本研究是根据《赫尔辛基宣言》原则进行的，并经中国人民解放军总医院机构伦理委员会批准。WA09（H9）电调线是从WiCell研究所获得的。
1.培养基和试剂制备
<ol><li>人胚胎干细胞培养基和传代溶液<ol><li>维持培养基 （MM）：无菌制备 500 mL 完全 MM（基础培养基 + 5x 补充剂;参见 材料表）。在室温（RT）下解冻5x补充剂或在2-8°C下过夜。 预热至室温，使用前充分搅拌，直到补充剂没有浑浊。</li>
		<li>1% 细胞外基质 （ECM）：使用预冷的移液管吸头和无菌试管在冰上每管分配 200 μL ECM（参见 材料表）。将试管储存在-20°C冰箱中。在冰上融化ECM，并用预冷的DMEM / F12以1：100稀释。</li>
		<li>0.5 mM pH = 8.0 EDTA：要制备 500 mL 0.5 mM EDTA，将 500 μL 0.5 M EDTA 和 0.9 g NaCl 加入 500 mL 1x Dulbecco 磷酸盐缓冲盐水 （DPBS） 中（参见 材料表）。充分混合并储存在2-8°C。</li>
	</ol></li>
	<li>视网膜分化培养基<ol><li>无生长因子化学成分培养基 （gfCDM）：通过结合 45% Iscove 改良的 Dulbecco 培养基 GlutaMAX （IMDM-GlutaMAX）、45% Ham 的 F12-GlutaMAX （F12-Glutamax）、10% 敲除血清替代物 （KSR）、1% 胆固醇脂质浓缩物和 450 μM 硫代甘油来制备 gfCDM（参见 材料表）。</li>
	</ol></li>
	<li>小分子化合物<ol><li>Y-27632 2HCl：将 50 mg Y-27632 粉末加入 3.122 mL 二甲基亚砜 （DMSO） 中。在-80°C下以50mM的浓度分配并储存Y-27632（参见 材料表）2年，远离光线。将原液稀释 2,500 倍 （20 μM） 以供使用，相当于每 mL gfCDM 含 0.4 μL Y-27632。</li>
		<li>IWR1-endo：加入2.4424mL DMSO溶解10mg IWR1-endo（参见 材料表），得到10mM储备溶液。分配等分试样并将其储存在-80°C长达2年。每 mL gfCDM 加入 0.3 μL 10 mM IWR1-endo，以获得 3 μM 浓度用于诱导。</li>
		<li>SB431542：要制备 50 mM SB431542，将 10 mg SB431542（参见 材料表）加入 0.5203 mL DMSO 并充分混合。在-80°C溶剂中储存最多2年。为了制备工作浓度为10μM的SB431542，将2μL的50mM储备溶液加入10mL gfCDM中。</li>
		<li>LDN-193189 2HCl：将 5 mg LDN-193189 2HCl（参见 材料表）溶于 10.4297 mL DMSO 中，得到 1 mM 储备溶液。储存在-20°C或-80°C（根据制造商的建议）。向每 10 mL gfCDM 中加入 0.1 μL 原液，得到 100 nM 的 LDN-193189 用于诱导。</li>
		<li>重组人骨形态发生蛋白4（BMP4）：在4 mM HCl中以50-200 μg/mL的比例复溶（参见 材料表）。复溶后在2°C至8°C下储存1个月或-20°C储存1年。使用 1.5 nM BMP4 进行 NR 诱导。</li>
	</ol></li>
	<li>长期NR培养基<ol><li>视黄酸 （RA）：量取 6 mg RA 粉末（见 材料表）并加入 3.9941 mL DMSO。在-80°C下以5mM的等分试样储存，并在3个月内使用。为了达到 0.5 μM 的工作浓度，将 10 μL 预混液加入 100 mL 神经视网膜分化培养基 （NRDM） 中。使用前添加。 注意： 在准备和储存过程中避免避光。</li>
		<li>牛磺酸：将称量200mg的牛磺酸粉末（见 材料表）加入7.9904mL DMSO中，得到200mM储备溶液。分配并储存在2-8°C。 通过每 100 mL NRDM 加入 50 μL 储备溶液，可达到 0.1 mM 牛磺酸的工作浓度。</li>
		<li>NRDM：用 DMEM/F12-GlutaMAX 培养基、1% N2 添加剂、10% 胎牛血清、0.5 mM RA 和 0.1 mM 牛磺酸组成 NRDM（参见 材料表）。在2-8°C下储存长达2周或在-20°C下储存6个月，以确保组分的活性。</li>
	</ol></li>
