Name: differentiation, maintenance, and analysis of human retinal pigment epithelium cells: a disease-in-a-dish model for best1 mutations
Uploaded: 2018-08-24T12:30:00.000+00:00
Duration: 6 min 39 s
Description: Watch this Scientific Journal Video about Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations at JoVE.com

该项目由 NIH 赠款 EY025290、GM127652 和罗切斯特大学开办资金资助。

1. hPSC 与视网膜色素的分化
<ol><li>维护和通过 hPSCs 如前所述18。 注: 所有细胞 (包括 hPSCs 和 hPSC-RPEs) 生长在37摄氏度, 在 5% CO2期间, 整个增长和分化协议。</li>
	<li>
在分化之前, 分裂汇合 hPSCs 到预涂覆的6井板。
	<ol>
<li>要涂板, 解冻基底膜基质在冰上约1小时, 悬浮液化基质在4°c DMEM 培养基1:50 稀释, 添加800µL 涂层混合物的每一个井, 并孵化至少1小时在37摄氏度。</li>
		<li>当涂层完成后, 吸入混合物并立即将1.5–2毫升介质加入水井中。</li>
		<li>从旧的培养皿中抬起 hPSCs, 用 PBS 加0.5 毫米 EDTA 孵化细胞, 室温5分钟, 吸出溶液, 用培养基将小团簇并用重悬细胞。</li>
		<li>种子细胞在20% 融合在新的板材。 注: 贮存基底膜基质在100µL 整除数在-20 摄氏度, 并使用一个整除涂层6井板。涂层的时间可以延长到4小时, 在37摄氏度, 或在室温过夜。重要的是暂停和种子 hPSCs 作为小团簇的3–5细胞, 而不是单个细胞。</li>
	</ol>
</li>
	<li>当细胞长成全融合时, 用分化培养基 (4 毫升/井) 取代培养基 (表 1, 不激活)。培养14天, 改变介质 (4 毫升/井) 3 x/周。 注: hPSC 板密度加倍大约每24小时. 在分化过程开始后, 明显的细胞死亡是预期的。在培养基变化过程中, 肉眼可以看到死细胞和碎片在水井中的聚集。</li>
	<li>从15天到28天, 补充分化培养基与 100 ng/毫升人类激活 (表 1, 与人类激活 a)。改变介质 (4 毫升/井) 3 x/周。</li>
	<li>停止激活-从29天开始补充 (表 1, 没有人激活)。继续细胞培养在分化培养基为8–10星期, 直到色素 hPSC-RPE 簇出现 (图 1A-B)。 注意: 染色细胞簇可以被肉眼观察到细胞培养板上的小黑点 (图 1A-B, 顶部)。在20X 显微镜下 (图 1A-B, 底部) 可以看到具有特征鹅卵石形状的个体色素细胞。</li>
</ol>2. 分化的视网膜色素上皮细胞的分离和培养
<ol><li>移除分化介质, 用 PBS 冲洗一次, 在每个井中加入1毫升0.05% 胰蛋白酶加 1 U/µL 胶原酶, 并在37摄氏度内孵育 20–30. 除去胰蛋白酶/胶原酶, 轻轻地将2毫升预热的 RPE 介质加入6井板的每一井 (c0>Table 2)。 注: 使用倒置显微镜监测 hPSC 视网膜色素上皮细胞的形态, 在胰蛋白酶/胶原酶治疗前是多边形, 并在充分消化后变成圆形。</li>
	<li>
使用本生燃烧器拉玻璃巴斯德吸管。
	<ol>
<li>用双手, 保持长 (9 英寸) 巴斯德吸管在两端, 所以它是水平的, 上面的本生燃烧器约2/3 的长度的薄部分的吸管。</li>
		<li>一旦玻璃从火焰中变软, 迅速把两端分开, 这样在吸管的新端形成一个微型刮刀状尖端 (图 2)。</li>
		<li>重复数次以创建多个 "单元格刮刀"。喷雾的拉吸管与70% 乙醇灭菌和空气干燥他们在细胞培养罩。</li>
	</ol>
</li>
	<li>
在显微镜下, 用微 "细胞刮刀" 轻轻地将色素 hPSC-RPE 簇从盘子中分离出来。
	<ol>
<li>轻轻地轻敲 (不要刮) 直接对色素细胞, 它将漂浮到培养基中, 一旦它们从井底离解。</li>
		<li>立即使用20µL 微轻轻吸入分离的 hPSC 视网膜色素细胞, 并将其转移到预涂6井板与视网膜色素中等24。</li>
		<li>收集 ~ 5 x 103细胞 (~ 10% 融合, 观察下20X 显微镜下) 在每一个12井板的预涂层井通过重复步骤 2.3. 1–2.3. 2 多次, 根据需要。把最后的音量从中到2毫升。 注: 为了防止游离 hPSC 视网膜色素上皮细胞漂浮, 避免在离解过程中板块的移动, 使离解细胞漂浮在培养基中, 但仍保持局部集中。</li>
	</ol>
</li>
	<li>培养在视网膜色素培养基上的12井板上新隔离的 hPSC 细胞 (P0), 使其形成染色单层 (图 1C)。</li>
