This work was supported by the Research Program funded by the Korea Centers for Disease Control and Prevention (HI13D2188).

伦理学声明:本研究方案被批准由韩国天主教大学（KC12TISI0861）的机构审查委员会。 1.滑膜细胞分离和扩增<ol><li>隔离滑膜<ol><li>消毒2双手术剪和一对钳。 </li><li>转移滑膜组织到100mm皿并用含有1％青霉素/链霉素5毫升磷酸盐缓冲盐水的（PBS）中洗涤。 </li><li>切断淡黄色脂肪组织和骨的残基。转移修整组织到一个6孔板的孔中加入5 ml Dulbecco改良的Eagle培养基（DMEM），用20％胎牛血清（FBS）的。 </li><li>剁用剪刀组织直至件小到足以穿透一次性吸。 </li><li>含组织媒体传送到50ml锥形管中。通过加入5ml DMEM中，用20％的FBS的6孔板收获剩余的材料，然后转移到管中。&lt;/ LI&gt; <li>在冰上解冻胶原酶。添加胶原酶到0.01％的终浓度和密封用封口膜的管。孵育在水浴在37℃振荡4小时。 </li><li>温育后，填充有20％FBS的DMEM管，直到总体积为50 ml和离心机在300 xg离心，室温10分钟。 </li><li>不干扰沉淀除去上清液并加40毫升培养基重悬沉淀。 </li><li>重复步骤1.1.10-1.1.11。 </li><li>重悬在25毫升的DMEM的沉淀，用20％FBS和等待组织的大的团块，以沉底。 </li><li>将上清转移至100毫米培养皿，并在5％CO 2在37℃下孵育14一天。 </li></ol></li><li>滑膜维护和扩展<ol><li>丢弃从板的使用的媒体，并用5毫升的PBS洗细胞。 </li><li>添加1ml的PBS / 1mM EDTA中，并在5％CO 2在37℃下孵育2分钟。 </li><li>轻轻点击菜和transfer中的细胞到15毫升锥形管中。离心细胞，在250 xg离心，室温2分钟。 </li><li>不干扰沉淀除去上清液和重悬沉淀在30ml DMEM中，用20％的FBS。 </li><li>转移细胞以3×100毫米培养皿，不留任何可见的边角料。 </li><li>新鲜传媒每3天更换介质。分裂细胞以用1ml的PBS / 1mM EDTA中80％汇合。保持，直到使用之前3代。分裂细胞的每道菜到每分3菜肴。 注:到达通道3后，即不会立即使用的细胞可以被冷冻。 </li></ol></li></ol> 2.重新编程FLSS使用编码慢病毒，山因素<ol><li>转导（D0） <ol><li>每在生长培养基的6孔板的种子3×10 4细胞（500毫升DMEM中补充有10％FBS和1％青霉素/链霉素）。孵育细胞O / N在37℃下在5％CO2。 </li><li>次日，除去含有4山因子慢病毒之一小瓶:Oct4的，Klf4的，Sox2的和c-Myc从冷冻和解冻，在4℃。注意:慢病毒通过在我们以前的研究11中描述的方法产生的。 </li><li>而解冻病毒，更改（在DMEM 20％胎牛血清加抗生素）含有10微克/毫升聚凝胺和50微克/毫升抗坏血酸媒体FLS生长培养基。 </li><li>更换介质后，加入30微升慢病毒的细胞，轻轻混匀。以提高感染，在680 XG，35℃30分钟离心板。 </li><li>离心后，孵育细胞在37℃下在5％CO 2。 </li></ol></li><li>维护直到重新编程可见<ol><li>为第3天，用含有0.1毫丁酸钠和50微克/毫升抗坏血酸FLS培养基每天更换介质。 </li><li>第二天，与FLS生长培养基的混合物代替媒体和含有0.1mM丁酸钠和50微克/毫升抗坏血酸:（1比1）的iPSC介质。 注意:iPSC的介质的组分中的材料/设备列表中给出。 </li></ol></li><li>拆分单元格的集落形成<ol><li>制备玻连蛋白包被的6孔板中。 <ol><li>加入60微升玻〜6毫升的PBS无钙+和Mg2 +。放2毫升混合物到每个孔中，并在室温下孵育至少1小时。注意:玻连蛋白的工作浓度为5微克/毫升。 </li></ol></li><li>上D5，洗涤细胞，用PBS。 </li><li>添加1ml的PBS / 1mM EDTA中分离细胞，并在37℃，5％CO 2孵育2分钟。 </li><li>收获细胞，离心，在250 xg离心，室温2分钟。 </li><li>分裂的细胞以3个不同的比例（1:3,1:6和1:9），以实现不同的汇合。添加900微升的媒体向细胞沉淀重悬。每孔中添加6- 300，150，和100微升细胞混合物的孔板来实现的比率为1:3,1:6和1:9，分别。 </li><li>直到克隆出现与媒体的iPSC每天更换介质。关于D18菌落后会出现。注意:在此阶段，的iPSC集落共存与非重新编程FLSS。 </li></ol></li><li>殖民地采摘<ol><li>通过加入500微升玻连至孔准备48孔玻连蛋白包被的板，并在室温下孵育至少1小时。 </li><li>放置一个净化台显微镜，并从培养箱取出6孔板。 </li><li>从48孔板中除去玻连蛋白溶液，并添加补充有含10mM的Rho相关，卷曲螺旋蛋白激酶（ROCK）抑制剂500微升的iPSC介质。 </li><li>用10便士的枪头，切断周围的殖民地。传送所拾取菌落至48孔板的一个孔中。 </li><li>采摘几个菌落后，孵育细胞在37℃，5％的CO 2。 </li><li>维持细胞直到菌落大ENO唉转移。注意:我们通常洒细胞时菌落失控显微镜的可见领域，当在100X放大率的观看。 </li></ol></li></ol> 3.免疫荧光染色<ol><li>细胞的制备<ol><li>放置无菌18毫米盖玻片至12孔板中。 </li><li>加入1毫升的PBS以冷却和冲洗玻璃盖。 </li><li>用1毫升10微克/毫升玻连蛋白溶液的更换。 </li><li>孵育在室温的板至少1小时。 </li><li>弃去玻连蛋白溶液和板的iPSC在玻连蛋白包被的12孔平板并培养7天，在37℃，5％CO 2，每日更换介质。 </li></ol></li><li>细胞染色<ol><li>弃去培养基，用PBS洗细胞一次。 </li><li>固定在0.4％多聚甲醛（PFA）将细胞在室温30分钟。 </li><li>透用0.1％的Triton X-100将细胞在室温下5分钟。 </li><li>取出透溶液和块与含有2％牛血清白蛋白（BSA）在室温30分钟的PBS。 </li><li>稀释按表1含2％BSA的PBS中的抗体。用在RT 2小时的初级抗体孵育的细胞。 </li><li>添加二次抗体（稀释1:200），孵育所述细胞在室温下1小时，以避免光。 </li><li>处理细胞用1微升/毫升的DAPI 10分钟。 </li><li>放置在载玻片与抗淬灭剂的顶部的盖玻璃，并在室温下孵育24小时，避光。 </li><li>验证用荧光显微镜表达。 </li></ol></li></ol> 4.实时聚合酶链式反应（RT-PCR） <ol><li>使用硫氰酸胍-苯酚-氯仿提取法11细胞沉淀中提取的mRNA。 </li><li>使用反转录11总RNA 2微克扩增基因。 </li><li>混合使用2微升cDNA的统的用于PCR所需的组件板11。 </li><li>进行RT-PCR和验证通过凝胶电泳11的结果。 </li></ol> 5.碱性磷酸酶（AP）染色<ol><li>培养的iPSC 5-7天，在37℃，5％CO 2的前染色。 </li><li>吸媒体和修复用4％PFA的细胞1分钟。 </li><li>丢弃固定液和冲洗用1X冲洗缓冲细胞。 </li><li>准备AP染色试剂。混合试剂在下列比率:快速红紫:酚AS-BI磷酸酯溶液:水= 2:1:1。 </li><li>孵育在室温染色溶液15分钟的细胞中，避光。 </li><li>丢弃染色液和冲洗用漂洗缓冲液将细胞。 </li><li>覆盖细胞用PBS以防止干燥，并用明场显微镜验证表达。 </li></ol>

