此处包含的意见或主张是作者的个人观点，并不应被理解为官方或反映了陆军部和国防部的意见。 这项研究进行的同时，作者惠特尼A.格林，阿尔贝托穆尼斯和拉梅什R.凯妮召开全国研究理事会博士后研究员院士在USAISR。 微阵列分析是由Greehey儿童癌症研究所基因芯片核心设备和生物信息学系UT健康科学中心圣安东尼奥进行。 这项工作是由美国陆军临床康复医学研究发展计划（CRMRP）和军事作战医学研究发展计划（MOMRP）的支持。

培养试剂和培养板的1。准备<ol><li> mTeSR1媒体:根据制造商的指示准备mTeSR1媒体。解冻百毫升mTeSR1 5倍的补充一夜之间在4°C。添加百毫升mTeSR1 5倍的补充至400ml mTeSR1基础培养基中拌匀。此介质是稳定的，在4℃下进行长达2周，并在-20℃下6个月。 </li><li>分化培养基:准备分化培养基中的DMEM/F12，得到0.1mM的β-巯基乙醇，0.1mM非必需氨基酸，2.0 2mM L-谷氨酰胺，10％敲除血清，和10微克/毫升庆大霉素。 </li><li>的iPS-RPE媒体:准备的iPS-RPE媒体在MEM中，得到1X N1的补充，0.1mM非必需氨基酸，250μg/ ml的牛磺酸，13毫微克/毫升三碘-L-甲状腺原氨酸钠盐，20毫微克/毫升氢化可的松，5微克/毫升庆大霉素和15％FBS的26。 </li><li>三碘-L-甲状腺原氨酸钠盐原液。为了准备储备液溶解1.0毫克三碘-L-甲状腺素的在1N氢氧化钠轻轻旋转钠钠盐。接下来添加49.0毫升基础介质，使50.0毫升的三碘-L-甲状腺原氨酸钠盐的20微克/毫升。准备分装和直到需要冻结在-20°C。使用适当的音量，以达到理想的浓度在最终培养基。 </li><li>氢化可的松原液:要准备氢化可的松原液，溶解1毫克的氢化可的松1毫升的无水乙醇轻轻搅动。接下来添加19.0毫升基础介质的为50微克/毫升20毫升氢化可的松原液。分装，冷冻在-20°C，直到需要。使用适当量以达到所需浓度在最终培养基中。 </li><li>胎儿的视网膜色素上皮生长培养基。通过将所有的试剂在RTEGM试剂盒中的基础培养基制备生长培养基，按照制造商指示除外FBS。 </li><li>胎儿视网膜色素上皮电镀媒体。通过将少量的生长培养基，以无菌容器和一个准备电镀媒体西元FBS到2％的终浓度。 </li><li>分散酶:准备分散酶的1毫克/毫升的DMEM/F12中的工作液。 </li><li> Matrigel包被板:在DMEM/F12准备基质胶到0.08毫克/毫升。 </li><li>外套每孔一个6孔板用1.0mL的基质胶溶液中。 </li><li>孵育包被的板在室温下搅拌1.0小时。 </li><li>在1.0小时孵育后，吸去多余的DMEM/F12。 </li><li>加入0.5毫升mTeSR1媒体，以避免干燥。基质胶涂层板现已准备就绪。 </li></ol> 2，培养的iPS <ol><li>之前的iPS细胞播种都管，暖mTESR1媒体至室温，并在基质胶包被的平板准备。 </li><li>迅速解冻在37℃水浴中IMR90-4 iPS细胞。然后从水浴中取出冷冻管和消毒用70％的乙醇。 </li><li>用2毫升的移液管，以减少细胞聚集体破碎细胞转移到15毫升锥形管中。 </li><li>逐滴加入5 mL水中的RM mTeSR1媒体向细胞并轻轻混匀。离心细胞，在300×g离心5分钟，在室温下，去除上清。 </li><li>小心地重悬细胞聚集体在2毫升mTeSR1介质和种子细胞到6孔板的一个孔中。 </li><li>将板放入37℃的培养箱中培养，并用5％CO 2，95％湿度。 </li><li>每天都在变化的iPS细胞培养基。播种后5-7天未分化克隆将出现。检查未分化的殖民地，它们准备好通道（与密集中心殖民地）。 </li></ol> iPS细胞的3。传代<ol><li>之前开始到通道中的iPS细胞，都管，分散酶溶液，DMEM/F12和mTERS1介质温热至室温，并在基质胶包被的平板准备。 </li><li>之前传代iPS细胞，通过刮区和抽吸介质除去分化的地方。分化应保持低于井20％的高质量文化。仔细冲洗的iPS含细胞以及用2ml的DMEM/F12的并加入1 ml的1毫克/毫升分散酶到每个孔中。 </li><li>在37℃下7分钟。吸出分散酶，轻轻冲洗细胞集落用2毫升的DMEM/F12的两倍。 </li><li>冲洗后，加入2ml mTSER1的每个孔中。 </li><li>用5毫升的移液管轻轻刮板的集落形成的聚集体。 </li><li>转移分离的菌落到15毫升锥形管中。加足够mTeSR1媒体的种子细胞的下一通道。 </li></ol> 4，分化的iPS细胞来的iPS RPE <ol><li>板块上，以在mTESR1媒体新涂层板的iPS细胞。 </li><li>允许iPS细胞集落，扩大在文化4-5天。 </li><li>替代mTeSR1媒体用4ml分化培养基。改变一半的媒体，每3天。 </li><li>让色素病灶增长足够大，可以手动解剖出来的文化（40-50天）。 </li><li>使用200μL枪头切割四周，抬起色素的菌落。 </li><li>收集和集中的解剖殖民地在一个15毫升的锥形管。 </li><li>然后加入5 ml DMEM/F12到汇集RPE细胞，离心300×g离心5分钟两次。 </li><li>通过孵育汇集的iPS-RPE用0.25％胰蛋白酶EDTA制备单细胞溶液。 </li><li>冲洗的iPS-RPE细胞与IPS RPE媒体和板将收集的细胞在新鲜的基质胶涂覆在4ml的iPS-RPE媒体良好。 </li><li>培养细胞3周前通过。更改介质每3天。在实验中，板的iPS-RPE在100,000个细胞/ cm 2。收集细胞于17日，播种当天计数为0天。 </li></ol> 5，样品采集<ol><li> iPS细胞采集<ol><li>培养的iPS细胞的未分化直至菌落观察到致密的中心。 </li><li>在冲洗DMEM/F12殖民地。 </li><li>使用accutas解离成单个细胞<html> Ë。收集细胞入15ml锥形管中并离心分离，在300×g离心5分钟。吸上清。重悬在2毫升的DMEM/F12。 </li></ol></li><li>胎儿视网膜色素上皮收藏<ol><li>冲洗胎儿视网膜色素上皮细胞培养用DMEM/F12并用0.25％胰蛋白酶EDTA解离成单细胞。 </li><li>收集细胞在15毫升锥形管。离心机在300×g离心5分钟。吸上清。重悬在2毫升的DMEM/F12。 </li></ol></li><li>的iPS-RPE细胞采集<ol><li>当上IPS-RPE达到第3代和第5代，收集通过后第17天的细胞。冲洗的iPS-RPE两次在PBS中，通过温育与1.0ml 0.25％胰蛋白酶制备单细胞悬浮液。 </li><li>胰蛋白酶中和用2毫升冰冷的iPS-RPE媒体。 </li><li>收集在15毫升锥形管中，离心分离机，在300×g离心5分钟，将细胞。吸上清。 </li><li>重悬细胞悬浮于3ml的染色缓冲液从微珠试剂盒。 </li></ol></li></ol>中的iPS-RPE ve_title"&gt; 6。阴性选择删除未分化的细胞<ol><li>保留所有试剂在4°C，直到使用，但不要在冰工作。从步骤5.3.4在300×g离心5分钟，离心分离细胞悬浮液。吸上清。 </li><li>将细胞再悬浮于由每2×10 6细胞微珠试剂盒100μl的染色缓冲液中。 </li><li>加10微升每2×10 6细胞抗TRA-1-60-PE抗体。 </li><li>并充分混匀，4℃10分钟。 </li><li>在1-2毫升缓冲液中每2×10 6细胞洗涤细胞。离心10分钟，在300×g下。吸上清。 </li><li>将细胞再悬浮于80微升的染色缓冲液中每2×10 6个细胞。每2×10 6个细胞加入20微升抗-PE标记的微珠（由试剂盒）。拌匀。孵育15分钟，在4℃下</li><li>加入1-2毫升染色缓冲液每2×10 6个细胞。离心10分钟，在300×g下。吸上清。 </li><li>重悬细胞在500＆＃956;升的染色缓冲液中。 </li><li>磁性细胞分离: <ol><li>将列在磁性细胞分选仪。冲洗柱用3毫升的染色缓冲液中。 </li><li> 6.8应用细胞悬液到列。 </li><li>放置一个管子在负端口收集TRA-1-60阴性细胞。 </li></ol></li></ol> 7。总RNA提取<ol><li>裂解细胞，通过使用市售的离心均化微型旋转柱设计用于核酸小量制备在运行样品。 </li><li>从iPS细胞的iPS-RPE和胎儿RPE与市售的RNA提取试剂盒设计为保留小分子RNA 27提取总RNA。 </li><li>测定总RNA浓度使用NanoDrop分光光度计。 </li></ol> 8，生物分析仪和芯片分析<ol><li>检查RNA的完整性与生物分析仪与市售RNA质量套件配合使用。 </li><li>孵育100纳克总RNA用小牛肠磷酸酶在37℃，30分钟</li><li>用100％DMSO中，在100℃下进行5分钟变性的RNA。 </li><li>标签使用PCP-Cy3的T4连接酶孵育在16℃，1小时的样品。 </li><li>杂交上的8_x_15K格式人类miRNA芯片。 </li><li>根据制造商的说明冲洗阵列和在5μm的使用扫描仪的分辨率进行扫描。 </li><li>使用微阵列特征提取软件与miRNA芯片兼容采集数据。 </li><li>处理所述miRNA的信号，以确定每个样品中表达的相对水平。 </li><li>比较样品组之间的每个miRNA的表达水平。选择的miRNA用于进一步的分析至少2倍差异表达。 </li><li>用市售软件对数据进行分析，按照指示生成的差异表达的miRNA 28可视化热图。 </li></ol> 9。途径分析<ol><li>在Excel格式8.9选择的miRNA的保存数据。 </li><li>登录到在线的RNA途径分析程序。上传Excel文件。选择"灵活的格式"的文件格式，并从标识符类型下拉列表中选择"安捷伦"。 </li><li>根据原始数据的标题，指定"ID"探测ID列，并指定"观察1"和"对数比"到日志口粮列。选择"忽略"对于所有其他列。 </li><li>选择如下的参数进行分析:信心:只有实验观察到;包括直接和间接的关系;并包括内源性化学物质;选择别出心裁的知识库作为参照组。选择参照分子的数据库，其中包括来自所有物种的数据，所有的细胞系和组织，以及所有的突变。 </li><li>运行网络和通路分析。 </li></ol>

