Name: generation of induced pluripotent stem cells from muscular dystrophy patients: efficient integration-free reprogramming of urine derived cells
Uploaded: 2015-01-28T13:00:00.000+00:00
Duration: 9 min 11 s
Description: Watch this Scientific Journal Video about Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells at JoVE.com

Development of this protocol was supported by the Advancing a Healthier Wisconsin and the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant Number 8UL1TR000055. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

注:患者和/或其监护人应知情同意参与机构审查委员会批准的研究。 1.缓冲区和媒体准备<ol><li>洗涤缓冲液:向制备100毫升的洗涤缓冲液，加入1 ml 100×青霉素/链霉素溶液（链霉素100U / ml青霉素+ 100微克/毫升）的99毫升磷酸盐缓冲盐水（PBS）。 </li><li>尿祖细胞（UPC）介质:要准备UPC中，混合的角质形成细胞无血清（KSF）中等+祖细胞培养基等体积。 <ol><li> KSF培养基:制备角质形成细胞培养基中，添加的表皮生长因子5纳克/毫升（EGF），牛垂体提取物50纳克/毫升（BPE），霍乱毒素的30纳克/毫升，和青霉素/链霉素溶液（100U / ml青霉素，100μg/ ml的链霉素）至500ml KSF介质。 </li><li>祖细胞培养基:添加四分之三DMEM中以四分之一的Ham-F12培养基，并补充有10％FBS，0.4微克/ ml氢化可的松，0.1纳米霍乱毒素，5纳克/毫升的胰岛素，1.8×10 -4 M腺嘌呤，5微克/毫升转铁蛋白，2×10 -9 M 的三碘甲腺氨酸，10纳克/毫升的EGF，和青霉素/链霉素溶液中。 </li></ol></li><li>的hES培养基:添加20％KO血清替代（K-SR），1×MEM非必需氨基酸，2mM的L-谷氨酰胺，100μM的β巯基乙醇，20纳克/ ml的bFGF和青霉素/链霉素至400毫升DMEM / F12培养基。 </li></ol> 2.尿液样品采集<ol><li>指示患者饮用液体30分钟之前，采尿，以确保尿液足够量（〜30-40毫升）可以被收集。 </li><li>得到患者和/或其监护人含有下列项目的尿收集试剂盒:（1）关于如何获得无菌或清洁渔获尿样的书面说明，（2）湿润抗菌洗手间，及（3）的100毫升的无菌标本采集杯。指导患者收集样品。 </li><li>立即将尿液样品在冰上并传送到labora保守党在凉爽。立即处理的尿细胞分离。如果延迟是不可避免的，储存在冰上的样品长达4小时与细胞存活力损失最小。 </li></ol> 3.分离和扩增尿细胞注意:请在一个BSL2生物安全柜在无菌条件下进行如下操作。 <ol><li>使用无菌移液管，移尿样到无菌的50毫升离心管中。离心机在400×g下在室温下10分钟。 </li><li>吸出上清液留下1毫升在管;要小心，不要扰乱细胞沉淀。 </li><li>重悬并结合粒料从多个管为7毫升的洗涤缓冲液，并重复离心，在400×g下在室温下10分钟。 </li><li>小心吸出上清液，留下细胞团块和〜0.2-0.5上清液毫升。重悬在2-3毫升尿祖细胞（UPC）的培养基中，该悬浮液转移到4-6孔的未中涂覆的24孔板中。 </li><li> 72小时后，补充的培养，用0.5毫升每孔新鲜培养基的UPC和之后每2-3天更换培养基。红细胞和鳞状上皮细胞不附着于板将与介质的变化被移除。 </li><li>经过4-6天监测的文化。 注:2-4小细胞集落将开始出现。细胞将是圆形的或细长代表的I型或肾上皮（RE）细胞分别为3的II型。 </li><li>改变介质每2-3天，因为一旦出现细胞，它们将经历快速膨胀阶段。 </li><li>一旦细胞达到80-90％汇合时，电镀后左右9-15天后，分离和传代的细胞（15,000-18,000细胞/ cm 2）到2-4孔中的24孔平板用于进一步扩展。