	<li>更改中等 3 x/周。当改变培养基时, 在每个井中留出大约1/3 体积的旧介质, 并添加2/3 体积的新鲜、预热的视网膜色素培养基。在这个阶段, 使用2毫升的 RPE 介质为每个井在一个12井板 (因此, 交换1.3 毫升的媒介每次)。 注: P0 hPSC-RPE 细胞的单层可能形成圆顶, 表明细胞正积极输送液体, 并由紧密连接25锚定。因此, 圆顶的形成是一个成熟的视网膜色素状态的指标。</li>
</ol>3. 视网膜色素免疫印迹法的命运验证
<ol><li>利用哺乳动物蛋白提取缓冲剂, 用蛋白酶抑制剂鸡尾酒, 制作 hPSC 细胞裂解物。在30分钟的冰上, 在 1.3万 x g的离心前孵育细胞裂解物, 以在4摄氏度处丢弃难溶细胞碎片。测定裂解液的总蛋白浓度。</li>
	<li>变性40µg 的总蛋白在 Laemmli SDS 样品缓冲区75摄氏度10分钟. 将10% 三甘氨酸 SDS 页凝胶上的蛋白质样品分离为90伏的恒定电压, 然后 15 v, 直到染料前端达到凝胶的底部。</li>
	<li>在预冷的三甘氨酸缓冲液中将蛋白质转移到硝化膜上, 再辅以10% 甲醇100伏, 1 小时。</li>
	<li>在室温下, 用0.1% 非离子洗涤剂 (TBST) 和5% 非脂肪干牛奶在三层缓冲盐水中阻断膜。</li>
	<li>在4摄氏度的阻塞缓冲液中, 用最初的抗体将细胞膜孵化出来。稀释针对 RPE 特异标记蛋白的抗体如下: RPE65, 1:1, 000;CRALBP, 1:1, 000;BEST1, 1:1, 000。稀释抗体对β肌动蛋白在 1:10,000, 以检测β肌动蛋白作为负荷控制。</li>
	<li>用 TBST 洗3次膜, 每次洗5分钟。</li>
	<li>用荧光共轭山羊抗小鼠 IgG 二次抗体在室温下以1小时的阻塞缓冲液稀释1:10,000。</li>
	<li>用 TBST 洗4次膜, 每次洗10分钟。</li>
	<li>使用红外成像系统检测蛋白质 (图 3)。</li>
</ol>4. 染色检测 BEST1 亚细胞定位
<ol><li>用2毫升的 PBS 清洗 hPSC 视网膜色素细胞, 并在室温下固定2毫升4% 多聚甲醛45分钟。</li>
	<li>用2毫升的 pbs 冲洗两次, 并在2毫升的 pbs 中孵化出0.1% 个非离子表面活性剂和2% 驴血清以45分钟来阻断非特定的结合部位。</li>
	<li>室温下用 BEST1 抗体 (1:200 稀释) 孵化细胞, 2 小时。</li>
	<li>用2毫升 PBS 冲洗细胞3次。用荧光共轭二级 IgG (1:1, 000) 在室温下孵育1小时。</li>
	<li>用2毫升 PBS 冲洗3次以去除未绑定的二级抗体。</li>
	<li>用共焦显微镜观察染色细胞 (图 4)。</li>
</ol>5. 用全细胞膜片钳记录 hPSC -RPE 中 Ca2 +相关 Cl 电流
<ol><li>24–72 h 在膜片钳之前, 分裂一个完全汇合的井 (在一个12井板) 成熟 P0 hPSC-RPE 细胞 (鹅卵石形状和着色)。吸入培养基, 用 PBS 冲洗一次, 并在井中加入1毫升0.05% 胰蛋白酶加 1 U/µL 胶原酶。 注: 预热的胰蛋白酶/胶原酶到37摄氏度的权利使用前。</li>
	<li>孵育37°c 8 分钟。 注意: 在这一步之后, hPSC 细胞仍然黏附和连接。</li>
	<li>用1毫升的微轻轻地将细胞从盘子里冲洗出来, 将消化混合物中的细胞转移到15毫升锥形管上。用1毫升预热的胰蛋白酶/胶原酶洗井, 收集残余细胞, 并将其组合成15毫升锥形管。 注意: 在这一点上, hPSC 视网膜上皮细胞仍在大团簇中。不要试图通过剧烈的吹打打破细胞团簇。有些团簇可能粘在1毫升尖端的内壁上。</li>
	<li>在37°c 干浴中孵化锥形管8分钟。</li>
	<li>用1毫升的微轻轻地吸收10–15倍的消化混合。 注意: 细胞团簇变得小得多, 但仍然可见。避免剧烈的吹打。</li>
	<li>重复前面的两个步骤。 注: 大部分细胞应在吹打后单细胞悬浮。可能还有一些微小的细胞团簇, 这是可以接受的。可选: 在37摄氏度孵育锥形管, 再加5分钟, 然后用温和的吹打。胰蛋白酶/胶原酶在37°c 的总时间不应超过30分钟 (8 + 8 + 8 + 5 = 29)。</li>
	<li>将5毫升预热的 RPE 培养基添加到含有所消化细胞的15毫升圆锥管中。旋转 200 x g 5 分钟收集细胞。在10–20% 融合上预涂上 35 mm 的菜肴上重新悬浮和种子细胞 (例如, 在12井板上的100% 汇合井, 35 融合的五10% 毫米菜肴)。 注意: 在任何时候, 避免剧烈的吹打, 这可能导致显着的细胞死亡, 由明显的黑细胞碎片在上清后, 离心。良好分离的单细胞播种是修补钳记录的关键。</li>
	<li>按照前面描述的18、26、27、28、29、30、31, 执行全细胞贴片钳。从一个阶梯电位族 (-100 到 +100 mv, 从持有电位 0 mv) 获取当前痕迹 (图 5)。 注: 内部和外部解决方案的食谱分别在表 3和表 4中进行了说明。</li>
</ol>

