Name: deriving retinal pigment epithelium (rpe) from induced pluripotent stem (ips) cells by different sizes of embryoid bodies
Uploaded: 2015-02-04T14:00:00.000+00:00
Duration: 9 min 18 s
Description: Watch this Scientific Journal Video about Deriving Retinal Pigment Epithelium RPE from Induced Pluripotent Stem iPS Cells by Different Sizes of Embryoid Bodies at JoVE.com

The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense. This research was performed while the authors Alberto Mu&#241;iz, Ramesh R Kaini, Whitney A Greene and Jae-Hyek Choi held a National Research Council Postdoctoral Research Associateship at the USAISR. This work was supported by U.S. Army Clinical Rehabilitative Medicine Research Program (CRMRP) and Military Operational Medicine Research Program (MOMRP).

1.准备培养试剂和培养皿<ol><li>通过加入100ml 5×无血清补充到400毫升干细胞基础培养基的制备无饲养干细胞培养基。该介质是稳定的，在4℃下进行最多2周，并在-20℃下6个月。 </li><li>加的Rho相关，卷曲螺旋包含蛋白激酶（岩）抑制剂（Y-27362），以市售胚状体（EB）形成介质10μM的溶液。 </li><li>通过加入0.1mMβ巯基乙醇，0.1mM非必需氨基酸，2mM的L-谷氨酰胺，10％敲除血清替代品（KSR）和10微克/毫升庆大霉素到的Dulbecco改良Eagle培养基/营养混合物F-12制备的分化培养基（DMEM / F12）。 </li><li>通过添加1×N 1的补充，0.1mM非必需氨基酸，250微克/毫升牛磺酸，13纳克/毫升的三碘左旋甲腺氨酸钠盐，20纳克/ ml氢化可的松，5微克/毫升庆大霉素和15％制备的iPS-RPE介质胎牛血清（FBS）的&lt;SUP&gt; 21到最低基本培养基Eagle（MEM）。将pH调节至7.4。 </li><li>制备碘左旋甲腺氨酸钠盐原液。溶解1毫克碘左旋甲腺氨酸钠盐在1N氢氧化钠通过轻轻摇动。添加49毫升的MEM使50毫升20微克/三碘左旋甲腺氨酸钠盐的溶液中。制备等分试样和，直到需要冷冻在-20℃。使用适当的音量，以达到最终的培养液所需浓度。 </li><li>制备氢化可的松原液。溶解1毫克的氢化可的松在1ml的100％乙醇通过温和搅拌。添加19毫升MEM，使20ml中为50μg/ ml氢化可的松原液。等分试样并冷冻在-20℃直到需要。使用适当的体积，以达到在最终培养基中的所需浓度。 </li><li>制备为1mg / ml，在DMEM / F12分散酶的工作溶液。 </li><li>矩阵涂层板的制备<ol><li>准备从Engelbreth - 霍尔姆 - 群（EHS）提取蛋白基质亩本身肉瘤细胞在DMEM / F12中，以0.08毫克/毫升。外套一个6孔板用1ml的基质溶液中的各孔中。孵育包被的板在RT 1小时。 </li><li>在1小时温育后，吸出过量的DMEM / F12。添加0.5毫升无饲养干细胞培养基，以防止井的干燥。基质包被的板都可以使用。 </li></ol></li><li>制备微孔板，通过用2ml的DMEM / F12的井漂洗每个。