这标志着作为细胞传代1（P1）。 </li><li>通过流式细胞仪分析表征尿细胞谱系规范，免疫组织的iCal染色或通过逆转录酶 - 聚合酶链反应（RT-PCR）。 </li><li>重新编程分离尿液细胞（第4节）或冷冻下来在冷冻培养基（DMEM补充有10％血清和10％DMSO），用于长期cryostorage。 </li></ol> 4.尿细胞重编程使用仙台病毒注:妥善处理和使用PPE（个人防护装备）的建议，同时操纵转染试剂。执行在一个BSL2生物安全柜在无菌条件下进行如下操作。适当处理转染试剂和/或转染细胞的建议，以避免对环境和健康的危害风险。尿细胞重编程，使用仙台重编程套件改装制造商的馈线相关的协议，详情如下。 <ol><li>为了确保使用仙台病毒高效率的重新编程，使用通道1-5尿液细胞是迅速LY分。如果细胞不复制好或成为衰老，放弃他们，因为他们将不能有效地重新编程。 </li><li>种子每孔60,000个细胞在两口井6孔板。马克这是日-2。调节细胞接种密度（5× 十月四日至9日×10 4个细胞每孔）根据需要通过第0天至达到80-90％汇合。 </li><li>第0天（电镀细胞后48小时），制备含四个OSKM因素的仙台重编程载体按季。每个向量添加到1ml预热UPC培养基。使用1-1.5的MOI（5-7.5×10 5 CIU），这足够用于重编程细胞的尿。吸出培养基，并慢慢地加入1ml的UPC + SeV载体对尿小区中的一个井。使用第二以及转导阴性对照。 </li><li>第1天，更换UPC +中的SeV新鲜UPC媒介。由于病毒的细胞毒性24小时后的一些细胞死亡被看见。 </li><li>根据重新编程酷龙效率Ë形成和紧凑的形态，保持每日更换介质，直到6天或7。 </li><li>前一天转导的细胞（第5天或6），通过电镀丝裂霉素-C（10微克/毫升3小时）处理-MEF中以5×10 4个细胞/ cm 2，密度在0.1制备MEF饲养板的离解％明胶涂层百毫米2培养皿。 </li><li>次日（第6天或7），解离的细胞用0.25％胰蛋白酶，将细胞重悬于培养基中的UPC和板5×10 4 - 2个10 5细胞/ 100mm 2 MEF饲养盘。 </li><li>翌日，切换到的hES培养基每天更换介质。观察在显微镜下的细胞来监测转化的细胞。 注意:这些细胞形成克隆聚集具有特色的鹅卵石形态，并具有较高的细胞核，细胞质比例（12-18天后转）。 </li><li>在两到三个星期转导后，挑殖民地和转移到新的板块为酷龙人扩张。 </li><li>选择的iPSC克隆与TRA-1-81抗体孵育细胞1小时37进行活细胞染色 ℃下的CO 2培养箱中，然后用特定的荧光染料缀合的二级抗体计数器染色1小时，在37℃的CO 2培养箱中培养。 </li><li>使用＆G 1.5寸毫针交叉孵化较大TRA-1-81阳性的iPSC克隆成小相等大小的块（每4-6克隆件）。 </li><li>使用无菌移液管尖端来接并转印10-20交叉影线件从多个克隆到基质胶的每个孔（10微克/厘米2）包被的24孔板用的hES培养基。 注:从单独镀成单井单克隆饮片不扩大或保持多能性。 </li><li>由hES改变的iPSC介质24-48小时后的重新编程克隆附着到基质胶的表面。 </li><li>在对基质胶涂层板，手动涉农供应链的维护PE客用吸管尖，以丰富的完全重新编程和多能性营养不良的iPSC（MD-IPSC）克隆任何分化的细胞或污染MEF饲养层细胞。 </li><li>后单个克隆已达到约100微米的大小，通过用0.48毫摩尔EDTA（乙二胺四乙酸）溶液解离3分钟和电镀的MD-iPS细胞的小细胞聚集体悬浮在介质的iPSC展开克隆（补充有5微米Y27632，Rho激酶抑制剂）在新鲜的基质胶（10微克/厘米2）涂布12孔平板。每日更换的iPSC中。 </li><li>每个克隆的完整的重新编程可通过OSKM基因和多能性标记物（的Oct3 / 4和TRA-1-81）免疫组织化学染色的两个基因表达分析来确定。多能性潜力的一个严格的评估应该被用来确认完全重新编程iPSC系能够分化成三个胚层。这可以通过胚状体（EB）形成和分化测定或之三来实现atoma形成实验。 </li></ol>

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The authors declare that they have no competing financial interests.