	<li>封闭缓冲液：用DPBS以1：9稀释，得到10%的工作溶液（参见 材料表）。在-20°C下储存长达5年。 注意： 在 II 类生物安全柜中执行所有其他程序以确保无菌，称重除外。如果在制备过程中需要添加称重试剂，请使用 0.22 μm 过滤器进行过滤。</li>
</ol>2. H9-胚胎干细胞的培养
<ol><li>H9-ESCs的解冻<ol><li>向 6 孔板的每个孔中加入 1 mL 1% ECM。在37°C的5%CO2 气氛中的培养箱中孵育1小时。 注意：避免沿井壁添加ECM，因为它可能会粘在表面。</li>
		<li>从液氮罐中取出H9-ESC低温管原液，并在37°C水中快速振荡30秒。 注意：不要让小瓶完全解冻。</li>
		<li>取出小瓶并使用 75% 的消毒酒精喷雾仔细消毒。将冻存管中解冻的 H9-ESC 加入含有 9 mL MM 和 10 μM Y-27632 的 15 mL 管中。</li>
		<li>在室温下以190× g 离心管5分钟。 用1mL移液管小心地除去大部分上清液，留下约50μL上清液以避免细胞损失。</li>
		<li>向细胞沉淀物中加入含有 10 μM Y27632 的 1 mL MM，并用 1 mL 移液管上下移液 5-10 次轻轻重悬细胞沉淀物。</li>
		<li>孵育1小时后去除ECM涂层。向每个孔中加入 2 mL 含有 10 μM Y-27632 的预热 MM。</li>
		<li>每孔分配 0.5 mL 细胞悬液。轻轻横向摇晃板以确保细胞均匀分布。</li>
		<li>将板在37°C下在5%CO2 下孵育至少24小时而不接触它。</li>
		<li>每天更换培养基。当克隆密度达到70%及以上时，需要传代。</li>
	</ol></li>
	<li>H9-ESCs的传代<ol><li>如上所述制备ECM包被板（步骤2.1.1），每孔加入2mL MM。</li>
		<li>从6孔板中取出用过的培养基。用 1 mL DPBS 洗涤每个孔两次。</li>
		<li>通过缓慢加入 1 mL EDTA，并与 1 mL EDTA 在室温下孵育 4-7 分钟，洗涤每个孔两次。 注意：在孵育过程中，在显微镜下检查6孔板3分钟。如果大多数细胞接近从培养皿中分离，请立即进行下一步。避免长时间孵育，以减少对细胞的负面影响。</li>
		<li>弃去 EDTA 并加入 1 mL MM 以中止消化。</li>
		<li>轻轻敲击孔板，直到大部分细胞分离。</li>
		<li>用 5 mL 移液管小心地将细胞悬液转移到 15 mL 离心管中。轻轻地将H9-ESC菌落上下重悬3-5次，以将它们与巴斯德皮特混合。将 20-100 μL 细胞悬液转移到新的 ECM 包被的 6 孔培养皿中并来回摇动。</li>
		<li>当在显微镜下观察到细胞密度足够时，将板在37°C下在5%CO2 下孵育至少24小时而不接触它。</li>
		<li>每天更换介质。在克隆密度达到 70% 及以上时，需要传代。</li>
	</ol></li>
</ol>3. 人类NR的产生
注意：一旦菌落达到大约 70% 的汇合度，就可以使用 图 1 中概述的程序步骤将它们定向分化为视网膜类器官 （RO）。
<ol><li>第 0 天 - 胚状体 （EB） 形成<ol><li>用 2 mL DPBS 洗涤细胞后，向孔中加入 0.5 mL 含有 20 μM Y27632 的 CDS（参见 材料表）。在37°C下在加湿的5%CO2 培养箱中孵育3分钟。 注意：在显微镜下检查。尽可能减少孵育时间，避免对细胞产生有害影响。</li>
		<li>通过加入含有 20 μM Y27632 的 3 mL MM 中止消化。以190× g 离心5分钟并弃去上清液。</li>
		<li>将细胞沉淀重悬于含有 20 μM Y27632 的 1 mL gfCDM 中，并计数细胞。将 1.2 × 106 个细胞（每孔 1.2 ×10 个 4 个细胞）的相应体积加入 10 mL gfCDM 中，该 gfCDM 预先掺入了 20 μM Y-27632、3 μM IWR1-endo、10 μM SB431542 和 100 nM LDN-193189（参见 材料表）。</li>
		<li>向 96 个 V 底锥形孔的每个孔中加入 100 μL 细胞悬液。将板放入加湿的 5% CO2 培养箱中，直到第 6 天。 注意： 至少 24 小时内不要移动培养皿，以增强 EB 的粘附性。</li>
	</ol></li>
	<li>第 6 天 - 视网膜诱导<ol><li>在第 6 天，加入 10 mL 含有 55 ng/mL BMP4 的 gfCDM，以完全更换 EB 上的培养基。将板放回培养箱。 注意： 移液器朝向 96 V 型底锥形孔的壁以避免气泡。更换培养基时避免吸收任何类器官。</li>