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L., Chen, M., Fang, K., Colecraft, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bio-inspired+voltage-dependent+calcium+channel+blockers.">Bio-inspired voltage-dependent calcium channel blockers.</a> Nat Commun. 4, 2540 (2013).</li><li>Yang, T., Hendrickson, W. A., Colecraft, H. 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作者没有什么可透露的。

最重要的程序, 在一个菜式的方法是区分 hPSCs 与疾病引起的突变的正确的细胞谱系, 这是视网膜色素BEST1。因此, 在每次分化实验后, 通过视网膜色素特异的形貌和蛋白质标记物16、17、18, 应仔细验证所产生的 hPSC 视网膜上皮细胞的成熟状态。为了最小化克隆伪影, 应尽可能使用同一患者或多个人类胚胎干细胞克隆具有相同突变的多个 hiPSC 克隆。
hiPSC 和人类胚胎干细胞线都可以用相同的协议区分为 RPE。hiPSCs 有或不突变的BEST1基因可以重新编程从皮肤成纤维细胞的 BEST1 患者或健康的捐赠者, 分别。干细胞在 BEST1 中的突变可以从父母人类胚胎干细胞线上进行基因工程, 这种基因具有野生型 (小波) BEST1。hiPSC 和人类胚胎干细胞-rpe 路线有各自的优缺点。前者更具病人特异和临床相关, 特别是对个性化医学。然而, 值得注意的是, 有几个限制 hiPSC-RPE 方法: 1) 它是在后勤困难, 以获得全面的BEST1病人样本, 因为病人的捐赠不能总是被授予, 一些突变是非常罕见的第一位;2) 非特异表型可能是由于不同的遗传背景和捐赠者的身体状况造成的;3) 不同的捐赠者对视网膜色素分化效率的 hiPSC 差异很大, 因此有些病例在技术上具有挑战性, 可以获得 hiPSC-RPEs。另一方面, 人类胚胎干细胞-RPE 路线, 虽然更人工, 提供了一个多功能的平台, 以产生等基因系人类胚胎干细胞 RPEs 与预期的突变需求的非偏见功能调查。
成熟的 RPE 细胞是色素, 多边形, 并通过紧密连接的单层。在分裂成一个单一的细胞群体的补丁钳, 新鲜 reseeded hPSC 视网膜细胞将失去多边形形状, 但仍保留色素沉着数天 (图 5A)。膜片钳是执行 24–72 h 细胞分裂后, 在这段时间的色素沉着仍然可以作为一个可见的标记选择细胞具有良好的视网膜色素状态。一个温和的细胞分裂导致大多数分离良好的单细胞没有显著的细胞死亡, 是成功的补丁钳的关键。
在文献21、32中记录了其他几种不同细胞培养培养基和生长因子的分化协议。hPSC 与 RPE 的分化所需的时间在所有这些协议中都是相似的, 其中包括我们的, 但很难判断在没有并排比较的情况下, 分化效率是否有显著差异。应该指出的是, 在目前的协议中, hPSC 视网膜上皮细胞生长在正常的扁平底板上, 而不是在平板上的膜上插入, 所以这个过程产生的 hPSC, 可能不能最好地重述视网膜色素细胞在体内的极性。因此, 上述技术大多适合于研究设置33, 虽然生成的 hPSC 视网膜色素上皮细胞也可以培养 Transwell 板, 形成单层视网膜色素表为临床目的17。

据记载, 至少五的视网膜退行性疾病是由基因突变引起的BEST1基因1,2,3,4,5,6,7,8, 与报告的变异的数量已经超过200并且仍然增加。这些BEST1相关的疾病, 也称为 bestrophinopathies, 导致视力逐渐丧失甚至失明, 目前还没有有效的治疗方法。BEST1的蛋白产物, 即 BESTROPHIN1 (BEST1), 是一种 Ca2 +活化的 Cl 通道 (CaCC), 特别表达在视网膜色素上皮 (RPE) 的眼睛5,6, 8,9。重要的是, 临床表型的BEST1相关疾病是减少视觉反应的光刺激, 称为光峰 (LP) 测量的 electrooculogram10,11;LP 被认为是介导的 CaCC 在视网膜色素12,13,14。为了更好地理解BEST1突变的病理机制, 并致力于潜在的治疗, 有必要研究突变 BEST1 通道内在表达的人视网膜色素细胞。
然而, 直接从活体患者获得视网膜色素上皮细胞是非常不切实际的。虽然本机视网膜色素细胞可以从人体尸体和胎儿的活检中获得, 但这些来源的难以获取却极大地限制了科学研究。因此, 除了人眼以外, 有其他的视网膜色素源是至关重要的。这一呼吁得到了最近的进展, 干细胞技术, 因为功能性视网膜色素细胞现在可以区别于人类多潜能干细胞 (hPSCs), 包括胚胎干细胞 (干细胞) 和诱导多潜能干细胞 (hiPSCs), 后者由捐赠者16,17,18的主要皮肤成纤维细胞重新编程产生。重要的是, hPSCs 的自我更新和干细胞确保了产生 RPEs 的可靠来源, 而干细胞的 hiPSCs 和基因组修饰电位的患者特异性 (如CRISPR) 提供了一种多功能的碟形疾病模型, 用于期望的BEST1突变。
hPSC 在小鼠视网膜色素模型上有几个优势: 1) BEST1敲除小鼠不显示任何视网膜异常19, 提高BEST1在视网膜色素瘤中对小鼠和人类的不同遗传需求的可能性;2) 只有3% 的人视网膜色素细胞是双核的, 与35% 在小鼠20;3) hiPSC 增强自体移植在视网膜疾病临床治疗中的应用21。然而, 动物模型仍然是必不可少的研究视网膜色素生理和病理在一个活体系统, 和致癌的潜力 hiPSC 不能忽视。
这里的过程描述了一个有用的和适度简单的 hPSC 的视网膜色素分化协议, 可用于研究和临床目的。该协议使用烟酰胺 (维生素 B3) 增加 hPSCs 分化为神经组织, 进一步诱导分化为 RPE 的治疗与激活。烟酰胺治疗已被证明增加色素细胞的数量 (一种分化为 RPE 的迹象), 可能通过减轻分化细胞的凋亡活动22。结果 hPSC 细胞显示相同的关键标记, 鹅卵石形态学和细胞功能作为本机人类视网膜色素上皮细胞22。因此, 在一项研究中, 所产生的 hPSC 视网膜色素细胞适用于下游功能分析, 包括免疫印迹法、染色和全细胞贴片钳, 并提供了详细的实验程序。临床上, 从干细胞中提取的视网膜色素上皮细胞在动物研究和人类试验中都显示了移植治疗黄斑变性的巨大潜力23。