造型采用iPSCs的心血管疾病已成为了解遗传贡献4-6的常用方法。获得细胞样品从病人的一些未预料到的困难，尤其是幼儿，能够避免通过提供一种非侵入性的方法的选择，如尿液收集。在这个年轻的患者群中，通常难以收集的尿量足够的体积，以产生足够的尿细胞进行重新编程。执教年轻患者饮用液体前尿液收集30分钟，提高了尿细胞分离成功。然而，重复尿收集可能需要在该人群中。我们已合并，并以从患者的肌营养不良优化UCS的分离和培养适应两种不同的协议3,7。 诱导多能干细胞已经从肌营养不良patien要么皮肤成纤维细胞或尿液细胞生成TS 1,2。然而，这两种重编程协议依靠慢病毒引入重编程因子进入体细胞。此整合型病毒递送方法可能会产生问题，如果转基因随机整合到宿主基因组中，并使转基因的或者再活化或诱变（在8中综述）。因此，我们提高了对这些技术的使用仙台病毒转导OSKM转基因进入尿液细胞的非整合方式9。 使用仙台病毒重新编程UCS此方法提供了具有更高的转导效率9,10-一个非整合方法的优点。从肌营养不良患者的尿液收集细胞在2-3周后，转导重新编程。这些重编程动力学与使用慢病毒转1 UC重新编程。虽然这个协议可以扩展到建立无饲养方法尿细胞重编程，是荷兰国际集团使用尿细胞肌营养不良患者中分离的仙台病毒转导的有效方法。描述此方法，以充分建立之前的多能性的MD克隆转移到无饲养条件通路2依赖的MEF作为饲养层。 肌营养不良蛋白的Dp71亚型的核分配早已确定在不同胚胎阶段的细胞模型11,12。 Dp71在早期祖/萌芽阶段细胞的核基质分数这种无处不在的表现表明他们在核结构和细胞周期调控12的作用。我们重新编程野生型iPS细胞表达Dp71;然而，其在营养不良的iPSCs的缺席不抑制该重新编程或营养不良的细胞的多能性潜能。总之，营养不良基因突变是保守的重新编程iPSCs的;使它成为一个有效的工具来模拟营养不良的心肌病。

心肌病死亡的患者Duchenne型和Becker型肌营养不良症（MD）的第二大原因。虽然在X连锁的抗肌萎缩蛋白基因突变发生在1:3,500男胎，很少有人知道分子和细胞事件，导致进行性心肌损害。从肌营养不良患者衍生的人诱导多能干细胞已成为一种新的工具来研究潜在的疾病的机制和用于药物筛选1,2。 皮肤活检或血液样本的预期可能不适劝阻青少年患者和/或其监护人放弃了参与研究的同意。尿液样本是体细胞是易于进行重新编程的方法的非侵入性的来源。我们最近已表明，从肌营养不良的患者采集的尿液细胞可以是培养的，高效地重新编程成使用逆转录病毒转导的山中因素iPSCs的（的Oct3 / 4，SOX2，Klf4和c-Myc的; OSKM）1。的逆转录病毒基因递送的缺点是随机整合的重新编程的基因到宿主染色体中。为了克服这个限制，我们已经使用了非整合型仙台病毒为尿细胞重编程。 这个协议的细节由肌营养不良患者中分离尿细胞然后可以分化成心肌细胞或其它细胞类型用于进一步研究的仙台病毒重新编程。该协议也可以适用于其他患者特定的疾病。