		<li>在第 9 天、第 12 天和第 15 天进行半中度更换。用新鲜的gfCDM替换一半的培养基，以逐渐稀释BMP4。</li>
	</ol></li>
	<li>第 18 天 - 长期 NR 培养<ol><li>在第 18 天，使用 5 mL Pasteur 移液器小心地将形成的 EB 转移到 15 mL 离心管中，并再次用 NRDM 轻轻冲洗。将 EB 转移到低吸附 6 孔或 24 孔板上（参见 材料表）。将板放回培养箱。</li>
		<li>每 3 天用新鲜的 NRDM 更换培养基。 注意： 在更换介质期间关闭灯，因为 RA 对光敏感。去除严重分化的类器官，并在显微镜下分离贴壁类器官。</li>
	</ol></li>
</ol>4. 人类NRs的分析
<ol><li>RO的安装<ol><li>选择不同世代的H9-ESCs诱导三批ROs。 注意：计算三种评估方法（Kuwahara 等人 12、Döpper 等人 27 和本研究中的改进方法）中每一种形成 NR 数量的三个板以评估诱导成功率。如果光学显微镜检查在第 30 天显示神经上皮样结构的形成，则认为诱导成功。</li>
	</ol></li>
	<li>RO的免疫荧光染色<ol><li>将 3-5 个 RO 转移到 1.5 mL 试管中。移液掉多余的培养基，在室温下用 1 mL DPBS 洗涤 RO 一次，让 RO 下沉并小心地取出 DPBS。每 1.5 mL 试管加入 1 mL 4% 多聚甲醛（参见 材料表），并固定在 4 °C。 注意：第 6 天和第 18 天的 RO，固定 2 小时。在第 30 天修复 12 小时，在第 60 天修复 14 小时。</li>
		<li>固定后，使用梯度酒精脱水。让 RO 在 50%、60% 和 70% 酒精中各静置 15 分钟，然后在 80%、90%、95%、100% 和 100% 酒精中各静置 10 分钟。然后，加入 100% 酒精和二甲苯的 1：1 混合物 10 分钟，然后加入二甲苯两次，每次 10 分钟。</li>
		<li>将RO放入装有熔化副质体的金属模具中（见 材料表）40分钟，然后将模具淬火在冰上以固定RO。将嵌入盒放入模具中，并在-20°C下冷冻过夜。随后，小心地拆下嵌入盒。最后，将它们存放在 RT 中。</li>
		<li>用石蜡切片机创建连续切片（5 μm 厚度）。将切片固定在粘附显微镜载玻片上，干燥并储存在室温下。</li>
		<li>在抗原修复之前进行脱蜡和补液12,27。</li>
		<li>将 10 mL 20x pH 6.0 柠檬酸盐抗原修复溶液（参见 材料表）加入 190 mL ddH2O（双蒸馏 H2O）中。在微波炉中以高模式加热 4 分钟直至沸腾。加入石蜡切片后，在低模式下加热切片20分钟以完成抗原修复。</li>
		<li>让石蜡切片在通风处自然冷却，然后将它们放入潮湿的腔室中。</li>
		<li>使用巴氏笔（见 材料表）勾勒出各部分的轮廓。在室温下用0.2%Triton X-100孵育30分钟以打破膜，然后用TPBS洗涤载玻片三次，每次5分钟。</li>
		<li>在每个染色面积为10μL的潮湿室中，用在室温下用DPBS稀释的10%驴血清封闭1小时。</li>
		<li>加入在 10% 驴血清中稀释的一抗。在4°C的湿度室中孵育过夜。用 TPBS 洗涤样品三次，每次 10 分钟以除去未结合的抗体。 注：一抗如下：抗PAX6（1：250），抗SOX2（1：200），抗KI67（1：200），抗CHX10（1：200），抗β微管蛋白III（1：250）和抗NESTIN（1：200）（见 材料表）。</li>
		<li>在加湿室中与在DPBS中稀释的二抗一起在室温下孵育1小时。用TPBS重复洗涤步骤三次，每次10分钟。 注意：在黑暗中保存以防止荧光二抗淬灭。二抗为与驴抗小鼠 IgG 偶联的 Alexa Fluor 488 和与驴抗兔 IgG 偶联的 Alexa Fluor 568，稀释度为 1：400（参见 材料表）。</li>
		<li>在黑暗中与含有DAPI（1：500;参见 材料表）的DPBS在室温下孵育10分钟。用 TPBS 洗涤 3 次，每次 10 分钟。</li>
		<li>将适量的抗荧光密封剂（见 材料表）滴到载玻片上，并用盖玻片盖住。</li>
		<li>使用流样组织细胞定量分析仪（参见 材料表）或类似方法进行可视化。</li>
		<li>显微镜分析后，将载玻片储存在-20°C的黑暗中。</li>
	</ol></li>
</ol>