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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=MicroRNA+profiling+of+human-induced+pluripotent+stem+cells.">MicroRNA profiling of human-induced pluripotent stem cells.</a> Stem Cells Dev. 18, 749-758 (2009).</li><li>Neveu, P., 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=MicroRNA+profiling+reveals+two+distinct+p53-related+human+pluripotent+stem+cell+states.">MicroRNA profiling reveals two distinct p53-related human pluripotent stem cell states.</a> Cell Stem Cell. 7, 671-681 (2010).</li><li>Maminishkis, 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=Confluent+monolayers+of+cultured+human+fetal+retinal+pigment+epithelium+exhibit+morphology+and+physiology+of+native+tissue.">Confluent monolayers of cultured human fetal retinal pigment epithelium exhibit morphology and physiology of native tissue.</a> Invest Ophthalmol Vis Sci. 47, 3612-3624 (2006).</li><li>Qiagen, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miRNeasy+Mini+Handbook.">miRNeasy Mini Handbook.</a> Sample and Assay Technologies. , (2013).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+System+with+miRNA+Complete+Labeling+and+Hyb+Kit.">Microarray System with miRNA Complete Labeling and Hyb Kit.</a> Agilent Technologies. , (2011).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+hESCs+and+hiPSCs+in+mTESR1+and+mTESR2.">Maintenance of hESCs and hiPSCs in mTESR1 and mTESR2.</a> Stem Cell Technologies. , (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Analysis+of+DNA+or+RNA+using+its+wavelengths:+230+nm,+260+nm,+280+nm.">The Analysis of DNA or RNA using its wavelengths: 230 nm, 260 nm, 280 nm.</a> About Biotechnology. , (2013).</li></ol>