<table><tbody><tr><td colspan="4">&lt;strong&gt;Materials&lt;/strong&gt;</td></tr><tr><td>4 Oz. Specimen Cup with Lid</td><td>STL Medical Supply</td><td>M9AMSAS340</td><td>For Urine Sample Collection</td></tr><tr><td>15 ml BD-Falcon Tubes</td><td>Fisher Scientific</td><td>352097</td><td></td></tr><tr><td>50 ml BD-Falcon Tubes</td><td>Fisher Scientific</td><td>352098</td><td></td></tr><tr><td>Round Glass Coverslips</td><td>Fisher Scientific</td><td>12-545-81</td><td></td></tr><tr><td>CytoTune-iPS Sendai Reprogramming Kit</td><td>Life Technologies</td><td>A1378-001</td><td></td></tr><tr><td>PBS, pH 7.4</td><td>Life Technologies</td><td>10010-023</td><td></td></tr><tr><td>Pen-Strep w/o Glutamine&amp;nbsp;</td><td>Life Technologies</td><td>15140-122</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>RPMI Medium 1640</td><td>Life Technologies</td><td>11875-093</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>B-27 Supplement w/Insulin (50x)</td><td>Life Technologies</td><td>17504-044</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>B-27 Supplement w/o Insulin (50x)</td><td>Life Technologies</td><td>0050129SA</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>DMEM/F12 (1:1)</td><td>Life Technologies</td><td>11330-032</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Versene, 1:5,000</td><td>Life Technologies</td><td>15040066</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>bFGF (10 &amp;micro;g)</td><td>Life Technologies</td><td>13256-029</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Cell Counter Cartridges</td><td>Life Technologies</td><td>C10228</td><td></td></tr><tr><td>Knockout Serum (500&amp;nbsp;ml Bottle)</td><td>Life Technologies</td><td>10828-028</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Recovery Cell Culture Freezing Medium</td><td>Life Technologies</td><td>12648-010</td><td></td></tr><tr><td>Keratinocyte-SFM (1x), Liquid</td><td>Life Technologies</td><td>17005-042</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>DMEM, High Glucose, Glutamax</td><td>Life Technologies</td><td>10566-016</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Ham&#039;s F-12 Nutrient Mix&amp;nbsp;</td><td>Life Technologies</td><td>11765-054</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>EGF Recombinant Human Protein, Liquid Form</td><td>Life Technologies</td><td>PHG0311L</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Insulin, Human Recombinant, Zinc Soln</td><td>Life Technologies</td><td>12585-014</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>TeSR-E8</td><td>Stem Cell Technologies</td><td>5940</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>ROCK Inhibitor (Y-27632)</td><td>Selleck</td><td>S1049</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Matrigel</td><td>BD Biosciences</td><td>354277</td><td>hESC Qualified Matrix, LVED Free</td></tr><tr><td>RNeasy Mini Kit&amp;nbsp;</td><td>Qiagen</td><td>74104</td><td></td></tr><tr><td>iScript cDNA Synthesis Kit</td><td>Bio-Rad</td><td>170-8890</td><td></td></tr><tr><td>DreamTaq Green PCR Master Mix (2x)</td><td>Thermo-Scientific</td><td>K1081</td><td></td></tr><tr><td>FBS-Qualified</td><td>Sigma Aldrich</td><td>F6178</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Adenine Bioreagent</td><td>Sigma Aldrich</td><td>A2786-5G</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Cholera Toxin from &lt;em&gt;Vibrio cholerae&lt;/em&gt;</td><td>Sigma Aldrich</td><td>C8052-.5MG</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Hydrocortisone</td><td>Sigma Aldrich</td><td>H0888-1G</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Holo-transferrin from human</td><td>Sigma Aldrich</td><td>T0665-50MG</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>3,3&#039;,5-Triiodo-L-thyronine sodium salt</td><td>Sigma Aldrich</td><td>T6397-100MG</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>DAPI&amp;nbsp;</td><td>Santa Cruz Biotechnology</td><td>SC-3598</td><td></td></tr><tr><td>Fluoromount Aquous mounting medium</td><td>Sigma Aldrich</td><td>F4680</td><td></td></tr><tr><td>16% Paraformaldehyde</td><td>Alfa-Aesar</td><td>43368</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Antibodies&lt;/strong&gt;</td></tr><tr><td>Mouse anti CD44 - labeled with FITC&amp;nbsp;</td><td>BD Biosciences</td><td>560977</td><td></td></tr><tr><td>Mouse anti CD146 - labeled with PE</td><td>BD Biosciences</td><td>561013</td><td></td></tr><tr><td>Mouse anti CD73 - labeled with PE</td><td>BD Biosciences</td><td>561014</td><td></td></tr><tr><td>Mouse anti-&amp;alpha;-smooth muscle actin</td><td>Sigma Aldrich</td><td>A2547</td><td></td></tr><tr><td>Mouse anti-Vimentin</td><td>Abcam</td><td>ab8978-100</td><td></td></tr><tr><td>Mouse Anti - TRA-1-81</td><td>Life Technologies</td><td>411100</td><td></td></tr><tr><td>Rabbit anti Oct 3/4</td><td>Santa Cruz Biotechnology</td><td>SC-9081</td><td></td></tr><tr><td>Mouse Anti Dystrophin</td><td>Leica Biosystems</td><td>NCL-DYSB</td><td></td></tr></tbody></table>

generation of induced pluripotent stem cells from muscular dystrophy patients: efficient integration-free reprogramming of urine derived cells