增强人胚胎干细胞的 3D 神经视网膜分化

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E., 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=Reproducibility+and+staging+of+3D+human+Retinal+organoids+across+multiple+pluripotent+stem+cell+lines.">Reproducibility and staging of 3D human Retinal organoids across multiple pluripotent stem cell lines.</a> Development. 146 (1), 171686 (2019).</li><li>Sanjurjo-Soriano, C., 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=RA+delays+initial+photoreceptor+differentiation+and+results+in+a+highly+structured+mature+Retinal+organoid.">RA delays initial photoreceptor differentiation and results in a highly structured mature Retinal organoid.</a> Stem Cell Res Ther. 13 (1), 478 (2022).</li><li>Li, X., Zhang, L., Tang, F., Wei, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+organoids:+cultivation,+differentiation,+and+transplantation.">Retinal organoids: cultivation, differentiation, and transplantation.</a> Front Cell Neurosci. 15, 638439 (2021).</li><li>Zerti, 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=Developing+a+simple+method+to+enhance+the+generation+of+cone+and+rod+photoreceptors+in+pluripotent+stem+cell-derived+Retinal+organoids.">Developing a simple method to enhance the generation of cone and rod photoreceptors in pluripotent stem cell-derived Retinal organoids.</a> Stem Cells. 38 (1), 45-51 (2020).</li><li>Kim, 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=transcriptome+profiling,+and+functional+validation+of+cone-rich+human+Retinal+organoids.">transcriptome profiling, and functional validation of cone-rich human Retinal organoids.</a> Proc Natl Acad Sci U S A. 116 (22), 10824-10833 (2019).</li><li>Yamasaki, 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=Addition+of+Chk1+inhibitor+and+BMP4+cooperatively+promotes+retinal+tissue+formation+in+self-organizing+human+pluripotent+stem+cell+differentiation+culture.">Addition of Chk1 inhibitor and BMP4 cooperatively promotes retinal tissue formation in self-organizing human pluripotent stem cell differentiation culture.</a> Regen Ther. 19, 24-34 (2022).</li><li>D&#246;pper, 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=Differentiation+protocol+for+3D+Retinal+organoids,+immunostaining+and+signal+quantitation.">Differentiation protocol for 3D Retinal organoids, immunostaining and signal quantitation.</a> Curr Protoc Stem Cell Biol. 55 (1), e120 (2020).</li><li>Norrie, J. 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所有作者都声明他们没有利益冲突。

人类RO可以在空间和时间上概括胎儿视网膜的发育，早期RO在同等发育阶段表现出与胎儿视网膜的高度相似性15。在组织形态和分子表达方面，人类RO密切反映了视网膜组织的实际生长状态，为疾病建模、药物筛选和再生医学领域提供了巨大而前所未有的机会。目前，已经建立了几种不同的方法在 体外从人PSCs中产生RO，并且正在进行持续的修改和优化，以进一步提高效率<sup...