作者什么都没有透露。

总之，这份报告描述了用于培养iPS细胞的方法，能干-RPE和胎儿的RPE。从视网膜色素上皮衍生的iPS在形态和功能上类似于胎儿视网膜色素上皮。上IPS-RPE也表达特性的RPE基因，包括RPE65，CRALBP，PEDF和LRAT 16。为了进一步表征这些细胞，提取RNA，并用于进行微阵列分析，以确定差异表达的miRNA。的信号强度的分析表明，在胎儿的RPE检测与的iPS-RPE比较的差异表达的miRNA的最大数目，暗示的iPS-RPE可?...

干细胞具有无限制和分化成任何体细胞类型的潜力来复制的能力。的技术，体细胞重编程为多能干细胞的发展，就引起了极大的热情在研究界和临床医生之间，作为个性化的组织再生的出现在地平线上1。诱导多能干细胞（iPS细胞）表现出无限的复制能力和多能性的相同的功能，胚胎干细胞（ES细胞），同时规避与胚胎干细胞相关的伦理困境。此外，病人的干细胞不会刺激免疫反应，大大增加了成功的治疗性应用2-3的概率。根据特定的培养条件下，iPS细胞已经显示出分化成体外几种不同的细胞类型，包括心肌细胞，神经细胞，胰腺β细胞，肝细胞和视网膜色素上皮（RPE）4-12。 在RPE是一个专门的层设在视网膜的后面，执行多种功能，这对于视力健康和功能，如杂散光，吞噬作用的光感受器外段的吸收必需的色素上皮细胞，和类视黄酸处理的生产视觉生色。视网膜色素上皮因损伤或疾病功能障碍深刻地影响感光健康和视觉功能就证明了致盲的疾病，是潜在的视网膜色素上皮病变，如年龄相关性黄斑变性（AMD），Stargardt病和色素性视网膜炎（RP）的结果13。可以恢复视力确实有效的治疗方法尚未实现，并更换视网膜色素上皮病变与正常RPE细胞可能是为了防止视力14-15损失的最佳选择。 RPE由iPS（IPS-RPE）是衍生的细胞的一个可能的来源，以取代受损视网膜色素上皮。的iPS-RPE表达characteris抽动RPE蛋白质LRAT，CRALBP，PEDF和RPE65;显示经典的高色素六角形的RPE形态;并执行RPE功能，如吞噬作用，视黄酸类化合物的处理，和11的分泌- 顺视网膜5,16。然而，之前的iPS-RPE可用于治疗中，IPS-RPE必须彻底特征。理解支配RPE分化的因素是必要的，以改善细胞用于临床应用的产率和纯度。 细胞分化是高度调节基因表达的结果。转录因子的基因组和协调的后生重构所需的分化和发育17期间发生的细胞命运的决定。信息核糖核酸（mRNA）的翻译者的microRNA（miRNA）的调控提出了调控的另一个层面，影响细胞命运1。 miRNA是短，约为22个核苷酸，核苷酸的长度，要么抑制翻译的结合到mRNA的3&#39;非编码区或靶mRNA的降解。的miRNA已经在几乎所有组织中被发现，而且迄今超过2,000人独特的microRNA已经注册在数据库中的miRBase。由于miRNA的要求只有部分互补结合到靶mRNA，单个的miRNA有可能绑定到几十或上百个目标，反之亦然， 即单个基因可以由几个不同的miRNA有针对性。这种混杂的结合特性大大提高了监管的复杂性，在细胞功能18-21各玩各的水平以及确定单个miRNA的功能的难易程度和作用。然而，研究表明，miRNA的通过影响DNA甲基化状态17细化分化过程中的基因表达。在一项研究更具体的RPE，miR-204/211被证明能促进视网膜色素上皮22的上皮细胞表型。另一组分析了miRNA的个人资料视网膜色素上皮的过程中由ES细胞分化，并透露组不同的miRNA在分化过程中23表示。事实上，miRNA表达谱可以明确区分的细胞类型，包括胚胎干细胞，前体细胞，和终末分化细胞24,25。超过250个miRNA的表达在视网膜上。基于这些研究，我们推测，RPE细胞或者由iPS分化过程中的miRNA中发挥重要作用。 本报告的目的是从IMR90-4的iPS描述为RPE的分化的协议细胞，收集的RNA的微阵列分析和微阵列数据的分析，以确定差异表达三种类型的细胞的iPS细胞的miRNA， ，的iPS-RPE细胞和胎儿的RPE。总RNA从每种细胞类型的培养物中提取和杂交的miRNA的微阵列，包含特异性针对1205人的miRNA和144病毒的miRNA探针。基因芯片结果进行比较d，来确定哪些miRNA的差异表达在不同的细胞类型。 miRNA的2倍或更大的表达倍数变化被选择用于进一步分析。甲miRNA的分析软件程序来鉴定差异表达的miRNA的潜在目标，并生成由所选择的miRNA调节的蜂窝网络。