营养不良性心肌病是肌营养不良症的了解甚少的后果。产生从患者肌营养不良诱导多能干细胞（iPS细胞）可以在体外疾病模型系统的宝贵的蜂窝源和可用于药物的筛选研究。患者来源的尿细胞已被用于在成功的重编程为诱导性多能干细胞，以模拟营养不良的心肌1。寻址整合载体系统的安全问题，我们提出了一个协议使用非整合型仙台病毒载体为山的转导因子成从患者肌营养不良采集的尿液细胞。该协议产生完全重新编程2-3周内克隆。所述多能干细胞是由通道-13载体的无。这些营养不良的iPSC可以分化成心肌细胞和使用，也可以研究疾病的机制或用于药物筛选。

最祖细胞从人尿中分离的阳性尿路上皮的祖和周细胞标记物如CD44，CD73，和CD146（97.37％，97.09％，和97.3％分别; 图1A和1B）。这些细胞还表达其他间充质标记物如α-平滑肌肌动蛋白和波形蛋白（ 图1B）。 RT-PCR分析提供了细胞中，所述培养物的混合群的证据存在是细胞角蛋白-7的弱表达（CK-7）和Uroplakin（UP）-Ia＆-IIIa，尿路上皮谱系的标记物（ 图1C）。 所述重新编程步骤在图2A概括示意图。代表性图像显示出细胞的形态在整个协议（ 图2B）的不同时间点。尿细胞转化的拉长，Ⅱ型细胞（0天）成鹅卵石图案（4日）期间重新编程。 BÝ第12天，典型的多能性克隆（IPSC）被看见，并且能够维持其无饲养条件下的多能干形态（iPSC的P2）。活细胞染色为TRA-1-81识别重新编程克隆（ 图2C）。 以确认载体和转基因的多能干自由克隆的产生，RT-PCR，使用引物对的SeV病毒基因组和各外源OSKM因素进行。上调的内源性重编程因子还可以在该步骤确认。外源基因的表达是由通道13不再检测到，但上调的内源性因子看出（ 图3A）。免疫荧光染色证实对iPS细胞的多能性标记物表达（的Oct3 / 4和TRA-1-81; 图3B）。见于尿细胞中的内源性重编程基因的表达可导致这些细胞重新编程以更高的效率1，相对于其他体细胞的来源。该EXPRES抗肌萎缩蛋白基因和蛋白质的锡安是由免疫荧光染色证实，免疫印迹（WB）和RT-PCR分析（ 图3C-E）。野生型UCS和iPS细胞的抗肌萎缩蛋白免疫荧光染色展示iPS细胞明显核定位11，但很少UCS表示阳性（ 图3C）。为了验证在细胞中肌营养不良蛋白的表达，免疫印迹分析为肌营养不良蛋白显示无处不肌营养不良蛋白同种型（Dp71）是野生型的iPSC产生的主要的异构体，而营养不良的iPS细胞缺乏肌营养不良蛋白基因的表达已检测不到Dp71表达（ 图3D）。抗肌萎缩蛋白的外显子缺失突变可以在使用特异性引物侧翼的外显子缺失的营养不良的iPSC来确认。无PCR扩增是在营养不良的iPSCs的检测，而野生型的iPSC表明肌营养不良蛋白转录物的扩增（ 图3E）。 内页="总是"&gt; <img alt="图1" src="/files/ftp_upload/52032/52032fig1highres.jpg" width="700px" /> 孤立的尿细胞（UCS）的图1所示的表征尿路上皮祖，间质细胞和平滑肌细胞系。（A）流量UCS的流式细胞仪分析与探讨对尿路上皮和周细胞标志物CD44，CD73和CD146标记的抗体。 UCS的（B）免疫荧光染色与CD44，αSMA和波形。 （C）UCS基因表达谱分析表明CK-7和UP-IA和UP-IIIA弱表达相比，阳性对照样品，而α-平滑肌肌动蛋白（αSMA）的基因的表达是媲美阳性对照， <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52032/52032fig1highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> 上传/ 52032 / 52032fig2highres.jpg"WIDTH ="700像素"/&gt; 图2.重编程UC的生成iPS细胞。重编程时间轴（A）示意图。 （B）SeV的传导过程中代表UCS的不同形态（多能克隆新兴的MEF中）的图像，在第0天（SeV的转），4日（形态转换），12日和无饲养条件下的特征的iPSC克隆（IPSC -P2在基底膜）。 （C）活细胞成像TRA-1-81，以确定多能干的殖民地，在17日。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52032/52032fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52032/52032fig3highres.jpg" width="700px" /> 图3.生成病毒基因组和转基因免费iPS细胞的。（A）基因表达分析表明，在第2代（iPSC的P2），并且通过通道13（iPSC的P13）的消失SeV的基因组和OSKM转基因的存在，而内源基因表达整个持续存在。 （B）相衬图像和免疫荧光UCS和由此产生的iPS细胞进行比较的Oct3 / 4和TRA-1-81染色。 （C）的抗肌萎缩蛋白通过免疫荧光检测在野生型的iPSC相比野生型UCS，和（D）的特定同种型肌营养不良蛋白（Dp71）可以通过WB确认。 （E）RT-PCR分析用于确认特定的抗肌萎缩蛋白的外显子缺失相比，野生型UCS和iPS细胞的iPS细胞营养不良。