眼睛是人类感觉器官的主要信息来源，视网膜是哺乳动物眼睛的主要视觉感觉组织1。视网膜病变是导致眼部疾病的全球主要原因之一，导致失明2.全世界约有 285 万人因视网膜病变而遭受不同程度的视力损害3.因此，研究其发病机制对于早期诊断和及时治疗至关重要。大多数关于人类视网膜病变的研究主要集中在动物模型上4,5,6。然而，人类视网膜是一个复杂的多层组织，由各种细胞类型组成。传统的二维 （2D） 细胞培养和动物模型系统通常无法忠实地概括人类天然视网膜的正常时空发育和药物代谢 7,8。
最近，3D 培养技术已经发展到可以从多能干细胞 （PSC） 生成组织样器官9,10。在3D悬浮培养系统中，由人PSC产生的视网膜类器官（ROs）不仅包含七种视网膜细胞类型，而且还表现出与体内人视网膜相似的独特分层结构11,12,13。人 PSC 衍生的 RO 已获得普及和广泛使用，是目前研究人视网膜发育和疾病的最佳体外模型14,15。在过去的几十年中，许多研究人员已经证明，人类PSC，包括胚胎干细胞（ESC）和诱导多能干细胞（iPSC），可以使用各种诱导方案分化为RO。这些进展在视网膜病变的疾病建模、药物筛选和干细胞疗法方面具有巨大的潜力16,17,18。
然而，从人类多能干细胞 （PSC） 生成神经视网膜 （NR） 是一个复杂、繁琐且耗时的过程。此外，组织类器官的批次间差异可能导致结果的重现性降低19,20。许多内在和外在因素可以影响视网膜类器官（ROs）的产量，例如起始细胞的数量或种类以及转录因子和小分子化合物的使用21,22,23。自从Sasai实验室11产生第一个人类RO以来，多年来已经提出了多种修改，以提高诱导过程的易用性和有效性13,21,24,25。不幸的是，迄今为止，尚未建立在所有实验室中生成RO的金标准协议。事实上，由于不同的诱导方法，以及视网膜标志物的表达及其结构的稳健性存在很大差异，RO存在一定程度的差异22,26。这些问题可能会使样本采集和研究结果的解释严重复杂化。因此，需要一种更巩固和更强大的分化方案，以最大限度地提高效率，同时将RO产生的异质性降至最低。
本研究描述了一种基于 Kuwahara 等人 12 和 Döpper 等人 27 组合的优化诱导方案，并附有详细说明。新方法显著降低了类器官囊泡化和融合的概率，提高了产生NR的成功率。这一发展为视网膜疾病的疾病建模、药物筛选和细胞治疗应用带来了巨大的希望。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.01 M TPBS</td>
			<td>Servicebio</td>
			<td>G0002</td>
			<td>Washing slices</td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde</td>
			<td>Servicebio</td>
			<td>G1101-500ML</td>
			<td>Fix retinal organoids</td>
		</tr>
		<tr>
			<td>5 mL Pasteur pipette</td>
			<td>NEST Biotechnology</td>
			<td>318516</td>
			<td>Pipette retinal organoids</td>
		</tr>
		<tr>
			<td>96 V-bottomed conical wells</td>
			<td>Sumitomo Bakelit</td>
			<td>MS-9096VZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesion Microscope Slides</td>
			<td>CITOTEST</td>
			<td>188105</td>
			<td>Fix slices</td>
		</tr>
		<tr>
			<td>AggreWell medium</td>
			<td>STEMCELL Technologies</td>
			<td>5893</td>
			<td>Medium</td>
		</tr>
		<tr>
			<td>Anhydrous ethanol</td>
			<td>SINOPHARM</td>
			<td>10009218</td>
			<td>Dehydrate&#160;</td>
		</tr>
		<tr>
			<td>Anti-CHX10</td>
			<td>Santa Cruz</td>
			<td>sc-365519</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>Antifade Solution</td>
			<td>ZSGB-BIO</td>
			<td>ZLI-9556</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-KI67</td>
			<td>Abcam</td>
			<td>ab16667</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>Anti-NESTIN</td>
			<td>Sigma</td>
			<td>N5413</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>Anti-Neuronal Class III &#946;-Tubulin(TUJ1)</td>
			<td>Beyotime</td>
			<td>AT809</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>Anti-PAX6</td>
			<td>Abcam</td>
			<td>ab195045</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>Cell dissociation solution(CDS)</td>
			<td>STEMCELL Technologies</td>
			<td>7922</td>
			<td>Cell dissociation</td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Selleckchem</td>
			<td>S2924</td>
			<td>GSK-3&#945;/&#946; inhibitor</td>
		</tr>
		<tr>
			<td>Cholesterol Lipid Concentrate</td>
			<td>Gibco</td>
			<td>12531018</td>
			<td>250&#215;</td>
		</tr>
		<tr>
			<td>Citrate Antigen Retrieval Solution</td>
			<td>Servicebio</td>
			<td>G1202-250ML</td>
			<td>20&#215;, pH 6.0</td>
		</tr>
		<tr>
			<td>CS10</td>
			<td>STEMCELL Technologies</td>
			<td>1001061</td>
			<td>Cell Freezing Medium</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Roche</td>
			<td>10236276001</td>
			<td>Nuclear counterstain</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide(DMSO)</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>11330032</td>