除去的DMEM / F12，并添加0.5毫升补充有ROCK抑制剂向每个孔EB形成培养基中。离心板在2000×g离心5分钟以除去任何气泡。 </li><li>制备FACS染色缓冲液通过混合2％FBS和0.09％叠氮化钠的磷酸盐缓冲盐水（PBS）。调节缓冲至pH 7.4。 </li><li>通过加入10％胎牛血清，以DMEM / F12制备的胰蛋白酶中和溶液。 </li></ol> 2.培养的iPS <ol><li>之前的iPS细胞接种，温无饲养干细胞培养基至37℃，并有马TRIX涂层板准备就绪。 </li><li>迅速解冻在37℃的水浴的IMR90-1 iPS细胞。然后取出冷冻瓶从水浴擦拭用70％乙醇的小瓶。使用2毫升吸管，以减少细胞团的断裂转移细胞到15ml锥形管中。 </li><li>加入5毫升温无饲养干细胞培养基的细胞滴，并轻轻混合。离心细胞，在300×g离心5分钟，在室温，去除上清。 </li><li>小心重悬细胞团块在2ml无饲养干细胞培养基和种子的细胞团块在基质被覆的板的孔中。将板放入37℃培养箱中，用5％的CO 2，95％的湿度。 </li><li>每天都在变化的iPS细胞培养液中。接种后观察未分化集落五至七天。检查未分化集落的，可用于通过在光学显微镜下（具有致密中心集落）。 </li></ol> 3.传代的iPS细胞的<ol><li>之前开始到通道中的iPS细胞，温分散酶溶液，DMEM / F12和无饲养干细胞培养基到37℃水浴中。 </li><li>前传代iPS细胞，通过刮擦面积和吸出培养基除去分化的任何区域。仔细冲洗iPS细胞用2ml的DMEM / F12。 </li><li>加入1毫升1毫克/毫升分散酶至每个孔，并孵育在37℃下7分钟。吸出分散酶，轻轻冲洗细胞菌落用2ml的DMEM / F12两次。漂洗后，加2ml无饲养干细胞培养基到每个孔中。 </li><li>使用5毫升吸管轻轻刮板的菌落，以形成细胞团块。转移拆卸的细胞团块到15ml锥形管中。添加足够无饲养干细胞培养基的种子细胞的下一个通道。 </li></ol> 4代的EB利用微孔板<ol><li> iPS细胞的单细胞悬浮液的制备<ol><li>取出mediu米的iPS细胞和冲洗iPS细胞用2ml的DMEM / F12的。添加750微升的Accutase到iPS细胞在6孔板的每个孔中。孵育所述细胞在37℃和5％的CO 2，以使细胞从板（约5-10分钟）分离。 </li><li>用移液管轻轻地分离任何剩余的细胞并分离任何剩余的细胞中的板表面上。转移细胞到50ml锥形管中。冲洗该板用5ml的DMEM / F12的并与细胞中的锥形管结合的冲洗的DMEM / F12。通过40微米的细胞滤网通过细胞悬浮液，以除去任何可能残留的细胞团块。 </li><li>离心细胞，在300×g离心5分钟，在室温以除去的Accutase。重悬在EB形成培养基中的细胞沉淀，以使细胞浓度为约0.5-1.0×10 7个细胞/ ml。 </li><li>通过计数细胞与锥虫蓝和血细胞计数器确定存活细胞的数量。 </li></ol></li><li> Adjustin在微孔G细胞的密度，形成规模控制的胚<ol><li>调节细胞数至微孔板的各孔中以产生期望的EB尺寸。添加细胞在步骤1.9中制备的板的微孔，均匀数倍轻轻吹打细胞分发细胞。 注:每口井所需的细胞数=所需的每很好微孔EB x个细胞的数量。 </li><li>调整补充有ROCK抑制剂在孔至2.0 ml的终体积在EB形成培养基中。轻轻每孔吹打重新分配细胞。离心微孔板在100×g离心3分钟。放置在培养箱板在37℃，5％的CO 2和95％湿度下24小时。 </li></ol></li><li>从微孔板收获胚<ol><li>收获的EB从微孔通过在井上下用1毫升移液器以除去大部分的EB的吹打介质。通过倒置40微米的细胞通过EB停牌过滤器上的50毫升锥形管的顶部以去除单细胞。 </li><li> 1毫升DMEM / F12的洗微孔板5次，除去所有的胚。收集清洗和越过倒细胞过滤。旋转细胞过滤右侧到一个新的50ml锥形管中。通过EB形成培养基清洗收集胚。算的EB，以确定产率。 </li></ol></li></ol> 5.