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52032/52032fig3highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 试剂/材料/设备名称 </td><td>公司</td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> GAPDH正向引物</td><td> IDT公司</td><td>在注释中给出序列</td><td> GTGGACCTGACCTGCCGTCT </td></tr><tr><td> GAPDH，反向引</td><td> IDT公司</td><td>在注释中给出序列</td><td> GGAGGAGTGGGTGTCGCTGT </td></tr><tr><td> CK7的正向引物</td><td> IDT公司</td><td>在注释中给出序列</td><td> TGGTGCTGAAGAAGGATGTG </td></tr><tr><td> CK7，反向引</td><td> IDT公司</td><td>在注释中给出序列</td><td> CACGCTGGTTCTTGATGTTG </td></tr><tr><td>截至-IA-正向引物</td><td> IDT公司</td><td>在注释中给出序列</td><td> ACGTCCTACACCCACCGTGA </td></tr><tr><td>截至-IA-反向引</td><td> IDT公司</td><td>在注释中给出序列</td><td> ACCCCACGTGTAGCTGTCGAT </td></tr><tr><td>向上-IIIA-正向引物</td><td> IDT公司</td><td>以次克伊芬在评论</td><td> ACAAACAGAGGGTGGGAGGA CAG </td></tr><tr><td>截至-IIIA-反向引</td><td> IDT公司</td><td>在注释中给出序列</td><td> AGAAGGGCAGGGAGCCCAGG </td></tr><tr><td> αSMA-正向引物</td><td> IDT公司</td><td>在注释中给出序列</td><td> ACCCACAATGTCCCCATCTA </td></tr><tr><td> αSMA，反向引</td><td> IDT公司</td><td>在注释中给出序列</td><td> TGATCCACATCTGCTGGAAG </td></tr><tr><td> OCT3 / 4（外源性）-forward底漆* </td><td> IDT公司</td><td> *生命科技，Cytotune套件</td><td> CCCGAAAGAGAAAGCGAACCAG </td></tr><tr><td> OCT3 / 4（外源性）-reverse底漆* </td><td> IDT公司</td><td> *生命科技，Cytotune套件</td><td> AATGTATCGAAGGTGCTCAA </td></tr><tr><td> SOX2（外源性）-forward底漆* </td><td> IDT公司</td><td> *生命科技，Cytotune套件</td><td> ATGCACCGCTACGACGTGAG CGC </td></tr><tr><td> SOX2（外源性）-ReversË底漆* </td><td> IDT公司</td><td> *生命科技，Cytotune套件</td><td> AATGTATCGAAGGTGCTCAA </td></tr><tr><td> KLF4（外源性）-forward底漆* </td><td> IDT公司</td><td> *生命科技，Cytotune套件</td><td> TTCCTGCATGCCAGAGGAGCCC </td></tr><tr><td> KLF4（外源性）-reverse底漆* </td><td> IDT公司</td><td> *生命科技，Cytotune套件</td><td> AATGTATCGAAGGTGCTCAA </td></tr><tr><td> cMYC的（外源性）-forward底漆* </td><td> IDT公司</td><td> *生命科技，Cytotune套件</td><td> TAACTGACTAGCAGGCTTGTCG </td></tr><tr><td> cMYC的（外源性）-reverse底漆* </td><td> IDT公司</td><td> *生命科技，Cytotune套件</td><td> TCCACATACAGTCCTGGATGAT GATG </td></tr><tr><td> SeV载体（外源性）-forward底漆* </td><td> IDT公司</td><td> *生命科技，Cytotune套件</td><td> GGATCACTAGGTGATATCGAGC </td></tr><tr><td> SeV载体（外源性）-reverse底漆* </td><td> IDT公司</td><td> *生命科技，Cytotune套件</td><td>一CCAGACAAGAGTTTAAGAGA TATGTATC </td></tr><tr><td> OCT3 / 4（内生）-forward底漆</td><td> IDT公司</td><td>在注释中给出序列</td><td> CAGTGCCCGAAACCCACAC </td></tr><tr><td> OCT3 / 4（内生）-reverse底漆</td><td> IDT公司</td><td>在注释中给出序列</td><td> GGAGACCCAGCAGCCTCAAA </td></tr><tr><td> SOX2（内生）-forward底漆</td><td> IDT公司</td><td>在注释中给出序列</td><td> CAAGATGCACAACTCGGAGA </td></tr><tr><td> SOX2（内生）-reverse底漆</td><td> IDT公司</td><td>在注释中给出序列</td><td> GTTCATGTGCGCGTAACTGT </td></tr><tr><td>肌营养不良蛋白 - 正向引物</td><td> IDT公司</td><td>在注释中给出序列</td><td> GGCAAAAACTGCCAAAAGAA </td></tr><tr><td>抗肌萎缩蛋白，反向引</td><td> IDT公司</td><td>在注释中给出序列</td><td> GACCTGCCAGTGGAGGATTA </td></tr></tbody></table> 小学的表1.名单滨海序列。