<table border="0" cellpadding="0" cellspacing="0" width="676">
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			<td>11330-032</td>
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			<td height="18" style="height:18px;width:235px;">Non essential amino acids</td>
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			<td height="21" style="height:21px;width:235px;">Human miRNA microarray v16</td>
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			<td style="width:119px;">G4471A</td>
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		</tr>
	</tbody>
</table>

microrna expression profiles of human ips cells, retinal pigment epithelium derived from ips, and fetal retinal pigment epithelium

本报告的目的是描述的协议，用于比较的microRNA（miRNA）的人类诱导多能干细胞（iPS）细胞，视网膜色素上皮细胞（RPE）从人类iPS细胞（iPS-RPE）导出，胎儿视网膜色素上皮的配置文件。该协议包括收集的RNA通过微阵列分析，以及微阵列数据的分析，找出差异表达三种细胞类型的miRNA。该方法iPS细胞和胎儿视网膜色素上皮的文化解释。用于从人iPS分化的RPE的协议也被描述。我们描述了RNA提取技术被选中，允许非常小RNA在一个miRNA芯片的使用极大的恢复。最后，细胞通路和微阵列数据的网络分析解释。这些技术将促进三种不同类型的细胞的miRNA表达谱进行比较。

iPS细胞（ 图1A）培养在分化条 ​​件以诱导分化成的iPS-RPE细胞。上IPS-RPE展出的六角形色素细胞形态（ 图1B）相似，胎儿视网膜色素上皮（ 图1C）经典的视网膜色素上皮表型。 为了更好地理解了miRNA可能分化过程中发挥由iPS来的iPS-RPE的作用，进行miRNA表达的微阵列分析。总RNA从iPS细胞，胎儿视网膜色素上皮，和iPS-RPE使用mirVana miRNA分离试...

Watch this Scientific Journal Video about MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium at JoVE.com

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

微小RNA（miRNA）的由人诱导多能干细胞（iPS）细胞（iPS-RPE）和胎儿视网膜色素上皮衍生的人诱导多能干细胞的访问（iPS细胞），视网膜色素上皮细胞（RPE），进行比较。

人类iPS细胞的微小RNA表达谱，视网膜色素上皮衍生自IPS和胎儿视网膜色素上皮

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

microrna-expression-profiles-human-ips-cells-retinal-pigment

Research

JoVE Journal

Biology

11.3K 次观看.  JBSA Fort Sam Houston. 微小RNA（miRNA）的由人诱导多能干细胞（iPS）细胞（iPS-RPE）和胎儿视网膜色素上皮衍生的人诱导多能干细胞的访问（iPS细胞），视网膜色素上皮细胞（RPE），进行比较。 

视频: MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006; Wernig, et al., 2007; Okita, et al., 2007; Maherali, et al., 2007). We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors (Brambrink et al., 2008). Using these inducible constructs, we can derive induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs). In this video, we demonstrate the procedure for the generation of inducible lentiviruses that express the four transcription factors and show how to infect MEFs with these viruses in order to produce iPS cells. By using inducible lentiviruses, the expression of the four factors in controlled by the addition of doxycyline to the culture medium. The advantage of this system over the traditional retroviral infection is the ability to turn the genes on and off so that the kinetics of reprogramming and gene expression requirements can be analyzed in detail.