电镀胚和启动分化<ol><li>板对蛋白基质涂覆6孔板中的EB（如在步骤1.8.1），在&lt;1000的EB /密度以及在EB形成培养基中加10μM的岩抑制剂。孵育所述胚在37℃，5％的CO 2和95％湿度下24小时。 </li><li> 24小时EB电镀后，更换分化培养基开始分化。改变一半分化培养基每隔一天，直至细胞被收集用于进一步分析。 </li><li>收集在第6天，17，29和60个样品进行的RT-PCR，IMMunocytochemistry和FACS来验证特性RPE基因和蛋白质的表达。 </li></ol> 6. RNA提取和PCR <ol><li>根据在市售的试剂盒提供的指令从电子束的样品中提取的RNA。通过使用分光光度计测定RNA浓度。 </li><li>根据市售的RNA到cDNA的反转录试剂盒100纳克的总RNA进行反转录。 </li><li>以10微摩尔/ 10纳克的cDNA的浓度进行PCR用的cDNA的10毫微克使用以下引物（ 表1）。设置的PCR如下:DNA变性在95℃下进行5分钟，用35个循环扩增在95℃下15秒，60℃，30秒，72℃1分钟，接着是最终循环的72°下进行10分钟。运行该PCR产物在1％琼脂糖凝胶。 </li></ol> 7.免疫细胞化学<ol><li>与PBS在RT洗胚两次。固定在胚胎发育RT在4％paraformaldehyde 10分钟。冲洗一次，用PBS于室温。直到用于染色在PBS中在4℃下存储区样品。 </li><li>在染色天，治疗固定细胞固定和通透试剂。孵育样品用封闭溶液（10％山羊血清中透化溶液）处理1小时，在室温。 </li><li>使用以下主要抗体以所示稀释度在透化溶液中的10％山羊血清进行免疫化学:抗的Pax6（1:10），抗RX（1:200），抗MITF（1:30），和抗ZO1（1:100）。孵育样品与相应抗体O / N在4℃。 </li><li>第二天，除去从细胞初级抗体和冲洗样品三次，用PBS。孵育荧光团共轭次级抗体的样品进行1小时，在室温。 </li><li>从细胞中取出二次抗体和冲洗样本三次PBS中。安装盖玻片载玻片上含安装中DAPI，并允许样品设置O / N在黑暗的室内。使用荧光显微镜来可视化染色。 </li></ol> 8.染色的FACS分析<ol><li>在PBS洗培养胚两次。孵育用0.5ml 0.25％胰蛋白酶的胚5-10分钟，使单细胞悬浮液。中和用胰蛋白酶中和溶液中的胰蛋白酶（1毫升/孔）。 </li><li>传送单细胞悬液至15ml锥形管中并离心，在600×g离心10分钟。 </li><li>弃去上清液，并加入10 mL的DMEM / F12向细胞在管中。轻轻颠倒混合细胞。离心机在600×g的。离心后，弃去上清并将细胞重新悬浮在FACS染色缓冲液中。 </li><li>计数细胞的总数。离心在600×g离心10分钟。离心后弃上清。立即通过加入0.5ml的4％的多聚甲醛固定细胞。轻轻旋转混匀。孵育细胞，在4℃进行20分钟。 </li><li>离心在600×g离心10分钟，并取出上清。轻轻涡旋破坏细胞沉淀。通过加入1ml的冷却溶液透透化细胞。轻轻涡旋混合并在冰上孵育30分钟。 </li><li>离心在600×g离心10分钟并除去上清液。加入3毫升FACS染色缓冲液重悬。重复一次这个步骤。将细胞重悬于FACS染色缓冲液中的5×10 6个细胞/ ml的终浓度。 </li><li>转移100微升细胞悬浮液（0.5×10 6个细胞）至各离心管并加入初级抗体的推荐量。对于Pax6的，在100微升细胞悬液加5微升PE防的Pax6的。对于MITF，在100微升细胞悬液加5微升抗MITF的。 </li><li>混合并在室温下孵育60分钟。加入3毫升FACS染色缓冲。混合和离心机在600×g离心10分钟。去除上清，额外的两次重复8.16。 </li><li>对于MITF染色，孵化细胞二次antiboDY，山羊抗小鼠的Alexa Fluor 488的稀释度为1:2000 1小时，在4℃。对于Pax6的染色，避免这一步。 </li><li>用5毫升的FACS染色缓冲液和离心机在600×g离心洗细胞10分钟。