<table><tbody><tr><td>Knock-Out (KO) DMEM</td><td>ThermoFisher</td><td>10829018</td><td></td></tr><tr><td>KO serum replacement</td><td>ThermoFisher</td><td>10829028</td><td></td></tr><tr><td>Nonessential amino acids</td><td>ThermoFisher</td><td>11140050</td><td></td></tr><tr><td>Glutamine</td><td>ThermoFisher</td><td>35050061</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>ThermoFisher</td><td>10378016</td><td></td></tr><tr><td>Nicotinamide</td><td>Sigma-Aldrich</td><td>N0636</td><td></td></tr><tr><td>Human activin-A</td><td>PeproTech</td><td>120-14</td><td></td></tr><tr><td>MEM (a modification)</td><td>Sigma-Aldrich</td><td>M4526</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>VWR</td><td>97068-085</td><td></td></tr><tr><td>N1 supplement</td><td>Sigma-Aldrich</td><td>N6530</td><td></td></tr><tr><td>Glutamine-penicillin-streptomycin</td><td>Sigma-Aldrich</td><td>G1146</td><td></td></tr><tr><td>Nonessential amino acids</td><td>Sigma-Aldrich</td><td>M7156</td><td></td></tr><tr><td>Taurine</td><td>Sigma-Aldrich</td><td>T0625</td><td></td></tr><tr><td>Hydrocortisone</td><td>Sigma-Aldrich</td><td>H0386</td><td></td></tr><tr><td>Triiodo-thyronin</td><td>Sigma-Aldrich</td><td>T5516</td><td></td></tr><tr><td>mTeSR-1 medium</td><td>Stemcell Technologies</td><td>5850</td><td></td></tr><tr><td>Matrigel</td><td>Corning</td><td>356230</td><td></td></tr><tr><td>Collagenase</td><td>Gibco</td><td>17104019</td><td></td></tr><tr><td>Trypsin</td><td>VWR</td><td>45000-664</td><td></td></tr><tr><td>M-PER mammalian protein extraction reagent</td><td>Pierce</td><td>78501</td><td></td></tr><tr><td>proteinase inhibitor cocktail</td><td>Sigma-Aldrich</td><td>4693159001</td><td></td></tr><tr><td>RPE65 antibody</td><td>Novus Biologicals</td><td>NB100-355</td><td></td></tr><tr><td>CRALBP antibody</td><td>Abcam</td><td>ab15051</td><td></td></tr><tr><td>BEST1 antibody</td><td>Novus Biologicals</td><td>NB300-164</td><td></td></tr><tr><td>Beta Actin antibody</td><td>ThermoFisher</td><td>MA5-15739</td><td></td></tr><tr><td>Alexa Fluor 488-conjugated donkey anti-mouse IgG</td><td>ThermoFisher</td><td>A-21202</td><td></td></tr><tr><td>Goat anti-mouse IgG</td><td>ThermoFisher</td><td>SA5-35521</td><td></td></tr><tr><td>Goat anti-Rabbit IgG</td><td>LI-COR Biosciences</td><td>926-68071</td><td></td></tr><tr><td>Hoechst 33342</td><td>ThermoFisher</td><td>62249</td><td></td></tr><tr><td>HEKA EPC10 patch clamp amplifier</td><td>Warner Instruments</td><td>895000</td><td></td></tr><tr><td>Patchmaster</td><td>Warner Instruments</td><td>895040</td><td></td></tr></tbody></table>

differentiation, maintenance, and analysis of human retinal pigment epithelium cells: a disease-in-a-dish model for best1 mutations

虽然人类BEST1基因中有超过200种基因突变被发现并与视网膜退行性疾病相关联, 但病理机制仍然难以捉摸, 主要是由于缺乏良好的体内模型来研究BEST1及其在生理条件下的突变。BEST1编码离子通道, 即 BESTROPHIN1 (BEST1), 其作用于视网膜色素上皮 (RPE);然而, 人类视网膜色素上皮细胞的极有限的可获得性是科学研究的一个重大挑战。该协议描述了如何通过诱导人类多潜能干细胞 (hPSCs) 的分化而产生人类 RPEs 轴承BEST1致病突变。由于 hPSCs 是可自我再生的, 这种方法允许研究人员有一个稳定的来源的 hPSC RPEs 的各种实验分析, 如免疫印迹法, 免疫荧光和补丁钳, 从而提供了一个非常强大的疾病在一碟模型BEST1相关的视网膜条件。值得注意的是, 这一策略可以应用于研究 rpe (病理) 生理学和本机所表达的兴趣的其他基因。