Watch this Scientific Journal Video about Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells at JoVE.com

Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells

This protocol entails detailed procedures for isolation of urine derived cells from muscular dystrophy patients; their efficient and rapid reprogramming through Sendai virus transduction.

从生成性肌营养不良患者诱导多能干细胞的:尿源性细胞的有效整合，免费重编程

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with ...

generation-induced-pluripotent-stem-cells-from-muscular-dystrophy

Nanyang Technological University

Research

JoVE Journal

Medicine

11.2K 次观看.  Medical College of Wisconsin. This protocol entails detailed procedures for isolation of urine derived cells from muscular dystrophy patients; their efficient and rapid reprogramming through Sendai virus transduction. 

视频: Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

使用非整合附加型质粒从冷冻血沉棕黄层诱导多能干细胞的产生

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

科研

发育生物学

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

在肌肉再生的老鼠模型中移植诱导多能干细胞衍生的中生代细胞样肌原体

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

生物工程

Exon Skipping in Directly Reprogrammed Myotubes Obtained from Human Urine-Derived Cells

Duchenne muscular dystrophy (DMD), a progressive and fatal muscle disease, is caused by mutations in the DMD gene that result in the absence of dystrophin protein. To date, we have completed an investigator-initiated first-in-human study at the National Center of Neurology and Psychiatry based on the systemic injection of the morpholino oligonucleotides which are prone to exon-53 skipping. For the effective treatment of DMD, in vitro testing with myoblasts derived from DMD patients to screen drugs and assess patient eligibility before undertaking clinical trials is thought to be essential. Very recently, we reported a new MYOD1-converted urine-derived cell (UDC) treated with the histone methyltransferase inhibitor (3-deazaneplanocin A hydrochloride), as a cellular model of DMD. The new autologous UDC might show phenocopy of the disease-specific phenotypes of DMD, leading to the application of precision medicine in a variety of muscle-related diseases. In this article, we describe a detailed protocol for efficient modelling of DMD muscle cells using MYOD1-converted UDCs along with reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting, and immunocytochemistry to evaluate the restoration of dystrophin mRNA and protein levels after exon skipping.

In this article, we describe a detailed protocol for efficient modelling of Duchenne muscular dystrophy muscle using MYOD1-converted urine-derived cells to evaluate the restoration of dystrophin mRNA and protein levels after exon skipping.

外子跳过直接重新编程的线管从人类尿衍生细胞获得

Duchenne muscular dystrophy (DMD), a progressive and fatal muscle disease, is caused by mutations in the DMD gene that result in the absence of dystrophin ...