重复该过程两次以上。 </li><li>将细胞重悬于500微升FACS染色缓冲液并涡旋轻轻以破坏细胞沉淀。样品准备流式细胞仪分析。 </li></ol> 9.的iPS-RPE分离和培养<ol><li>在第29天，小心地从周围用200μl的移液管尖的培养板中的所选着色菌落切割。传送浮动菌落到15ml锥形管中。离心菌落在600×g离心在室温10分钟，并除去上清液。 </li><li>重悬在10ml的DMEM / F12和离心机的细胞集落，在600×g离心10分钟。去除上清。 </li><li>通过温育与2ml的0.25％胰蛋白酶的菌落在37℃下为7-10分钟制得单细胞悬浮液。轻轻涡旋细胞悬液分离的殖民地。 </li><li>加入2毫升的iPS-RPE介质中和胰蛋白酶。离心在600×g离心10分钟并弃去上清液。重悬在2ml的iPS-RPE介质的单细胞。转移细胞进入一种蛋白质基质包被的6孔板并置于培养箱中在37℃，5％CO 2和95％的湿度。 </li></ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+the+embryoid+body+microenvironment+to+direct+embryonic+stem+cell+differentiation.">Engineering the embryoid body microenvironment to direct embryonic stem cell differentiation.</a> Biotechnol Prog. 25 (1), 43-51 (2009).</li><li>Kurosawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+inducing+embryoid+body+formation:+in+vitro+differentiation+system+of+embryonic+stem+cells.">Methods for inducing embryoid body formation: in vitro differentiation system of embryonic stem cells.</a> J Biosci Bioeng. 103 (5), 389-398 (2007).</li><li>Itskovitz-Eldor, J., 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+of+human+embryonic+stem+cells+into+embryoid+bodies+compromising+the+three+embryonic+germ+layers.">Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers.</a> Mol Med. 6 (2), 88-95 (2000).</li><li>Ng, E. 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=Forced+aggregation+of+defined+numbers+of+human+embryonic+stem+cells+into+embryoid+bodies+fosters+robust,+reproducible+hematopoietic+differentiation.">Forced aggregation of defined numbers of human embryonic stem cells into embryoid bodies fosters robust, reproducible hematopoietic differentiation.</a> Blood. 106 (5), 1601-1603 (2005).</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 (8), 3612-3624 (2006).</li></ol>