技术上最具挑战性的一步是手工隔离, 目的是实现高纯度的差异 P0 hPSC-RPE 种群。经过成功的隔离, > 90% 细胞在 P0人口将增长和成熟, 以显示签名的 RPE 形态 (图 1C)。在 P0的人群中, 非 rpe 或未成熟的视网膜色素上皮细胞的存在几乎是不可避免的, 但不会干扰下游实验, 只要色素和鹅卵石状 hPSC 细胞的数量是压倒性的大多数。
以 hPSC-RPE 为基础的疾病在 a 盘方法, 每个患者特异的BEST1变异可以全面地被描述在体内为它的蛋白质表达 (免疫印迹法,图 3), 膜贩运 (染色,图 4) 和离子通道功能 (全细胞贴片钳,图 5)。这些结果将为阐明经络突变的病理机制和发展个性化医学提供重要的信息。
由于 BEST1 主要表现在视网膜色素上皮细胞, 它可以作为一个细胞标记, 以确认成熟的视网膜色素状态。应该指出的是, 一些BEST1突变可能会影响其蛋白表达, 所以其他已建立良好的 RPE 标记, 如 RPE65 和 CRALBP 仍然需要评估 (图 3)。
成熟的 p0 hPSC-RPE 细胞可以维持2-3 月前分裂 (对 P1) 的全细胞贴片钳。0个以上3月以上的细胞不推荐用于补丁钳, 但仍可用于免疫印迹法和染色实验。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57791/57791fig1.jpg"> 图 1: 不同阶段分化的 hPSC 视网膜色素上皮细胞.色素 hPSC-RPE 簇在培养板上的代表性图像 (A), 在隔离 (B) 之前, 以及在成长到成熟后的隔离 (C)。眼睛和20X 相位对比图像分别显示在顶部和底部板上。<a href="/files/ftp_upload/57791/57791fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57791/57791fig2.jpg"> 图 2: 从巴斯德吸管中拉出的微细胞刮刀的代表性图像.<a href="/files/ftp_upload/57791/57791fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57791/57791fig3v2.jpg"> 图 3: 免疫印迹法鉴别 hPSC 视网膜色素上皮细胞成熟的 rpe 状态的验证.BEST1、RPE65 和 CRALBP 在 hPSC 和 hPSC 视网膜色素上皮细胞中表达的视网膜色素特异蛋白标记物。<a href="/files/ftp_upload/57791/57791fig3v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57791/57791fig4v3.jpg"> 图 4: BEST1 的染色 hPSC RPEs.(A) 具有代表性的免疫荧光图像, 显示了 BEST1 在鹅卵石状 hPSC-RPE 细胞中的膜定位。(B) 染色阴性对照, 省略 BEST1 原抗体。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57791/57791fig5v3.jpg"> 图 5: hPSC-RPEs 的全细胞贴片钳记录.(a) 全细胞贴片钳的单个 hPSC 视网膜细胞。(B) 从BEST1 hPSC-rpe 和BEST1突变的 hPSC-视网膜色素在峰值 Ca2 +上记录的现有痕迹。在插入中显示用于引出电流的电压协议。刻度条 = 1 nA, 150 毫秒. 请参阅表 3和表 4以了解补丁夹的准备细节。<a href="/files/ftp_upload/57791/57791fig5v3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>试剂</td>
			<td>量</td>
		</tr>
<tr>
<td>敲出 (高) DMEM</td>
			<td>500毫升</td>
		</tr>
<tr>
<td>高血清置换术</td>
			<td>15% (75 毫升)</td>
		</tr>
<tr>
<td>非必需氨基酸</td>
			<td>1% (5 毫升)</td>
		</tr>
<tr>
<td>谷 氨 酰 胺</td>
			<td>1% (5 毫升)</td>
		</tr>
<tr>
<td>青霉素-链霉素</td>
			<td>1% (5 毫升)</td>
		</tr>
<tr>
<td>烟 酰 胺</td>
			<td>10毫米</td>
		</tr>
<tr>
<td>人类激活</td>
			<td>100 ng/毫升</td>
		</tr>
<tr>
<td colspan="2">* 人类激活在15–28的差异性协议的天数内得到补充。</td>
		</tr>
</tbody></table>表 1: 视网膜色素分化培养基。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>试剂</td>
			<td>量</td>
		</tr>
<tr>
<td>记忆 (α修改)</td>
			<td>500毫升</td>
		</tr>
<tr>
<td>胎牛血清</td>
			<td>5% (25 毫升)</td>
		</tr>
<tr>
<td>N1 补充剂</td>
			<td>1% (5 毫升)</td>
		</tr>
<tr>
<td>谷氨酰胺-青霉素-链霉素</td>
			<td>1% (5 毫升)</td>
		</tr>
<tr>
<td>非必需氨基酸</td>
			<td>1% (5 毫升)</td>
		</tr>
<tr>
<td>牛磺酸</td>
			<td>125毫克</td>
		</tr>
<tr>
<td>松</td>
			<td>10µg</td>
		</tr>
<tr>
<td>Triiodo-thyronin</td>
			<td>0.0065 µg</td>
		</tr>
</tbody></table>表 2: 视网膜色素培养培养基。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>试剂</td>
			<td>浓度</td>
		</tr>
<tr>
<td>Cscl</td>
			<td>130毫米</td>
		</tr>
<tr>
<td>氯化镁2
</td>
			<td>1毫米</td>
		</tr>
<tr>
<td>EGTA</td>
			<td>10毫米</td>
		</tr>
<tr>
<td>镁 ATP</td>
			<td>2毫米</td>
		</tr>
<tr>
<td>HEPES (pH 值 7.4)</td>
			<td>10毫米</td>
		</tr>
<tr>
<td>CaCl2*</td>
			<td>不同</td>
		</tr>
<tr>
<td>葡萄糖 **</td>
			<td>~ 5 毫米</td>
		</tr>
<tr>
<td colspan="2">* 添加 CaCl2以获得所需的 Ca2 +浓度</td>
		</tr>
<tr>
<td colspan="2">** 使用葡萄糖调节渗透为 290–295 mOsm/升</td>
		</tr>
</tbody></table>表 3: 膜片钳内部解决方案。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>试剂</td>
			<td>浓度</td>
		</tr>
<tr>
<td>Nacl</td>
			<td>140毫米</td>
		</tr>
<tr>
<td>氯化钾</td>
			<td>5毫米</td>
		</tr>
<tr>
<td>氯化镁2
</td>
			<td>1毫米</td>
		</tr>
<tr>
<td>CaCl2
</td>
			<td>2毫米</td>
		</tr>
<tr>
<td>HEPES (pH 值 7.4)</td>
			<td>10毫米</td>
		</tr>
<tr>
<td>葡萄糖</td>
			<td>~ 5 毫米</td>
		</tr>
<tr>
<td colspan="2">* 使用葡萄糖调节渗透至 300–305 mOsm。</td>
		</tr>
</tbody></table>表 4: 贴片钳外部解决方案。