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The authors have nothing to disclose.

iPS细胞的发现之前，科学家主要用于胚胎干细胞通过差异化来研究干细胞生物学和其他细胞系。然而，胚胎干细胞从胚泡，这是一个早期胚胎的内质起源。为了分离胚胎干细胞，囊胚破坏是不可避免的，提高是不可能克服的伦理问题。此外，尽管胚胎干细胞具有干性特征和多能性，它们不能从个体获得的，并且有时不进行个性化的分析和疾病筛查的理想工具。 在2007年，高桥...

多能性干细胞是在不同的临床和生物领域的下一代平台。它们是一种很有前途的工具，可以在疾病建模，药物筛选，并再生医疗治疗中使用。人类胚胎干细胞（胚胎干细胞）主要用于研究和理解多能细胞。然而，由人胚泡的破坏分离，人类胚胎干细胞与几个伦理问题相关联。 2007年，山中伸弥博士和他的团队逆转细胞编程过程和成人的体细胞1,2开发干细胞。因此，与人类胚胎干细胞，诱导多能干细胞（iPS细胞），可以从成熟的体细胞中产生，避免了伦理障碍。 的Oct4，Sox2的，Klf4和c-Myc的:通常，由四个外源基因递送产生的iPSC。这些因素山中正在使用慢病毒和逆转录病毒系统最初交付。第一iPS细胞是从鼠体细胞衍生Ç厄尔3。然后，将技术应用于人皮肤成纤维1,2。随后的研究成功生成从各种来源，如尿4，血液5,6- iPSCs的，角质7和其他一些类型的细胞。然而，也有没有在重新编程中使用的一些体细胞，并从在疾病状态特异组织的各种细胞类型的重新编程能力筛选，仍然需要。 类风湿关节炎（RA）是一种可以攻击所有关节，并导致在其他器官自身免疫疾病的疾病。 RA影响在发达国家成年人约1％。这是一种相当常见的疾病和其发病率逐年增加8。然而，RA是很难在早期识别和摧毁oncebone发生没有治疗，可以恢复的损害。此外，药物的功效，从患者的不同而不同病人，这是很难预测的军医所需INE。因此，药物筛选方法的开发是必要的，并且需要能反映RA的条件的细胞的材料。 成纤维样滑膜（FLSS）均在9,10 RA发病的积极参与者的细胞。 FLSS在关节囊和腔，其也被称为滑膜之间滑膜内膜衬里存在。通过支持接头结构和周围软骨提供营养，FLSS通常发挥关节功能和维护了至关重要的作用。然而，在FLSS RA具有侵入性的表型。 RA FLSS有一个肿瘤样表型，最终被无限增殖10摧毁了周围的骨骼。这种独特的特性，FLSS可以用作一个有前途的材料，可反映RA的病理生物学。然而，这些细胞很少产生，细胞表型改变的细胞经过在体外几段</eM&gt;的条件。因此，它可以使用复杂的RA FLSS作为一种工具，可以代表病人的状况。 从理论上讲，RA患者来源的iPS细胞（RA-iPSCs的），可以成为药物筛选和进一步研究的理想工具。产生的iPSC具有自我更新的能力，并且可以保持和体外扩展。与多能性，这些细胞可以分化为成熟软骨细胞和骨细胞谱系，其可为在RA和其它骨相关疾病11具体的研究有助于细胞材料。 在这项研究中，我们将演示如何隔离和从手术切除滑膜，以及如何扩大FLSS使用含有山因素慢病毒从FLSS产生RA-iPSCs的。