The authors have nothing to disclose.

为了实现多能干细胞用于细胞疗法的充分承诺，有必要调节其分化以一致和可再现的方式。这份报告描述了协议，形成利用微孔板技术尺寸控制EBS，开始分化为RPE，并确定RPE的蛋白质和基因标记。同步在体外分化，胚的大小均匀已知数字的iPS细胞在微孔板强迫聚集离心的形成。免疫细胞化学和逆转录聚合酶链反应（RT-PCR），用于监视所述表达式的RPE的蛋白质和基因说明。最后，分化的不同?...

诱导多能干细胞（iPS细胞）是由重编程体细胞与外在因素1派生的类型的多能干细胞。与此相反，胚胎干细胞（ESCs），另一种类型的多能干细胞，从胚泡2-3的内细胞团生成。尽管他们的不同的起源，iPS细胞和胚胎干细胞是可比在其无限的容量在体外和在其分化成任何细胞类型的能力4-5进行复制。 iPS细胞的这些特性使其成为理想的候选人在个性化的再生医学应用。最近的研究工作集中在开发健壮的分化协议用 ​​于生产特殊成年细胞包括视网膜色素上皮（RPE）6-11。 于iPS来源的细胞潜在的临床应用中，定向分化为特定的细胞类型是至关重要的。有多种方法公布为胚胎干细胞和iPS细胞定向分化为RPE的良莠不齐的工作效率6-7，12-16。我们仍然不知道有多少，发展和分化过程中执政细胞/组织命运的分子事件。在最近几年，已经作出努力来开发该分化方案，可以模仿的胚胎发育尽可能。在胚泡阶段，干细胞的未提交的人口都一起在三维微环境。因此，采用了不同的策略，使ESC / iPS细胞聚集在一起，在三个维度成长他们。这些干细胞聚集体被称为胚体（EB）。有研究表明，干细胞的分化的EB模仿胚胎发育的早期阶段，并且可以自发地产生原始内胚层在其外表面上。后来，随着EB开发的进展，所有三个胚谱系分化细胞的表型出现17-18。日erefore，胚基分化协议已经吸引了大量的关注在体外分化ESC / iPS细胞，是一个很好的候选人RPE代从多能干细胞13。 胚可以使用多种方法从ESC / iPS细胞进行。最初，胚被刮去附着的菌落，并保持它们在非粘附悬浮培养制成。然而，这种方法产生的EB，导致低再现性的异质群体。悬滴细胞培养和微孔基EBS形成其他流行的技术胚形成而产生定义尺寸的高度重复性的同质胚。另外，微孔的技术可以产生大量的聚集体以较少的努力。 内的EB细胞分化是通过从细胞外和细胞内的微形态线索多路复用调节。与此相反，以分化的纹olayer格式的EB为细胞的复杂的组装和细胞间信号发生17的平台。有趣的是，观察到用来制作个别的EB多能干细胞的数量来影响细胞的命运。例如，在人类胚胎干细胞的造血分化的研究中，观察到500个细胞EB对促进髓系分化，而​​1000细胞EB推向红系20。在另一项研究中，较小的EB青睐定形内胚层分化而促进向神经外胚层分化11,17更大的EB。 这些过去的研究有力地表明，用于制造单个的EB ESC / iPS细胞的数量的影响根据分化成任何细胞类型的胚。然而，据我们所知，有一些已经阐明其倾向胚大小的影响，区分对RPE目前没有研究。这项研究的目的是表征的影响EB大小对诱导多能干细胞（iPS细胞） - 视网膜色素上皮细胞（IPS-RPE）的分化，并确定最佳的细胞数量，使胚的对RPE细胞系定向分化。