This video shows the procedure for generating induced pluripotent stem cells using inducible lentivirus that express Oct4, Sox2, c-Myc and Klf4.

从MEFS iPS细胞生成通过强制SOX - 2的表达，10月4，c - Myc基因和Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006;  Wernig, et al., ...

科研

生物学

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

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

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

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

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, ...

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the eye. It is a single sheet of pigmented cells that are hexagonally packed and connected by tight junctions. The main functions of the RPE include absorption of light, phagocytosis of the shed photoreceptor outer segments, spatial buffering of ions, transport of nutrients, ions and water as well as active involvement in the visual cycle. With such important and diverse functions, it is critically important to study the biology of RPE cells. A number of RPE cell lines have been established; however, passaged and immortalized cells are known to quickly lose some of the morphological and physiological characteristics of natural RPE cells. Thus, primary cells are more suitable for studying different aspects of RPE cell biology and function. Mouse primary RPE cell culture is very useful to researchers since mouse models are widely used in biological studies, however collecting RPE cells from mouse is also very challenging due to their small size. Here, we present a protocol for establishing primary mouse RPE cell cultures which includes enucleation and dissection of the eyes and isolation of the RPE sheets to yield the cells for culturing. This method enables efficient cell recovery. The RPE cells obtained from two mice&#160;can reach confluency on one 12 mm polyester membrane insert pre-loaded in culture plate after one week of culture and display some of the original properties of bona fide RPE cells such as hexagonal shape and pigmentation after two weeks of culture.

The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE.

小鼠视网膜色素上皮细胞的原代培养

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the ...

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 ...

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform (RpEGEN) for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent retinal and vascular tissues. At present, the limited reparative capacity of mammalian RPE, which is restricted to small injuries, has hindered progress to understanding in vivo RPE regenerative processes. Here, a detailed methodology is provided to facilitate the study of in vivo RPE repair utilizing the zebrafish, a&#160;vertebrate model capable of robust tissue regeneration. This protocol describes a transgenic nitroreductase/metronidazole (NTR/MTZ)-mediated injury paradigm (rpe65a:nfsB-eGFP), which results in ablation of the central two-thirds of the RPE after 24 h treatment with MTZ, with subsequent tissue recovery. Focus is placed on RPE ablations in larval zebrafish and methods for testing the effects of pharmacological compounds on RPE regeneration are also outlined. Generation and validation of RpEGEN, a MATLAB script created to automate quantification of RPE regeneration based on pigmentation, is also discussed. Beyond active RPE repair mechanisms, this protocol can be expanded to studies of RPE degeneration and injury responses as well as the effects of RPE damage on adjacent retinal and vascular tissues, among other cellular and molecular processes. This zebrafish system holds significant promise in identifying genes, networks, and processes that drive RPE regeneration and RPE disease-related mechanisms, with the long-term goal of applying this knowledge to mammalian systems and, ultimately, toward therapeutic development.

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform RpEGEN for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

This protocol describes the methodology to genetically ablate the retinal pigment epithelium (RPE) using a transgenic zebrafish model. Adapting the protocol to incorporate signaling pathway modulation using pharmacological compounds is extensively detailed. A MATLAB platform for quantifying RPE regeneration based on pigmentation was developed and is presented and discussed.

硝基还原酶/甲硝唑介导的消融和MATLAB平台（RpEGEN）研究斑马鱼视网膜色素上皮的再生

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent ...