			<td>Medium</td>
		</tr>
		<tr>
			<td>DMEM/F12-GlutaMAX</td>
			<td>Gibco</td>
			<td>10565018</td>
			<td>Medium</td>
		</tr>
		<tr>
			<td>Donkey anti-Mouse Alexa Fluor Plus 488</td>
			<td>Invitrogen</td>
			<td>A32766</td>
			<td>Secondary Antibody</td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit Alexa Fluor 568</td>
			<td>Invitrogen</td>
			<td>A10042</td>
			<td>Secondary Antibody</td>
		</tr>
		<tr>
			<td>Ethylene Diamine Tetraacetic Acid (EDTA)</td>
			<td>Biosharp</td>
			<td>BL518A</td>
			<td>0.5 M, pH 8.0, cell dissociation</td>
		</tr>
		<tr>
			<td>Extracellular matrix (ECM)</td>
			<td>Corning</td>
			<td>354277</td>
			<td>Coating plates</td>
		</tr>
		<tr>
			<td>F12-Glutamax</td>
			<td>Gibco</td>
			<td>31765035</td>
			<td>Medium</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>A5669701</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow-like tissue cell quantitative analyzer</td>
			<td>TissueGnostics</td>
			<td>TissueFAXS Plus</td>
			<td>Scan sections</td>
		</tr>
		<tr>
			<td>IMDM-GlutaMAX</td>
			<td>Gibco</td>
			<td>31980030</td>
			<td>Medium</td>
		</tr>
		<tr>
			<td>IWR1-endo</td>
			<td>Selleckchem</td>
			<td>S7086</td>
			<td>Wnt-inhibitor</td>
		</tr>
		<tr>
			<td>KnockOut Serum Replacement</td>
			<td>Gibco</td>
			<td>10828028</td>
			<td></td>
		</tr>
		<tr>
			<td>LDN-193189 2HCl</td>
			<td>Selleckchem</td>
			<td>S7507</td>
			<td>BMP-inhibitor</td>
		</tr>
		<tr>
			<td>Low-adhesion 24-well Plates</td>
			<td>Corning</td>
			<td>3473</td>
			<td></td>
		</tr>
		<tr>
			<td>Low-adhesion 6-well Plates</td>
			<td>Corning</td>
			<td>3471</td>
			<td></td>
		</tr>
		<tr>
			<td>Maintenance medium (MM)</td>
			<td>STEMCELL Technologies</td>
			<td>85850</td>
			<td>Medium</td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Gibco</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Donkey Serum</td>
			<td>Solarbio</td>
			<td>SL050</td>
			<td>Blocking buffer</td>
		</tr>
		<tr>
			<td>Paraplast</td>
			<td>Leica</td>
			<td>39601006</td>
			<td>Tissue embedding</td>
		</tr>
		<tr>
			<td>PBS pH 7.4 basic (1x)</td>
			<td>Gibco</td>
			<td>C10010500BT</td>
			<td>Without Ca+,Mg+</td>
		</tr>
		<tr>
			<td>Reconbinant human bone morphogenetic protein-4(rhBMP4)</td>
			<td>R&#38;D</td>
			<td>314-BP</td>
			<td>Key protein factor</td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Sigma</td>
			<td>R2625</td>
			<td>Powder, keep out of light</td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Selleckchem</td>
			<td>S1067</td>
			<td>ALK5-inhibitor</td>
		</tr>
		<tr>
			<td>SU5402</td>
			<td>Selleckchem</td>
			<td>S7667</td>
			<td>Tyrosine kinase inhibitor</td>
		</tr>
		<tr>
			<td>Super PAP Pen</td>
			<td>ZSGB-BIO</td>
			<td>ZLI-9305</td>
			<td></td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Sigma</td>
			<td>T0625-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Thioglycerol</td>
			<td>Sigma</td>
			<td>M1753</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td>Permeabilization</td>
		</tr>
		<tr>
			<td>WA09 embryonic stem cell line</td>
			<td>WiCell Research Institute</td>
			<td></td>
			<td>Cell line</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>SINOPHARM</td>
			<td>10023418</td>
			<td>Dewaxing</td>
		</tr>
		<tr>
			<td>Y-27632 2HCL</td>
			<td>Selleckchem</td>
			<td>S1049</td>
			<td>ROCK-inhibitor</td>
		</tr>
	</tbody>
</table>