<table><tbody><tr><td>mTeSR1 media + 5x supplement</td><td>Stem Cell Technologies</td><td>5850</td><td></td></tr><tr><td>Y-27632 (Rock Inhibitor)</td><td>Stem Cell Technologies</td><td>72304</td><td></td></tr><tr><td>DMEM/F12</td><td>Life Technologies</td><td>11330-032</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma</td><td>M-7154</td><td></td></tr><tr><td>Non essential amino acids</td><td>Hyclone(Fisher)</td><td>SH30853.01</td><td></td></tr><tr><td>Knockout serum replacement</td><td>Life Technologies</td><td>10828-028</td><td></td></tr><tr><td>Gentamicin&amp;nbsp;</td><td>Life Technologies</td><td>15750-060</td><td></td></tr><tr><td>L-Glutamine</td><td>Life Technologies</td><td>25030-081</td><td></td></tr><tr><td>MEM media</td><td>Life Technologies</td><td>10370-021</td><td></td></tr><tr><td>N1 supplement</td><td>Sigma</td><td>N-6530-5ML</td><td></td></tr><tr><td>Taurine</td><td>Sigma</td><td>T-8691-25G</td><td></td></tr><tr><td>Hydrocortisone</td><td>Sigma</td><td>H0888-1G</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Hyclone(Fisher)</td><td>SH3008803HI</td><td></td></tr><tr><td>Triiodo-L-thyronine sodium&amp;nbsp; salt</td><td>Sigma</td><td>T6397</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Sigma</td><td>S5881</td><td></td></tr><tr><td>Dispase</td><td>Life Technologies</td><td>17105-041</td><td></td></tr><tr><td>Matrigel</td><td>BD Biosciences</td><td>354277</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Hyclone(Fisher)</td><td>10010-023</td><td></td></tr><tr><td>Aggrewell 400 plate</td><td>Stem Cell Technologies</td><td>27940</td><td></td></tr><tr><td>AggreWell medium</td><td>Stem Cell Technologies</td><td>5893</td><td></td></tr><tr><td>Accutase</td><td>Stem Cell Technologies</td><td>7920</td><td></td></tr><tr><td>BD Cytofix/Cytoperm Fixation/Permeabilization Kit</td><td>BD Biosciences</td><td>554714</td><td></td></tr><tr><td>Mouse Anti-PAX6 antibody</td><td>Developmental Studies Hybridoma Bank</td><td></td><td></td></tr><tr><td>Rabbit Anti-RX antibody</td><td>Abcam</td><td>Ab23340</td><td></td></tr><tr><td>Mouse&amp;nbsp; Anti-MITF antibody</td><td>Thermo Scientific</td><td>MS-772-P</td><td></td></tr><tr><td>Rabbit Anti-ZO-1 antibody</td><td>Invitrogen</td><td>40-2200</td><td></td></tr><tr><td>RNeasy plus mini kit</td><td>Qiagen</td><td>74134</td><td></td></tr><tr><td>PCR master mix</td><td>Promega</td><td>M7502</td><td></td></tr><tr><td>High capacity RNA to cDNA kit</td><td>Life Technologies</td><td>4387406</td><td></td></tr></tbody></table>

deriving retinal pigment epithelium (rpe) from induced pluripotent stem (ips) cells by different sizes of embryoid bodies

Pluripotent stem cells possess the ability to proliferate indefinitely and to differentiate into almost any cell type. Additionally, the development of techniques to reprogram somatic cells into induced pluripotent stem (iPS) cells has generated interest and excitement towards the possibility of customized personal regenerative medicine. However, the efficiency of stem cell differentiation towards a desired lineage remains low. The purpose of this study is to describe a protocol to derive retinal pigment epithelium (RPE) from iPS cells (iPS-RPE) by applying a tissue engineering approach to generate homogenous populations of embryoid bodies (EBs), a common intermediate during in vitro differentiation. The protocol applies the formation of specific size of EBs using microwell plate technology. The methods for identifying protein and gene markers of RPE by immunocytochemistry and reverse-transcription polymerase chain reaction (RT-PCR) are also explained. Finally, the efficiency of differentiation in different sizes of EBs monitored by fluorescence-activated cell sorting (FACS) analysis of RPE markers is described. These techniques will facilitate the differentiation of iPS cells into RPE for future applications.

在这个实验中，iPS细胞进行培养，分化成从胚的视网膜色素上皮谱系。利用微孔板形成控制大小胚。如从图1可见EB形成为同质的微孔板。这些胚然后收集并接种在6孔板（ 图2）。 RPE可以通过经典六角形态，色素沉着，及表达的视网膜色素上皮标志物来鉴定。 12周文化后，200细胞胚制定了星形胶质细胞和成纤维细胞的形态。无色素沉着，观察这些细胞...