Primary Culture of Porcine Retinal Pigment Epithelial Cells

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. Multiple functions, including phagocytosis, nutrient/metabolite transportation, vitamin A metabolism, etc., are conducted by the RPE on a daily basis. RPE cells are terminally differentiated epithelial cells with little or no regenerative capacity. Loss of RPE cells results in multiple eye diseases leading to visual impairment, such as age-related macular degeneration. Therefore, the establishment of an in vitro culture model of primary RPE cells, which more closely resembles the RPE in vivo than cell lines, is critical for the characteristic and mechanistic studies of RPE cells. Considering the fact that the source of human eyeballs is limited, we create a protocol to culture primary porcine RPE cells. By using this protocol, RPE cells can be easily dissociated from adult porcine eyeballs. Subsequently, these dissociated cells attach to culture dishes/inserts, proliferate to form a confluent monolayer, and quickly re-establish key features of epithelial tissue in vivo within 2 wks. By qRT-PCR, it is demonstrated that primary porcine RPE cells express multiple signature genes at comparable levels with native RPE tissue, while the expressions of most of these genes are lost/highly reduced in human RPE-like cells, ARPE-19. Moreover, the immunofluorescence staining shows the distribution of tight junction, tissue polarity, and cytoskeleton proteins, as well as the presence of RPE65, an isomerase critical for vitamin A metabolism, in cultured primary cells. Altogether, we have developed an easy-to-follow approach to culture primary porcine RPE cells with high purity and native RPE features, which could serve as a good model to understand RPE physiology, study cell toxicities, and facilitate drug screenings.

Here, an easy-to-follow method to culture primary porcine retinal pigment epithelial cells in vitro is presented.

猪视网膜色素上皮细胞原代培养

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. ...

Isolation of Primary Mouse Retinal Pigmented Epithelium Cells

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical roles in maintaining the photoreceptors' function. Thus, the RPE structure and function are essential to sustain normal vision. This manuscript presents an established protocol for primary mouse RPE cell isolation. RPE isolation is a great tool to investigate the molecular mechanisms underlying RPE pathology in the different mouse models of ocular disorders. Furthermore, RPE isolation can help in comparing primary mouse RPE cells isolated from wild-type and genetically modified mice, as well as testing drugs that can accelerate the development of therapy for visual disorders. The manuscript presents a step-by-step RPE isolation protocol; the entire procedure, from enucleation to seeding, takes approximately 4 hours. The media shouldn't be changed for 5-7 days after seeding, to allow the growth of the isolated cells without disturbance. This process is followed by the characterization of morphology, pigmentation, and specific markers in the cells via immunofluorescence. Cells can be passaged a maximum of three or four times.

This manuscript describes a simplified protocol for the isolation of retinal pigmented epithelium (RPE) cells from mouse eyes in a stepwise manner. The protocol includes the enucleation and dissection of mouse eyes, followed by the isolation, seeding, and culturing of RPE cells.

小鼠原代视网膜色素上皮细胞的分离

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical ...

Efficient and Consistent Generation of Retinal Pigment Epithelium/Choroid Flatmounts from Human Eyes for Histological Analysis

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and vision. Proteins on the RPE apical surface are tightly associated with proteins on the photoreceptor outer segment surface, making it difficult to consistently separate the RPE from the photoreceptors/retina. We developed a method to efficiently separate the retina from the RPE of human eyes to generate complete RPE/choroid and retina flatmounts for separate cellular analysis of the photoreceptors and RPE cells. An intravitreal injection of a high-osmolarity solution of D-mannitol, a sugar not transported by the RPE, induced the separation of the RPE and retina across the entire posterior chamber without causing damage to the RPE cell junctions. No RPE patches were observed attached to the retina. Phalloidin labeling of actin showed RPE shape preservation and allowed morphometric analysis of the entire epithelium. An artificial intelligence (AI)-based software was developed to accurately recognize and segment the RPE cell borders and quantify 30 different shape metrics. This dissection method is highly reproducible and can be easily extended to other animal models.

We describe a method to efficiently separate retinal pigment epithelium (RPE) from the retina in human eyes and generate whole RPE/choroid flatmounts for histological and morphometric analyses of the RPE.

从人眼高效、一致地生成视网膜色素上皮/脉络膜平面安装，用于组织学分析

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and ...