<table><tbody><tr><td>100 mm Dish</td><td>TPP</td><td>93100</td><td></td></tr><tr><td>6-well Plate</td><td>TPP</td><td>92006</td><td></td></tr><tr><td>50 ml Cornical Tube</td><td>SPL</td><td>50050</td><td></td></tr><tr><td>15 mlL Cornical Tube</td><td>SPL</td><td>50015</td><td></td></tr><tr><td>10 ml Disposable Pipette</td><td>Falcon</td><td>7551</td><td></td></tr><tr><td>5 ml Disposable Pipette</td><td>Falcon</td><td>7543</td><td></td></tr><tr><td>12-well Plate</td><td>TPP</td><td>92012</td><td></td></tr><tr><td>&lt;strong&gt;FLS Isolation Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Surgical Scissors</td><td></td><td></td><td></td></tr><tr><td>Surgical Forcep</td><td></td><td></td><td></td></tr><tr><td>DPBS</td><td>Life Technologies</td><td>14190-144</td><td></td></tr><tr><td>DMEM</td><td>Life Technologies</td><td>11995-073</td><td></td></tr><tr><td>Penicilin Streptomycin</td><td>Sigma Aldrich</td><td>P4333</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Life Technologies</td><td>16000-044</td><td></td></tr><tr><td>Collagenase</td><td>Sigma Aldrich</td><td>C6885-100MG</td><td></td></tr><tr><td>Parafilm</td><td>Sigma Aldrich</td><td>54956</td><td></td></tr><tr><td>PBS/1 mM EDTA</td><td>Life Technologies</td><td>12604-039</td><td></td></tr><tr><td>&lt;strong&gt;iPSC Generation Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DMEM</td><td>Life Technologies</td><td>11885</td><td></td></tr><tr><td>MEM Non-Essential Amino Acids Solution (100x)</td><td>Life Technologies</td><td>11140-050</td><td></td></tr><tr><td>&amp;beta;-Mercaptoethanol</td><td>Sigma Aldrich</td><td>M3148</td><td></td></tr><tr><td>Polybrene</td><td>Chemicon</td><td>TR-1003-G</td><td></td></tr><tr><td>Penicilin Streptomycin</td><td>Life Technologies</td><td>P4333</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Life Technologies</td><td>16000-044</td><td></td></tr><tr><td>DPBS</td><td>Life Technologies</td><td>14190-144</td><td></td></tr><tr><td>Lentivirus</td><td></td><td></td><td></td></tr><tr><td>DMEM/F12, HEPES</td><td>Life Technologies</td><td>11330-057</td><td>iPSC media ingredient (500 ml)</td></tr><tr><td>Sodium Bicarbonate</td><td>Life Technologies</td><td>25080-094</td><td>iPSC media ingredient (Conc.: 543 &amp;mu;g/ml)</td></tr><tr><td>Sodium Selenite</td><td>Sigma Aldrich</td><td>S5261</td><td>iPSC media ingredient&amp;nbsp; (Conc.: 14 ng/mL)</td></tr><tr><td>Human Transfferin</td><td>Sigma Aldrich</td><td>T3705</td><td>iPSC media ingredient (Conc.: 10.7 &amp;mu;g/ml)</td></tr><tr><td>Basic FGF2</td><td>Peprotech</td><td>100-18B</td><td>iPSC media ingredient&amp;nbsp; (Conc.: 100 ng/ml)</td></tr><tr><td>Human Insulin</td><td>Life Technologies</td><td>12585-014</td><td>iPSC media ingredient (Conc.: 20 &amp;mu;g/ml)</td></tr><tr><td>Human TGF&amp;beta;1</td><td>Peprotech</td><td>100-21</td><td>iPSC media ingredient (Conc.: 2 ng/ml)</td></tr><tr><td>Ascorbic Acid</td><td>Sigma Aldrich</td><td>A8960</td><td>iPSC media ingredient&amp;nbsp; (Conc.: 64 &amp;mu;g/ml)</td></tr><tr><td>Polybrene</td><td>Chemicon</td><td>TR-1003</td><td></td></tr><tr><td>Sodium Butyrate</td><td>Sigma Aldrich</td><td>B5887</td><td></td></tr><tr><td>Vitronectin</td><td>Life Technologies</td><td>A14700</td><td></td></tr><tr><td>ROCK Inhibitor</td><td>Sigma Aldrich</td><td>Y0503</td><td></td></tr><tr><td>&lt;strong&gt;Guality Control Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>18 mm Cover Glass</td><td>Superior</td><td>HSU-0111580</td><td></td></tr><tr><td>4% Paraformaldyhyde (PFA)</td><td>Tech &amp;amp; Innovation</td><td>BPP-9004</td><td></td></tr><tr><td>Triton X-100</td><td>BIOSESANG</td><td>9002-93-1</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Vector Lab</td><td>SP-5050</td><td></td></tr><tr><td>Anti-SSEA4 Antibody</td><td>Millipore</td><td>MAB4304</td><td></td></tr><tr><td>Anti-Oct4 Antibody</td><td>Santa Cruz</td><td>SC9081</td><td></td></tr><tr><td>Anti-TRA-1-60 Antibody</td><td>Millipore</td><td>MAB4360</td><td></td></tr><tr><td>Anti-Sox2 Antibody</td><td>Biolegend</td><td>630801</td><td></td></tr><tr><td>Anti-TRA-1-81 Antibody</td><td>Millipore</td><td>MAB4381</td><td></td></tr><tr><td>Anti-Klf4 Antibody</td><td>Abcam</td><td>ab151733</td><td></td></tr><tr><td>Alexa Fluor 488 goat anti-mouse IgG (H+L) antibody</td><td>Molecular Probe</td><td>A11029</td><td></td></tr><tr><td>Alexa Fluor 594 goat anti-rabbit IgG (H+L) antibody</td><td>Molecular Probe</td><td>A11037</td><td></td></tr><tr><td>DAPI</td><td>Molecular Probe</td><td>D1306</td><td></td></tr><tr><td>Prolong gold antifade reagent</td><td>Invitrogen</td><td>P36934</td><td></td></tr><tr><td>Slide Glass, Coated</td><td>Hyun Il Lab-Mate</td><td>HMA-S9914</td><td></td></tr><tr><td>Trizol</td><td>Invitrogen</td><td>15596-018</td><td></td></tr><tr><td>Chloroform</td><td>Sigma Aldrich</td><td>366919</td><td></td></tr><tr><td>Isoprypylalcohol</td><td>Millipore</td><td>109634</td><td></td></tr><tr><td>Ethanol</td><td>Duksan</td><td>64-17-5</td><td></td></tr><tr><td>RevertAid First Strand cDNA Synthesis kit</td><td>Thermo Scientfic</td><td>K1622</td><td></td></tr><tr><td>i-Taq DNA Polymerase</td><td>iNtRON BIOTECH</td><td>25021</td><td></td></tr><tr><td>UltraPure 10x TBE Buffer</td><td>Life Technologies</td><td>15581-044</td><td></td></tr><tr><td>loading star</td><td>Dyne Bio</td><td>A750</td><td></td></tr><tr><td>Agarose</td><td>Sigma-Aldrich</td><td>9012-36-6</td><td></td></tr><tr><td>1 kb (+) DNA ladder marker</td><td>Enzynomics</td><td>DM003</td><td></td></tr><tr><td>Alkaline Phosphatase</td><td>Millipore</td><td>SCR004</td><td></td></tr></tbody></table>