Watch this Scientific Journal Video about Deriving Retinal Pigment Epithelium RPE from Induced Pluripotent Stem iPS Cells by Different Sizes of Embryoid Bodies at JoVE.com

Deriving Retinal Pigment Epithelium RPE from Induced Pluripotent Stem iPS Cells by Different Sizes of Embryoid Bodies

此报告的目的是描述协议来导出从诱导的多能干（iPS细胞）使用不同尺寸的胚状体的视网膜色素上皮细胞（RPE）。

从诱导多能干得出视网膜色素上皮（RPE）（IPS），由胚体大小不同的细胞

Pluripotent stem cells possess the ability to proliferate indefinitely and to differentiate into almost any cell type. Additionally, the development of ...

deriving-retinal-pigment-epithelium-rpe-from-induced-pluripotent-stem

Research

JoVE Journal

Developmental Biology

Deriving Retinal Pigment Epithelium (RPE) from Induced Pluripotent Stem (iPS) Cells by Different Sizes of Embryoid Bodies

10.5K 次观看.  U.S. Army Institute of Surgical Research. 此报告的目的是描述协议来导出从诱导的多能干（iPS细胞）使用不同尺寸的胚状体的视网膜色素上皮细胞（RPE）。 

视频: Deriving Retinal Pigment Epithelium RPE from Induced Pluripotent Stem iPS Cells by Different Sizes of Embryoid Bodies

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

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

人胚胎或诱导多能干细胞对视网膜色素上皮细胞的快速定向分化

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

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 ...

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

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

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

从人胚胎干细胞中提取合适的视网膜色素上皮组织的工程移植

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

Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment epithelium (RPE) cells. RPE provide critical support for photoreceptors and death or dysfunction of RPE cells is characteristic of age-related macular degeneration (AMD), the leading cause of permanent vision loss in people age 55 and older. While no cure for AMD has been identified, implantation of healthy RPE in diseased eyes may prove to be an effective treatment, and large numbers of RPE cells can be readily generated from pluripotent stem cells. Several interesting questions regarding the safety and efficacy of RPE cell delivery can still be examined in animal models, and well-accepted protocols used to inject RPE have been developed. The technique described here has been used by multiple groups in various studies and involves first creating a hole in the eye with a sharp needle. Then a syringe with a blunt needle loaded with cells is inserted through the hole and passed through the vitreous until it gently touches the RPE. Using this injection method, which is relatively simple and requires minimal equipment, we achieve consistent and efficient integration of stem cell-derived RPE cells in between the host RPE that prevents significant amount of photoreceptor degeneration in animal models. While not part of the actual protocol, we also describe how to determine the extent of the trauma induced by the injection, and how to verify that the cells were injected into the subretinal space using in vivo imaging modalities. Finally, the use of this protocol is not limited to RPE cells; it may be used to inject any compound or cell into the subretinal space.

Here we present a community accepted protocol in multimedia format for subretinally injecting a bolus of RPE cells in rats and mice. This approach can be used for determining rescue potentials, safety profiles, and survival capacities of grafted RPE cells upon implantation in animal models of retinal degeneration.

视网膜下进行注射的鼠害提供视网膜色素上皮细胞悬浮

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

医学

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 ...

增强人胚胎干细胞的 3D 神经视网膜分化

Generating Neural Retina from Human Pluripotent Stem Cells

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.

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

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.

从人类多能干细胞生成神经视网膜

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

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

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

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, ...

从诱导多能干得出视网膜色素上皮（RPE）（IPS），由胚体大小不同的细胞

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视网膜色素上皮细胞的干细胞高效的推导

人胚胎或诱导多能干细胞对视网膜色素上皮细胞的快速定向分化

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

从人胚胎干细胞中提取合适的视网膜色素上皮组织的工程移植

视网膜下进行注射的鼠害提供视网膜色素上皮细胞悬浮

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

从人类多能干细胞生成神经视网膜

从人类多能干细胞生成神经视网膜

从人类多能干细胞生成神经视网膜

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