Watch this Scientific Journal Video about Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations at JoVE.com

Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations

人视网膜色素上皮细胞的分化、维持和分析: 一种BEST1突变的碟形模型

在这里, 我们提出了一个协议, 以区分视网膜色素上皮 (RPE) 细胞的人多潜能干细胞与患者衍生的突变。突变细胞系可用于功能分析, 包括免疫印迹法、免疫荧光和贴片钳。这一碟形疾病的方法绕过了获取本族人类视网膜色素细胞的困难。

Although over 200 genetic mutations in the human BEST1 gene have been identified and linked to retinal degenerative diseases, the pathological mechanisms ...

differentiation-maintenance-analysis-human-retinal-pigment-epithelium

Nanyang Technological University

Research

JoVE Journal

Genetics

Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations

6.7K 次观看.  University of Rochester, School of Medicine and Dentistry. 在这里, 我们提出了一个协议, 以区分视网膜色素上皮 (RPE) 细胞的人多潜能干细胞与患者衍生的突变。突变细胞系可用于功能分析, 包括免疫印迹法、免疫荧光和贴片钳。这一碟形疾病的方法绕过了获取本族人类视网膜色素细胞的困难。

视频: Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations

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

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium (hfRPE) cultures with morphological, physiological and genetic characteristics of native human RPE. These hfRPE cell cultures exhibit heavy pigmentation, and electron microscopy show extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied mammalian models of native RPE, including human. These results were extended by the development of therapeutic interventions in several animal models of human eye disease. We have focused on strategies for the removal of abnormal fluid accumulation in the retina or subretinal space. The extracellular subretinal space separates the photoreceptor outer segments and the apical membrane of the RPE and is critical for maintenance of retinal attachments and a whole host of RPE/retina interactions.

We provide a reproducible method for culturing confluent monolayers of human fetal retinal pigment epithelial cells (hfRPE) cells that exhibit morphology, physiology, polarity, and protein and gene expression patterns of adult native tissue. This work has been extended to an animal model of several eye diseases.

视网膜色素上皮的生理与病理生理学研究的实验模型

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium ...

神经科学

Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of human-induced pluripotent stem cells (iPSCs) is particularly attractive for neurodegenerative disease studies, including retinal dystrophies, where iPSC-derived retinal cell models mark a major step forward to understand and fight blindness. In this paper, we describe a simple and scalable protocol to generate, mature, and cryopreserve retinal organoids. Based on medium changing, the main advantage of this method is to avoid multiple and time-consuming steps commonly required in a guided differentiation of iPSCs. Mimicking the early phases of retinal development by successive changes of defined media on adherent human iPSC cultures, this protocol allows the simultaneous generation of self-forming neuroretinal structures and retinal pigmented epithelial (RPE) cells in a reproducible and efficient manner in 4 weeks. These structures containing retinal progenitor cells (RPCs) can be easily isolated for further maturation in a floating culture condition enabling the differentiation of RPCs into the seven retinal cell types present in the adult human retina. Additionally, we describe quick methods for the cryopreservation of retinal organoids and RPE cells for long-term storage. Combined together, the methods described here will be useful to produce and bank human iPSC-derived retinal cells or tissues for both basic and clinical research.

The production of specialized retinal cells from pluripotent stem cells is a turning point in the development of stem cell-based therapy for retinal diseases. The present paper describes a simple method for an efficient generation of retinal organoids and retinal pigmented epithelium for basic, translational, and clinical research.

定义的无异和无饲养培养条件的人 iPSC 视网膜细胞模型的生成

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of ...