generating neural retina from human pluripotent stem cells

视网膜病变是全球失明的主要原因之一。研究其发病机制对于视网膜病变的早期诊断和及时治疗至关重要。不幸的是，道德障碍阻碍了从人类那里收集证据。最近，大量研究表明，人类多能干细胞（PSCs）可以使用不同的诱导方案分化为视网膜类器官（RO），这在视网膜病变中具有巨大的潜力，可用于疾病建模、药物筛选和基于干细胞的治疗。本研究描述了一种优化的诱导方案，以产生神经视网膜 （NR），显着降低囊泡和融合的可能性，提高生产成功率，直到第 60 天。基于PSCs解离后自重组的能力，结合某些互补因子，这种新方法可以特异性地驱动NR分化。此外，该方法简单、具有成本效益、具有显着的可重复性和效率，为视网膜疾病的个性化模型提供了令人鼓舞的前景，并为细胞治疗、药物筛选和基因治疗测试等应用提供了丰富的细胞库。

The eye serves as the primary source of information among human sensory organs, with the retina being the principal visual sensory tissue in mammalian eyes1. Retinopathy stands as one of the primary global causes of eye diseases, leading to blindness2. Approximately 2.85 million people worldwide suffer from varying degrees of vision impairment due to retinopathy3. Consequently, investigating its pathogenesis is crucial for early diagnosis and timely treatment. Most studies on human retinopathy have primarily focused on animal models4,5,6. However, the human retina is a complex, multi-layered tissue comprising various cell types. Traditional two-dimensional (2D) cell culture and animal model systems typically fail to faithfully recapitulate the normal spatiotemporal development and drug metabolism of the native human retina7,8.
Recently, 3D culture techniques have evolved to generate tissue-like organs from pluripotent stem cells (PSCs)9,10. Retinal organoids (ROs) generated from human PSCs in a 3D suspension culture system not only contain seven retinal cell types but also exhibit a distinct stratified structure similar to the human retina in vivo11,12,13. Human PSC-derived ROs have gained popularity and widespread availability and are currently the best in vitro models for studying the development and disease of the human retina14,15. Over the past few decades, numerous researchers have demonstrated that human PSCs, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), can differentiate into ROs using various induction protocols. These advancements hold enormous potential in retinopathy for disease modeling, drug screening, and stem cell-based therapies16,17,18.
However, the generation of neural retina (NR) from human pluripotent stem cells (PSCs) is a complex, cumbersome, and time-consuming process. Furthermore, batch-to-batch variations in tissue organoids may lead to lower reproducibility of results19,20. Numerous intrinsic and extrinsic factors can influence the yield of retinal organoids (ROs), such as the number or species of starting cells and the use of transcription factors and small-molecule compounds21,22,23. Since the first human RO was generated by the Sasai laboratory11, multiple modifications have been proposed over the years to enhance the ease and effectiveness of the induction process13,21,24,25. Unfortunately, to date, no gold standard protocol has been established for generating ROs in all laboratories. Indeed, there is a certain degree of discrepancy in ROs resulting from different induction methods, as well as wide variation in the expression of retinal markers and the robustness of their structure22,26. These issues may severely complicate sample collection and the interpretation of study findings. Therefore, a more consolidated and robust differentiation protocol is needed to maximize efficiency with minimal heterogeneity of RO generation.
This study describes an optimized induction protocol based on a combination of Kuwahara et al.12 and D&#246;pper et al.27 with detailed instructions. The new method significantly reduces the probability of organoid vesiculation and fusion, increasing the success rate of generating NR. This development holds great promise for disease modeling, drug screening, and cell therapy applications for retinal disorders.

作者聚焦：用于疾病建模的简单高效的神经视网膜类器官生产

从人类多能干细胞生成神经视网膜

Our group focuses on understanding the pathogenesis and the treatment of human retinal diseases. Our protocol describes a optimized induction technique to generate a neural retina that significantly reduce the probability of vasculation and fusion to increase the success rate of retinal production until day 60. The modified method is simpler and more efficient in generating neural retina, which can prove to be a better disease model for drug screening and cell therapy.In the future, we aim to study the effects of physicochemical factors on retinal development and underlying mechanism using our retinal organoid model.

修改后的协议的图形概述如 图 1 所示。当细胞生长到70%-80%的密度时，使用H9-ESCs产生RO。H9-ESCs在96个V型底锥形孔中的单细胞悬浮液在第1天聚集，并在第6天形成边界良好的圆形EB。随着培养时间的增加，EB的体积逐渐增加。第30天，神经上皮样结构在长期NR分化过程中明显形成并增厚。
此外，将改进的方法与其他两种方法（Kuwahara 等人 12 ...

Watch this Scientific Journal Video about Generating Neural Retina from Human Pluripotent Stem Cells at JoVE.com

本方案描述了一种优化的3D神经视网膜诱导系统，该系统以高可重复性和效率减少视网膜类器官的粘附和融合。

Retinopathy is one of the main causes of blindness worldwide. Investigating its pathogenesis is essential for the early diagnosis and timely treatment of ...

我们小组专注于了解人类视网膜疾病的发病机制和治疗。我们的方案描述了一种优化的诱导技术，以产生神经视网膜，显着降低血管形成和融合的可能性，以提高视网膜产生的成功率，直到第 60 天。改进的方法在生成神经视网膜方面更简单、更有效，可以证明是药物筛选和细胞治疗的更好疾病模型。未来，我们的目标是使用我们的视网膜类器官模型研究物理化学因素对视网膜发育的影响和潜在机制。

generating-neural-retina-from-human-pluripotent-stem-cells

Research

JoVE Journal

Developmental Biology

Generating Neural Retina from Human Pluripotent Stem Cells

816 次观看.  Medical School of Chinese PLA. 我们小组专注于了解人类视网膜疾病的发病机制和治疗。我们的方案描述了一种优化的诱导技术，以产生神经视网膜，显着降低血管形成和融合的可能性，以提高视网膜产生的成功率，直到第 60 天。改进的方法在生成神经视网膜方面更简单、更有效，可以证明是药物筛选和细胞治疗的更好疾病模型。未来，我们的目标是使用我们的视网膜类器官模型研究物理化学因素对...

