手术后给予10毫升生理盐水皮下注射, 以防止脱水。不要给手术后的外用类固醇或抗生素。镇痛药给0.5 毫升丁丙诺啡0.3 毫克/毫升皮下手术后, 以及手术后的一天。</li>
	<li>使用后, 先在70% 乙醇中浸泡几秒钟, 然后用45% 乙醇, 最后在蒸馏水中冲洗所有仪器。用纸巾把它们擦干。</li>
	<li>参加动物, 直到他们恢复足够的意识, 并把他们所有的单一笼。如果需要 (例如, 为免疫组化目的), 安乐动物静脉注射100毫克/千克戊巴比妥 (见材料表)。</li>
	<li>等待7天, 进行干细胞-RPE 细胞的移植。</li>
</ol>2. 干细胞-RPE 细胞在治疗动物中的视网膜移植
<ol><li>管理2毫克 (100 µL) 的玻璃曲安在麻醉动物使用30克注射针插入1-2 毫米从角膜缘在下颞象限1周前移植干细胞-RPE, 并重新管理它每3月。确保指向眼睛后极的尖端, 以避免镜头接触。</li>
	<li>干细胞-RPE 单层的培养, 如前所述1。</li>
	<li>移除细胞分化介质 (参见材料表) 的24井汇合干细胞-RPE 单层和洗涤每井与500µL PBS 没有 Ca2 +和镁2 +。再次重复此操作, 共2次洗涤。</li>
	<li>丢弃上清液, 每井添加500µL 胰蛋白酶, 在37° c 孵育12分钟。</li>
	<li>倾斜板和小心地去除胰蛋白酶 (细胞应该保持连接到板材)。收集细胞在800µL 新鲜37° c prewarmed 分化培养基由温和的移, 甚至刮如果需要, 获得一个单细胞悬浮。 注: 如果观察到细胞团簇, 请使用40µm 细胞过滤器。</li>
	<li>根据制造商的指示, 用0.2% 台台台蓝来计数例室中的细胞。</li>
	<li>在室温下加入5毫升的分化培养基和离心机, 300 x g 5 分钟。</li>
	<li>在新鲜的过滤消毒 PBS 中丢弃上清和重颗粒 (通过 25 mm 注射器过滤器), 最终浓度为1000细胞/µL。</li>
	<li>分前细胞悬浮成600µL 等分, 并保持在冰, 直到和手术期间。</li>
	<li>麻醉动物通过肌肉注射在大腿与35毫克/千克氯胺酮和5毫克/千克嗪在盐水中使用30克注射器的混合物。用混合0.75% 喷和2.5% 肾上腺素的局部眼药液扩张瞳孔。如果动物对背部腿部的硬捏没有反应, 适当的麻醉就会被证实。</li>
	<li>把兔子放在头上, 面对外科医生。使用盖子牵引器去除眼睑和 nictitans 膜与无菌布, 以尽量减少污染的风险。用 BSS 预防两眼麻醉时的干燥。</li>
	<li>
对于显微外科, 使用2端口 (或可选的3端口) 25 G transvitreal 部扁平技术与套管放置在相同的位置, 如步骤1.3 和图 2所述。如果以前的 sclerotomies 是可见的, 执行套管针插入只是相邻的, 但不是通过这些, 以尽量减少术后渗漏的风险。
	<ol>
<li>在角膜上放置一个单一的使用平板隐形眼镜来可视化视网膜, 用合成的泪水作为眼睛和隐形眼镜之间的接触凝胶 (图 1A)。</li>
		<li>经过适当的尖端定位 (见步骤1.3.5 和 1.3.6), 注入50µL 轻度混合干细胞-RPE 悬浮液 (5万细胞) 下。目的为 NaIO3预处理区域的中心, 由特征 "金属" 内照射反射来区分。感觉神经视网膜应分离容易形成清晰可见的疱疹。用不育的 H2O 在家兔之间冲洗针头, 避免因细胞结块引起的针头堵塞。当发现堵塞时, 更换针头。</li>
		<li>在注射过程中, 慢慢缩回针头, 但要确保在疱疹中保持尖端, 以减少回流。</li>
		<li>去除 endoillumation 和注射套管后, 取出套管使用爪钳, 并适用于三十年代的光压力自密封缝合-少 sclerotomies 使用尖端或钝端的镊子。</li>
	</ol>
</li>
	<li>手术后给予10毫升生理盐水皮下注射, 以防止脱水。不要给手术后的外用类固醇或抗生素。镇痛药给0.5 毫升丁丙诺啡0.3 毫克/毫升皮下手术后, 以及手术后的一天。</li>
	<li>使用后, 把所有的仪器浸泡几秒钟, 首先在70% 乙醇, 其次在45% 乙醇, 最后在蒸馏水。用纸巾把它们擦干。</li>
	<li>参加动物, 直到他们恢复足够的意识, 并把他们所有的单一笼。如果需要 (例如为免疫组织化学目的), 安乐动物通过静脉注射100毫克/千克戊巴比妥。</li>
</ol>3.活体视网膜和视网膜成像
<ol><li>
根据制造商的说明, 使用带有随附软件的光谱域光学相干层析 (SD OCT) 设备 (参见材料表), 获得经过处理的动物的横截面 b 扫描。为了避免图像模糊, 确保保持角膜湿润的冲洗与局部生理盐水每30-60 秒。
	<ol>
<li>放置麻醉和瞳孔扩张的动物 (见步骤1.1 和 2.10) 在一个可调节的坐骑获得一个畅通无阻的路径从仪器光源到兔视网膜。</li>
		<li>获得至少 3 OCT 扫描与同时红外共焦扫描激光眼底 (IR-cSLO) 反射参考图像代表的上部, 中央, 和较低的部分注入区域。</li>
		<li>获得 cSLO 蓝色, 绿色, 红外线和多色激光反射 (即, 多个同时激光颜色) 的面部眼底图像分别。</li>
		<li>利用 SD OCT 设备的蓝光激光能力捕获蓝光自荧光 (曝气生物滤池) 图像。</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>

没有一个作者有竞争利益或利益冲突。

在本协议中, 对遗传算法的大眼模型的生成及其在评价干细胞-RPE 积分中的临床应用进行了描述。
为了将 GA 及相关疾病的再生疗法翻译为临床7, 必须开发和优化方法, 以忠实地捕获移植和影像学的临床方法。兔子在这方面有吸引力: 它有一个相对较大的眼睛, 允许眼内手术和标准成像的使用, 是便宜和容易安置相比, 其他大眼睛动物。
<p class="jove_conten...