Generation of Human Patient iPSC-derived Retinal Organoids to Model Retinitis Pigmentosa

Retinitis pigmentosa (RP) is a rare and inherited retinal degenerative disease with a prevalence of approximately 1/4,000 people worldwide. The majority of RP patients have progressive photoreceptor degeneration leading to peripheral vision loss, night blindness, and finally, total blindness. To date, thousands of mutations in more than 90 genes have been reported to be associated with RP. Currently, there are few animal models available for all the affected genes and different types of mutations, which largely hampers the deciphering of the mechanisms underlying the gene/mutation pathology and limits treatment and drug development. Patient induced pluripotent stem cell (iPSC)-derived 3D retinal organoids (ROs) have provided a better system to model the human early-onset disease than cells and animals. In order to study RP, those patient-derived 3D retinal organoids were utilized to recapitulate the clinical phenotypes of RP. In the RP patient-derived ROs, Rhodopsin mislocalization was clearly displayed. Compared with other animal models, patient iPSC-derived retinal organoid models more closely recapitulated RP features and represent an ideal approach for investigating the disease pathogenesis and for drug development.

In this protocol, retinitis pigmentosa patient induced pluripotent stem cell (iPSC)-derived 3D retinal organoids were generated. Those organoids successfully recapitulated some clinical phenotypes of the retinitis pigmentosa disease.

Retinitis pigmentosa (RP) is a rare and inherited retinal degenerative disease with a prevalence of approximately 1/4,000 people worldwide. The majority ...

Generation of Retinal Organoids from Healthy and Retinal Disease-Specific Human-Induced Pluripotent Stem Cells

Pluripotent stem cells can generate complex tissue organoids that are useful for in vitro disease modeling studies and for developing regenerative therapies. This protocol describes a simpler, robust, and stepwise method of generating retinal organoids in a hybrid culture system consisting of adherent monolayer cultures during the first 4 weeks of retinal differentiation till the emergence of distinct, self-organized eye field primordial clusters (EFPs). Further, the doughnut-shaped, circular, and translucent neuro-retinal islands within each EFP are manually picked and cultured under suspension using non-adherent culture dishes in a retinal differentiation medium for 1-2 weeks to generate multilayered 3D optic cups (OC-1M). These immature retinal organoids contain PAX6+ and ChX10+ proliferating, multipotent retinal precursors. The precursor cells are linearly self-assembled within the organoids and appear as distinct radial striations. At 4 weeks after suspension culture, the retinal progenitors undergo post-mitotic arrest and lineage differentiation to form mature retinal organoids (OC-2M). The photoreceptor lineage committed precursors develop within the outermost layers of retinal organoids. These CRX+ and RCVRN+ photoreceptor cells morphologically mature to display inner segment-like extensions. This method can be adopted for generating retinal organoids using human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). All steps and procedures are clearly explained and demonstrated to ensure replicability and for wider applications in basic science and translational research.

This protocol describes an efficient method of differentiating hiPSCs into eye field clusters and generating neuro-retinal organoids using simplified culture conditions involving both adherent and suspension culture systems. Other ocular cell types, such as the RPE and corneal epithelium, can also be isolated from mature eye fields in retinal cultures.

从健康和视网膜疾病特异性人诱导的多能干细胞中生成视网膜类器官

Pluripotent stem cells can generate complex tissue organoids that are useful for in vitro disease modeling studies and for developing regenerative ...

Toxicity Screens in Human Retinal Organoids for Pharmaceutical Discovery

Organoids provide a promising platform to study disease mechanism and treatments, directly in the context of human tissue with the versatility and throughput of cell culture. Mature human retinal organoids are utilized to screen potential pharmaceutical treatments for the age-related retinal degenerative disease macular telangiectasia type 2 (MacTel).
We have recently shown that MacTel can be caused by elevated levels of an atypical lipid species, deoxysphingolipids (deoxySLs). These lipids are toxic to the retina and may drive the photoreceptor loss that occurs in MacTel patients. To screen drugs for their ability to prevent deoxySL photoreceptor toxicity, we generated human retinal organoids from a non-MacTel induced pluripotent stem cell (iPSC) line and matured them to a post-mitotic age where they develop all of the neuronal lineage-derived cells of the retina, including functionally mature photoreceptors. The retinal organoids were treated with a deoxySL metabolite and apoptosis was measured within the photoreceptor layer using immunohistochemistry. Using this toxicity model, pharmacological compounds that prevent deoxySL-induced photoreceptor death were screened. Using a targeted candidate approach, we determined that fenofibrate, a drug commonly prescribed for the treatment of high cholesterol and triglycerides, can also prevent deoxySL toxicity in the cells of the retina.
The toxicity screen successfully identified an FDA-approved drug that can prevent photoreceptor death. This is a directly actionable finding owing to the highly disease-relevant model tested. This platform can be easily modified to test any number of metabolic stressors and potential pharmacological interventions for future treatment discovery in retinal diseases.

Here we present a step-by-step protocol to generate mature human retinal organoids and utilize them in a photoreceptor toxicity assay to identify pharmaceutical candidates for the age-related retinal degenerative disease macular telangiectasia type 2 (MacTel).

用于药物发现的人视网膜类器官毒性筛选

Organoids provide a promising platform to study disease mechanism and treatments, directly in the context of human tissue with the versatility and ...