遗传学

Direct Reprogramming of Human Fibroblasts into Myoblasts to Investigate Therapies for Neuromuscular Disorders

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Several mouse models have been created but they either do not truly represent the human physiopathology of the disease or are not representative of the broad spectrum of mutations found in humans. The immortalization of human primary myoblasts is an alternative to this limitation; however, it is still dependent on muscle biopsies, which are invasive and not easily available. In contrast, skin biopsies are easier to obtain and less invasive to patients. Fibroblasts derived from skin biopsies can be immortalized and transdifferentiated into myoblasts, providing a source of cells with excellent myogenic potential. Here, we describe a fast and direct reprogramming method of fibroblast into a myogenic lineage. Fibroblasts are transduced with two lentiviruses: hTERT to immortalize the primary culture and a tet-inducible MYOD, which upon the addition of doxycycline, induces the conversion of fibroblasts into myoblasts and then mature myotubes, which express late differentiation markers. This quick transdifferentiation protocol represents a powerful tool to investigate pathological mechanisms and to investigate innovative gene-based or pharmacological biotherapies for neuromuscular disorders.

This protocol describes the conversion of skin fibroblasts into myoblasts and their differentiation into myotubes. The cell lines are derived from patients with neuromuscular disorders and can be used to investigate pathological mechanisms and to test therapeutic strategies.

将人类成纤维细胞直接重新编程到肌细胞中，以研究神经肌肉疾病的治疗方法

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of ...

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 ...

CRISPR/Cas9 Technology in Restoring Dystrophin Expression in iPSC-Derived Muscle Progenitors

Duchenne muscular dystrophy (DMD) is a severe progressive muscle disease caused by mutations in the dystrophin gene, which ultimately leads to the exhaustion of muscle progenitor cells. Clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) gene editing has the potential to restore the expression of the dystrophin gene. Autologous induced pluripotent stem cells (iPSCs)-derived muscle progenitor cells (MPC) can replenish the stem/progenitor cell pool, repair damage, and prevent further complications in DMD without causing an immune response. In this study, we introduce a combination of CRISPR/Cas9 and non-integrated iPSC technologies to obtain muscle progenitors with recovered dystrophin protein expression. Briefly, we use a non-integrating Sendai vector to establish an iPSC line from dermal fibroblasts of Dmdmdx mice. We then use the CRISPR/Cas9 deletion strategy to restore dystrophin expression through a non-homologous end joining of the reframed dystrophin gene. After PCR validation of exon23 depletion in three colonies from 94 picked iPSC colonies, we differentiate iPSC into MPC by doxycycline (Dox)-induced expression of MyoD, a key transcription factor playing a significant role in regulating muscle differentiation. Our results show the feasibility of using CRISPR/Cas9 deletion strategy to restore dystrophin expression in iPSC-derived MPC, which has significant potential for developing future therapies for the treatment of DMD.

Here, we present a Cas9-based exon23 deletion protocol to restore dystrophin expression in iPSC from Dmdmdx mouse-derived skin fibroblasts and directly differentiate iPSCs into myogenic progenitor cells (MPC) using the Tet-on MyoD activation system.

CRISPR/Cas9在iPSC衍生肌肉预祖中恢复肌营养不良素表达的技术

Duchenne muscular dystrophy (DMD) is a severe progressive muscle disease caused by mutations in the dystrophin gene, which ultimately leads to the ...

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

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 ...

生物学

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

使用非整合方法分离成人人体皮肤纤维细胞与诱导多能干细胞的生成

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

Generation of Myospheres From hESCs by Epigenetic Reprogramming

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based therapies and for a "disease in a dish" model of human neuromuscular diseases. Major hurdles, such as low abundance and heterogeneity of the population of interest, as well as a lack of protocols for the formation of three-dimensional contractile structures, have limited the applications of stem cells for neuromuscular disorders. We have designed a protocol that overcomes these limits by ectopic introduction of defined factors in hESCs - the muscle determination factor MyoD and SWI/SNF chromatin remodeling complex component BAF60C - that are able to reprogram hESCs into skeletal muscle cells. Here we describe the protocol established to generate hESC-derived myoblasts and promote their clustering into tridimensional miniaturized structures (myospheres) that functionally mimic miniaturized skeletal muscles7.

Here, we describe a protocol based on epigenetic reprogramming of human embryonic stem cells (hESCs) toward generating a homogeneous population of skeletal muscle progenitors that under permissive culture conditions form three-dimensional clusters of contractile myofibers (myospheres), which recapitulate biological features of human skeletal muscles.

Myospheres的产生从人类胚胎干细胞通过表观遗传修饰

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based ...