视频: 作者聚焦：用于疾病建模的简单高效的神经视网膜类器官生产

Retinopathy is one of the main causes of blindness worldwide. Investigating its pathogenesis is essential for the early diagnosis and timely treatment of retinopathy. Unfortunately, ethical barriers hinder the collection of evidence from humans. Recently, numerous studies have shown that human pluripotent stem cells (PSCs) can be differentiated into retinal organoids (ROs) using different induction protocols, which have enormous potential in retinopathy for disease modeling, drug screening, and stem cell-based therapies. This study describes an optimized induction protocol to generate neural retina (NR) that significantly reduces the probability of vesiculation and fusion, increasing the success rate of production until day 60. Based on the ability of PSCs to self-reorganize after dissociation, combined with certain complementary factors, this new method can specifically drive NR differentiation. Furthermore, the approach is uncomplicated, cost-effective, exhibits notable repeatability and efficiency, presents encouraging prospects for personalized models of retinal diseases, and supplies a plentiful cell reservoir for applications such as cell therapy, drug screening, and gene therapy testing.

The present protocol describes an optimized 3D neural retina induction system that reduces the adhesion and fusion of retinal organoids with high repeatability and efficiency.

科研

发育生物学

Culturing H9-Embryonic Stem Cells for Generation of Human Neural Retina

To begin, add one milliliter of 1%extracellular matrix to each well of a six well plate. Incubate the plate at 37 degrees Celsius in a 5%carbon dioxide atmosphere. After one hour, add two milliliters of prewarm maintenance medium containing 0.4 micromolar Y-27632 to each well.To thaw the H9 embryonic stem cells, shake the cryovial stock in a 37 degree Celsius water bath for 30 seconds. Carefully sterilize the cryovial with a 75%disinfectant alcohol spray. Then transfer the thawed cells to a 15 milliliter tube containing the maintenance medium and Y-27632.Centrifuge the tube at 190 G for five minutes at room temperature. Then carefully remove most of the supernatant and resuspend the cells in one milliliter of maintenance medium containing Y-27632. Dispense 0.5 milliliters of the cell suspension to each well of the extracellular matrix coated plate containing maintenance medium.Shake the plate laterally and incubate the plate at 37 degrees Celsius under 5%carbon dioxide for at least 24 hours. Change the medium every day. Once the clone density reaches 70%remove the spent medium.After 2D PBS washes, incubate the cells with one milliliter of EDTA for four to seven minutes at room temperature and discard EDTA completely. Add one milliliter of maintenance medium to the cells and tap the plate gently. Then transfer the dislodged cells to a 15 milliliter tube and mix them three to five times.Add 20 to 100 microliters of cell suspension to each well of a previously prepared ECM-coated dish and mix well. Using a microscope, confirm the cell density and incubate the cells at 37 degrees Celsius under 5%carbon dioxide.

培养H9胚胎干细胞以生成人类神经视网膜 

Inducing 3D Neural Retina Formation from Cultured H9-Embryonic Stem Cells for Stem-Cell Based Therapies

To begin, thaw and culture H9-embryonic stem cells in maintenance medium containing Y-27632. On day zero, once the colonies become 70%confluent, wash them with 2 milliliters of DPBS, then incubate the cells with 0.5 milliliters of cell dissociation solution containing 20 micromoles per liter Y-27632. To abort the digestion, add 3 milliliters of maintenance medium containing 20 micromoles per liter Y-27632.To pellet the cells, centrifuge the tube at 190 G for five minutes and discard the supernatant. Resuspend the pellet in one milliliter of growth factor-free chemically-defined medium with Y-27632. After counting the cells, add 1.2 million cells to 10 milliliters of growth factor-free chemically-defined medium and mix well.Then dispense 100 microliters of cell suspension to each well of a 96 well conical bottom plate. Incubate in a humidified 5%carbon dioxide atmosphere. To induce retina formation on day six, add medium containing BMP4 to each well of a 96 well V-bottom plate and incubate again.On day nine, 12, and 15, replace half of the spent medium with fresh medium without BMP4. On day 18, carefully transfer the formed embryoid bodies to a 15 milliliter centrifuge tube. After the natural precipitation of the embryoid bodies, gently remove the supernatant and rinse them with the neural retina differentiation medium.Then place the embryoid bodies in a low absorption six well plate and incubate the plate again. On day 21, Brightfield images showed continuous neuroepithelial structure in the neural retina generated using this method. The retinal induction success rate was significantly higher and retinal organoids generated using this protocol were similar in size and morphology compared to other methods.