生物工程

Characterization of a Novel Human Organotypic Retinal Culture Technique

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal pigment epithelium-choroid (RPE-choroid) and sclera, the integrity of the fragile retina can easily be compromised. The aim of this study was to develop a novel HORC model that contains the retina, RPE-choroid and sclera to maintain retinal integrity when culturing retinal explants.
After cutting circumferentially along the limbus to remove iris and lens, four deep incisions were made to flatten the eyecup. In contrast to previous HORC protocols, a trephine was used to cut through not only the retina but also the RPE-choroid and sclera. The resultant triple-layered explants were cultured for 72 h. Hematoxylin and Eosin staining (H&#38;E) was used to assess anatomical structures and retinal explants were further characterized by immunohistochemistry (IHC) for apoptosis, M&#252;ller cell integrity and retinal inflammation. To confirm the possibility of disease induction, explants were exposed to high glucose (HG) and pro-inflammatory cytokines (Cyt), to mimic diabetic retinopathy (DR). The Luminex magnetic bead assay was used to measure DR-related cytokines released into the culture medium.
H&#38;E staining revealed distinct retinal lamellae and compact nuclei in retinal explants with the underlying RPE-choroid and sclera, while retinas without the underlying structures exhibited reduced thickness and severe nuclei loss. IHC results indicated absence of apoptosis and retinal inflammation as well as preserved M&#252;ller cell integrity. The Luminex&#160;assays showed significantly increased secretion of DR-associated pro-inflammatory cytokines in retinal explants exposed to HG + Cyt relative to baseline levels at 24 h.
We successfully developed and characterized a novel HORC protocol in which retinal integrity was preserved without apoptosis or retinal inflammation. Moreover, the induced secretion of DR-associated pro-inflammatory biomarkers when exposing retinal explants to HG + Cyt suggests that this model could be used for clinically translatable retinal disease studies.

This study aims to develop a novel human organotypic retinal culture (HORC) model that prevents compromising retinal integrity during explant handling. This is achieved by culturing the retina with the overlying vitreous and the underlying retinal pigment&#160;epithelium-choroid (RPE-choroid) and sclera.

新型人类组织特征视网膜培养技术的特征

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal ...

医学

Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a multifactorial disease influenced by environmental and genetic factors, which lead to functional impairment of the retina due to retinal pigment epithelial (RPE) cell degeneration followed by photoreceptor degradation. An ideal treatment would include the transplantation of healthy RPE cells secreting neuroprotective factors to prevent RPE cell death and photoreceptor degeneration. Due to the functional and genetic similarities and the possibility of a less invasive biopsy, the transplantation of iris pigment epithelial (IPE) cells was proposed as a substitute for the degenerated RPE. Secretion of neuroprotective factors by a low number of subretinally-transplanted cells can be achieved by Sleeping Beauty (SB100X) transposon-mediated transfection with genes coding for the pigment epithelium-derived factor (PEDF) and/or the granulocyte macrophage-colony stimulating factor (GM-CSF). We established the isolation, culture, and SB100X-mediated transfection of RPE and IPE cells from various species including rodents, pigs, and cattle. Globes are explanted and the cornea and lens are removed to access the iris and the retina. Using a custom-made spatula, IPE cells are removed from the isolated iris. To harvest RPE cells, a trypsin incubation may be required, depending on the species. Then, using RPE-customized spatula, cells are suspended in medium. After seeding, cells are monitored twice per week and, after reaching confluence, transfected by electroporation. Gene integration, expression, protein secretion, and function were confirmed by qPCR, WB, ELISA, immunofluorescence, and functional assays. Depending on the species, 30,000-5 million (RPE) and 10,000-1.5 million (IPE) cells can be isolated per eye. Genetically modified cells show significant PEDF/GM-CSF overexpression with the capacity to reduce oxidative stress and offers a flexible system for ex vivo&#160;analyses and in vivo studies transferable to humans to develop ocular gene therapy approaches.

Here, a protocol to isolate and transfect primary iris and retinal pigment epithelial cells from various mammals (mice, rat, rabbit, pig, and bovine) is presented. The method is ideally suited to study ocular gene therapy approaches in various set-ups for ex vivo&#160;analyses and&#160;in vivo studies transferable to humans.

哺乳动物原色素上皮细胞的分离、培养和基因工程用于非病毒基因治疗

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a ...

生物学

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

原发性小鼠视网膜色素上皮细胞的有效解剖与培养

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...

<meta charset="utf8"></head><body>评估啮齿动物眼球的视网膜厚度和神经节细胞层计数

Preparation and Analysis of Histological Slides of Rat and Mouse Eyeballs to Evaluate the Retina

A rodent eyeball is a powerful tool for researching the pathomechanisms of many ophthalmic diseases, such as glaucoma, hypertensive retinopathy, and many more. Preclinical experiments enable researchers to examine the efficacy of novel drugs, develop new methods of treatment, or seek new pathomechanisms involved in the disease's onset or progression. A histological examination provides a lot of information necessary to assess the effects of the conducted experiments and can reveal degeneration, tissue remodeling, infiltration, and many other pathologies. In clinical research, there is rarely any chance of obtaining eye tissue suitable for a histological examination, which is why researchers should take advantage of the opportunity offered by the examination of eyeballs from rodents. This manuscript presents a protocol for the histological preparation of rodent eyeballs' sections. The procedure is presented for the eyeballs of mice and rats and has the following steps: (i) harvesting the eyeball, (ii) preserving the eyeball for further analysis, (iii) processing the tissue in paraffin, (iv) preparing slides, (v) staining with hematoxylin and eosin, (vi) assessing the tissue under a light microscope. With the proposed method, the retina can be easily visualized and assessed in detail.

<meta charset="utf8"></head><body>大鼠和小鼠眼球组织学载玻片的制备和分析以评估视网膜

This manuscript presents a protocol for the histological preparation of slides from rodent eyeballs. With the proposed method, the inner retina can be easily visualized and assessed under a light microscope. The procedure is presented for the eyeballs of mice and rats.

大鼠和小鼠眼球组织学载玻片的制备和分析以评估视网膜

A rodent eyeball is a powerful tool for researching the pathomechanisms of many ophthalmic diseases, such as glaucoma, hypertensive retinopathy, and many ...