generation of induced-pluripotent stem cells using fibroblast-like synoviocytes isolated from joints of rheumatoid arthritis patients

成熟的体细胞可以使用一组已定义的重编程因子被逆转成多潜能干细胞样的状态。的Oct4，Sox2的，Klf4的和c-Myc:大量的研究已经通过转导4山中转录因子产生的各种体细胞类型的诱导多能干细胞（iPS细胞）。 iPS细胞的研究仍然是生物学和临床研究的前沿。特别是，患者特异性iPSCs的可以被用作疾病病理学的研究开拓工具，因为iPSCs的可以从任何个体的组织进行诱导。类风湿关节炎（RA）是一种慢性炎性疾病，软骨和骨结构中的关节的破坏分类。滑膜增生是导致这些结果在RA中的主要原因或症状之一。成纤维细胞样滑膜细胞（FLSS）是在增生的滑膜的主要成分的细胞。 FLSS在合资无限增殖，最终侵入相邻cartil年龄和骨骼。目前，增生性滑膜只能通过外科手术去除。移除的滑膜用于RA的研究作为反映关节的炎性病症的材料。如在RA的发病机理的主要参与者，FLSS可用作一种材料，以产生并调查RA患者的iPSCs的。在这项研究中，我们使用了RA患者的FLSS产生的iPSC。使用慢病毒系统，我们发现FLSS可以生成RA患者特异性的iPSC。从FLSS产生的iPS细胞可以进一步用来作为一种工具来研究RA的病理生理的未来。

在这项研究中，我们描述了一种协议使用的慢病毒系统生成从FLSS的iPSC。 图1A示出了FLS隔离协议的一个简单的方案。继手术切除滑膜，组织被切成采用手术剪小块。加入胶原酶向细胞从组织的团块隔离。细胞进一步处理前温育14天。 图 1B示出的分离的FLSS的形态。细胞保持在使用前3的通道。 FLSS共享一般的成纤维细胞的类似特性。我们?...