该协议描述了一种大眼睛的地理萎缩 (GA) 的临床前模型的产生, 允许评价移植干细胞-RPE 在视网膜空间的整合。本文详细介绍的方法已在3份最近的出版物中使用, 它们展示了干细胞1的视网膜色素上皮细胞的丰富、纯净和功能性种群的产生, 以及产生外视损伤和类似 GA 的表型由视网膜注射的生理盐溶液 (即, BSS 和 PBS) 或 NaIO3在兔子眼中2,3。我们进一步证明, 亚视网膜悬吊移植的干细胞-RPE 形成广泛功能单分子膜与感光剂抢救能力2。
利用兔眼的几个优点来产生一种 GA 模型的疾病。首先, 兔眼的大小, 这是70% 的成人眼睛的体积, 允许临床有意义的移植使用的细胞密度远远低于常规使用的小鼠眼 (1000 细胞/µL vs. 5万细胞/µL)4,5. 其次, 啮齿类动物的手术通常是通过脉络膜巩膜的, 这会损害视网膜屏障, 并可能引发炎症反应和可能的拒绝6。这两个因素一起可能导致移植细胞的多层和结块, 以及移植细胞在一个被破坏的本地视网膜组织中整体的不良整合。然而, 大眼睛的兔子模型允许执行一项与临床设置完全相同的手术技术。第三, 大眼睛模型也允许高分辨率的在体内成像和监测移植细胞和上覆视网膜通过时间1,2,3。因此, 我们描述了一个临床相关和成本效率的临床前模型, 应该是一个有吸引力的替代啮齿目动物的人有兴趣的研究正常和病变的视网膜和亚视网膜空间。

<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

地理萎缩 (GA), 晚期与干年龄相关的黄斑变性的特点是失去视网膜色素上皮 (RPE) 层, 这导致后退化的重要视网膜结构 (如, 光) 导致严重视力损害。类似地, 视网膜色素上皮的丧失和视力减退见于长期随访的晚期与湿性黄斑变性 (AMD) 接受玻璃抗血管内皮生长因子 (VEGF) 治疗的患者。因此, 一方面, 这是至关重要的有效地从一个无限的来源, 可以作为替代治疗的视网膜色素上皮细胞。另一方面, 重要的是要评估的行为和整合的模型中衍生细胞的疾病所涉及的外科和影像学方法尽可能接近于那些应用于人类。在这里, 我们提供了一个详细的协议的基础上, 我们以前的出版物, 描述了使用白化兔眼的 GA 前体细胞的生成, 用于评价人类胚胎干细胞衍生的视网膜色素上皮细胞 (干细胞-RPE) 在临床相关的设置。分化干细胞-RPE 被移植到天真的眼睛或眼睛与 NaIO3诱导的 GA 样视网膜变性使用25克 transvitreal 部扁平技术。采用多模态高分辨率非侵入实时成像技术对退化和移植区进行评价。

在图 2中显示了典型的活体图像, 如生物滤池、IR-cSLO 和正常白化兔视网膜的 SD OCT。注意不同的视网膜层与他们的独特水平的光反射捕获的 SD OCT 仪器。
在数字 1A和图 1B中, 创建子视网膜泡的设置如下所示: 一个盖子牵引器定位, 使眼睛的盖子, 允许插入3?...

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

人胚胎干细胞源性视网膜色素上皮细胞移植视网膜大眼模型的地理萎缩

视网膜色素上皮细胞可作为晚期相关黄斑变性的细胞替代疗法。该协议描述了大眼睛的地理萎缩模型的产生和人类胚胎干细胞源性视网膜色素上皮细胞视网膜移植到这种疾病模型。

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 次观看.  Karolinska Institutet. 视网膜色素上皮细胞可作为晚期相关黄斑变性的细胞替代疗法。该协议描述了大眼睛的地理萎缩模型的产生和人类胚胎干细胞源性视网膜色素上皮细胞视网膜移植到这种疾病模型。

视频: 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.

视网膜色素上皮细胞的干细胞高效的推导

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 ...

科研

发育生物学

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.

MACS纯化光感受器前体细胞的亚视移植到成年小鼠视网膜中

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 ...

医学

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.

从人胚胎干细胞中提取合适的视网膜色素上皮组织的工程移植

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.

视网膜下进行注射的鼠害提供视网膜色素上皮细胞悬浮

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.

一种用于兔视网膜下手术分步协议

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.

人视网膜色素上皮细胞向大鼠眼巩膜视网膜移植精制方案的研制

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

神经科学

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.

小型猪载体上 RPE 的视网膜下植入:术前准备、手术技术和术后护理指南

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

生物工程

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.

猫大型动物模型中人类胚胎干细胞来源的视网膜组织的视网膜下移植

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.

非人灵长类动物模型中视网膜色素上皮移植治疗退行性视网膜疾病

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.

经瞳孔引导下视网膜下移植物的视网膜下移植物经巩膜移植视网膜变性小鼠模型

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