Real-Time Analysis of Bioenergetics in Primary Human Retinal Pigment Epithelial Cells Using High-Resolution Respirometry

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). Since RPE are highly metabolically-active cells, alterations in their metabolic status reflect changes in their health and function. High-resolution respirometry allows for real-time kinetic analysis of the two major bioenergetic pathways, glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), through quantification of the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), respectively. The following is an optimized protocol for conducting high-resolution respirometry on primary human retinal pigment epithelial cells (H-RPE). This protocol provides a detailed description of the steps involved in producing bioenergetic profiles of RPE to define their basal and maximal OXPHOS and glycolytic capacities. Exposing H-RPE to different drug injections targeting the mitochondrial and glycolytic machinery results in defined bioenergetic profiles, from which key metabolic parameters can be calculated. This protocol highlights the enhanced response of BAM15 as an uncoupling agent compared to carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) to induce the maximal respiration capacity in RPE. This protocol can be utilized to study the bioenergetic status of RPE under different disease conditions and test the efficacy of novel drugs in restoring the basal metabolic status of RPE.

The metabolic status of human retinal pigment epithelial cells (H-RPE) reflects their health and function. Presented here is an optimized protocol for examining the real-time metabolic flux of H-RPE using high-resolution respirometry.

使用高分辨率呼吸测定法实时分析原代人视网膜色素上皮细胞中的生物能量学

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration ...

量化视网膜色素上皮吞噬作用和脂褐素积累

Improved Lipofuscin Models and Quantification of Outer Segment Phagocytosis Capacity in Highly Polarized Human Retinal Pigment Epithelial Cultures

The daily phagocytosis of photoreceptor outer segments by the retinal pigment epithelium (RPE) contributes to the accumulation of an intracellular aging pigment termed lipofuscin. The toxicity of lipofuscin is well established in Stargardt's disease, the most common inherited retinal degeneration, but is more controversial in age-related macular degeneration (AMD), the leading cause of irreversible blindness in the developed world. Determining lipofuscin toxicity in humans has been difficult, and animal models of Stargardt's have limited toxicity. Thus, in vitro models that mimic human RPE in vivo are needed to better understand lipofuscin generation, clearance, and toxicity. The majority of cell culture lipofuscin models to date have been in cell lines or have involved feeding RPE a single component of the complex lipofuscin mixture rather than fragments/tips of the entire photoreceptor outer segment, which generates a more complete and physiologic lipofuscin model. Described here is a method to induce the accumulation of lipofuscin-like material (termed undigestible autofluorescence material, or UAM) in highly differentiated primary human pre-natal RPE (hfRPE) and induced pluripotent stem cell (iPSC) derived RPE. UAM accumulated in cultures by repeated feedings of ultraviolet light-treated OS fragments taken up by the RPE via phagocytosis. The key ways that UAM approximates and differs from lipofuscin in vivo are also discussed. Accompanying this model of lipofuscin-like accumulation, imaging methods to distinguish the broad autofluorescence spectrum of UAM granules from concurrent antibody staining are introduced. Finally, to assess the impact of UAM on RPE phagocytosis capacity, a new method for quantifying outer segment fragment/tips uptake and breakdown has been introduced. Termed "Total Consumptive Capacity", this method overcomes potential misinterpretations of RPE phagocytosis capacity inherent in classic outer segment "pulse-chase" assays. The models and techniques introduced here can be used to study lipofuscin generation and clearance pathways and putative toxicity.

作者聚焦：改进的脂褐素模型和高度极化人类视网膜色素上皮培养物中外段吞噬作用能力的定量  

This protocol describes a lipofuscin accumulation model in highly differentiated and polarized human retinal pigment epithelial (RPE) cultures and an improved outer segment (OS) phagocytosis assay to detect the total OS consumption/degradation capacity of the RPE. These methods overcome the limitations of previous lipofuscin models and classical pulse-chase outer segment phagocytosis assays.

改进脂褐素模型和高极化人视网膜色素上皮培养物中外段吞噬能力的定量

The daily phagocytosis of photoreceptor outer segments by the retinal pigment epithelium (RPE) contributes to the accumulation of an intracellular aging ...