Watch this Scientific Journal Video about Generation of Induced-pluripotent Stem Cells Using Fibroblast-like Synoviocytes Isolated from Joints of Rheumatoid Arthritis Patients at JoVE.com

Generation of Induced-pluripotent Stem Cells Using Fibroblast-like Synoviocytes Isolated from Joints of Rheumatoid Arthritis Patients

在这里，我们描述了从患者来源的成纤维细胞样滑膜细胞产生的诱导的人多能干细胞，使用慢病毒系统无饲养细胞的协议。

诱导多能干细胞使用成纤维样滑膜生成类风湿关节炎患者的关节分离

Mature somatic cells can be reversed into a pluripotent stem cell-like state using a defined set of reprogramming factors. Numerous studies have generated ...

generation-induced-pluripotent-stem-cells-using-fibroblast-like

Research

JoVE Journal

Developmental Biology

9.3K 次观看.  Seoul St. Mary's Hospital, Republic of Korea. 在这里，我们描述了从患者来源的成纤维细胞样滑膜细胞产生的诱导的人多能干细胞，使用慢病毒系统无饲养细胞的协议。 

视频: Generation of Induced-pluripotent Stem Cells Using Fibroblast-like Synoviocytes Isolated from Joints of Rheumatoid Arthritis Patients

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative therapeutic requires a safe, robust, and expedient reprogramming protocol. Here, we present a clinically relevant, step-by-step protocol for the extremely high-efficiency reprogramming of human dermal fibroblasts into iPSCs using a non-integrating approach. The core of the protocol consists of expressing pluripotency factors (SOX2, KLF4, cMYC, LIN28A, NANOG, OCT4-MyoD fusion) from in vitro transcribed messenger RNAs synthesized with modified nucleotides (modified mRNAs). The reprogramming modified mRNAs are transfected into primary fibroblasts every 48 h together with mature embryonic stem cell-specific microRNA-367/302 mimics for two weeks. The resulting iPSC colonies can then be isolated and directly expanded in feeder-free conditions. To maximize efficiency and consistency of our reprogramming protocol across fibroblast samples, we have optimized various parameters including the RNA transfection regimen, timing of transfections, culture conditions, and seeding densities. Importantly, our method generates high-quality iPSCs from most fibroblast sources, including difficult-to-reprogram diseased, aged, and/or senescent samples.

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

基于 rna 的人原代成纤维细胞重组成诱导多能干细胞

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...

科研

发育生物学

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing the pluripotent state in mouse fibroblasts.1 The same group also reported the successful reprogramming of human somatic cells into induced pluripotent stem (iPS) cells using human versions of the same transcription factors delivered by retroviral vectors.2 Additionally, James Thomson et al. reported that the lentivirus-mediated co-expression of another set of factors (Oct4, Sox2, Nanog and Lin28) was capable of reprogramming human somatic cells into iPS cells.3
iPS cells are similar to ES cells in morphology, proliferation and the ability to differentiate into all tissue types of the body. Human iPS cells have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction. The generation of patient-specific iPS cells circumvents an important roadblock to personalized regenerative medicine therapies by eliminating the potential for immune rejection of non-autologous transplanted cells.
Here we demonstrate the protocol for reprogramming human fibroblast cells using the Stemgent Human TF Lentivirus Set. We also show that cells reprogrammed with this set begin to show iPS morphology four days post-transduction. Using the Stemolecule Y27632, we selected for iPS cells and observed correct morphology after three sequential rounds of colony picking and passaging. We also demonstrate that after reprogramming cells displayed the pluripotency marker AP, surface markers TRA-1-81, TRA-1-60, SSEA-4, and SSEA-3, and nuclear markers Oct4, Sox2 and Nanog.

We demonstrate the protocol for the generation of induced pluripotent stem cells from human somatic cells using lentivirus-mediated delivery of the human factors Oct4, Sox2, Nanog, and Lin28. Pluripotency was confirmed by morphology and the presence of embryonic stem (ES) cell-specific markers.

由Stemgent人类TF慢病毒重新编程人类成纤维细胞生成诱导多能干细胞

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing ...

生物学

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides fascinating prospects for the derivation of autologous transplants that circumvent histocompatibility barriers. However, progression through a pluripotent state and subsequent complete differentiation into desired lineages remains a roadblock for the clinical translation of iPSC technology because of the associated neoplastic potential and genomic instability. Recently, we and others showed that somatic cells cannot only be converted into iPSCs but also into different types of multipotent somatic stem cells by using defined factors, thereby circumventing progression through the pluripotent state. In particular, the direct conversion of human fibroblasts into induced neural progenitor cells (iNPCs) heralds the possibility of a novel autologous cell source for various applications such as cell replacement, disease modeling and drug screening. Here, we describe the isolation of adult human primary fibroblasts by skin biopsy and their efficient direct conversion into iNPCs by timely restricted expression of Oct4, Sox2, Klf4, as well as c-Myc. Sox2-positive neuroepithelial colonies appear after 17 days of induction and iNPC lines can be established efficiently by monoclonal isolation and expansion. Precise adjustment of viral multiplicity of infection and supplementation of leukemia inhibitory factor during the induction phase represent critical factors to achieve conversion efficiencies of up to 0.2%. Thus far, patient-specific iNPC lines could be expanded for more than 12 passages and uniformly display morphological and molecular features of neural stem/progenitor cells, such as the expression of Nestin and Sox2. The iNPC lines can be differentiated into neurons and astrocytes as judged by staining against TUJ1 and GFAP, respectively. In conclusion, we report a robust protocol for the derivation and direct conversion of human fibroblasts into stably expandable neural progenitor cells that might provide a cellular source for biomedical applications such as autologous neural cell replacement and disease modeling.

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

成人人成纤维细胞的推导和他们直接转换成可扩展的神经祖细胞

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

神经科学

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

干细胞衍生的抗原特异性调节性T细胞对自身免疫的发展

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

免疫与感染

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

选择和隔离殖民地的人类诱导多能干细胞重新编程成年成纤维细胞

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

人类成人皮肤成纤维细胞诱导神经元高产培养的简单生成

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

利用 iPSC 来源的 iTenocytes 进行有效的基于细胞的治疗

Generation of Induced Pluripotent Stem Cell-Derived iTenocytes via Combined Scleraxis Overexpression and 2D Uniaxial Tension

Today&#39;s challenges in tendon and ligament repair necessitate the identification of a suitable and effective candidate for cell-based therapy to promote tendon regeneration. Mesenchymal stromal cells (MSCs) have been explored as a potential tissue engineering strategy for tendon repair. While they are multipotent and have regenerative potential in vivo, they are limited in their self-renewal capacity and exhibit phenotypic heterogeneity. Induced pluripotent stem cells (iPSCs) can circumvent these limitations due to their high self-renewal capacity and unparalleled developmental plasticity. In tenocyte development, Scleraxis (Scx) is a crucial direct molecular regulator of tendon differentiation. Additionally, mechanoregulation has been shown to be a central element guiding embryonic tendon development and healing. As such, we have developed a protocol to encapsulate the synergistic effect of biological and mechanical stimulation that may be essential for generating tenocytes. iPSCs were induced to become mesenchymal stromal cells (iMSCs) and were characterized with classic mesenchymal stromal cell markers via flow cytometry. Next, using a lentiviral vector, the iMSCs were transduced to stably overexpress SCX (iMSCSCX+). These iMSCSCX+ cells can be further matured into iTenocytes via uniaxial tensile loading using a 2D bioreactor. The resulting cells were characterized by observing the upregulation of early and late tendon markers, as well as collagen deposition. This method of generating iTenocytes can be used to assist researchers in developing a potentially unlimited off-the-shelf allogeneic cell source for tendon cell therapy applications.

作者聚焦:肌腱修复的细胞和组织工程进展 

This article describes a procedure to produce iTenocytes by generating iPSC-derived mesenchymal stromal cells with combined overexpression of Scleraxis using a lentiviral vector and uniaxial stretching via a 2D bioreactor.

通过联合巩膜过表达和 2D 单轴张力产生诱导多能干细胞衍生的 iTenocytes

Today&#39;s challenges in tendon and ligament repair necessitate the identification of a suitable and effective candidate for cell-based therapy to ...

诱导多能干细胞使用成纤维样滑膜生成类风湿关节炎患者的关节分离

摘要

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此视频中的章节

基于 rna 的人原代成纤维细胞重组成诱导多能干细胞

由Stemgent人类TF慢病毒重新编程人类成纤维细胞生成诱导多能干细胞

人诱导多能干细胞体外生成体细胞衍生物的研究

成人人成纤维细胞的推导和他们直接转换成可扩展的神经祖细胞

干细胞衍生的抗原特异性调节性T细胞对自身免疫的发展

选择和隔离殖民地的人类诱导多能干细胞重新编程成年成纤维细胞

人类成人皮肤成纤维细胞诱导神经元高产培养的简单生成

通过联合巩膜过表达和 2D 单轴张力产生诱导多能干细胞衍生的 iTenocytes

通过联合巩膜过表达和 2D 单轴张力产生诱导多能干细胞衍生的 iTenocytes

通过联合巩膜过表达和 2D 单轴张力产生诱导多能干细胞衍生的 iTenocytes