Funding Sources: The University of Arizona, The Jim Himelic Foundation, and the Arizona Center for the Biology of Complex Diseases.

人iPS细胞是从从4毫米皮肤钻取活组织检查分离并通过仙台病毒介导的重编程11在内部重新编程的人真皮成纤维细胞衍生的。亚利桑那州的机构审查委员会的批准大学为对象招募和活检集合中的所有程序。 1. IPSC的文化细胞外基质表面涂层的制备<ol><li> hiPSC文化上的iMEFs日益合流前一天，准备外基质（基质胶）涂层板。 </li><li>缓慢解冻的细胞外基质的等分试样（批号相关的，按照该规格表的说明）在冰上在4℃2小时。 </li><li>在生物安全罩，使用冷枪头（储存在-20℃），加入1等份的细胞外基质的冷Dulbecco氏改良Eagle培养基/营养混合物F-12（DMEM / F12 / HEPES）根据许多特定稀释因子。 </li><li>免除〜60081;每每个新的细胞培养物井外基质L的处理的12孔板需要的。 </li><li>在室温下孵育该板进行几个小时。在这里，用3小时的培养时间。 注:将板可以立即使用或储存于4℃多达两个星期。 </li></ol> 2.将iPS细胞对IMEF饲养细胞生长到细胞外基质的传播<ol><li>关于传代当天，除去从4℃的矩阵涂板，并允许所述板到适应室温在组织培养罩1小时。 </li><li>吸从1井hiPSC培养物的6孔板上的iMEFs生长培养基并用500μL的胶原酶分解试剂代替（1毫克/毫升在基础培养基）。 </li><li>在37℃，10〜 - 30分钟，直到的iPSC菌落边缘出现小幅提升。 注:孵化时间是线依赖性和将变化。显示器在显微镜下分离。 </li><li>汽车efully吸出解离试剂和用磷酸盐缓冲盐水（PBS，pH7.4）中洗轻轻2次。 </li><li>添加1毫升室温mTeSR1完全培养基与10毫微克/毫升Rho相关的蛋白激酶抑制剂（Y-27632 ROCK抑制剂）到孔中。 注:此初始通路期间使用IMEF空调介质和mTeSR1的50:50混合物，建议并且可以是用于人iPS细胞的这种媒体转换期间的存活益处。 </li><li>使用部分放置在罩内的小相差显微镜，手动得分并挑选1 - 2 hiPSC菌落（〜700 - 1000微米直径）使用注射器和针（25克，1.5英寸）。蹬殖民地的得分件与P200的枪头的媒介。 </li><li>吸并从新盘基体处置，小心不要去打扰涂层。 </li><li>转移1ml得自老井含有得分的iPSC成涂好新的矩阵的细胞悬浮液。 </li><li>解放军在37℃CE在孵化器的板，5％ 的 CO 2。岩石在几个快，背部的往复板和侧到另一侧的运动，以均匀地分布的细胞。请勿打扰板24小时。 </li><li>执行与mTeSR1完全培养基（除了Y-27632 ROCK抑制剂不需要在步骤2.5初始电镀后），每日完整的媒体的变化。 </li></ol>矩阵涂层钢板iPS细胞3.小型传代注意:一般来说，人iPS细胞的初始传送到矩阵包被的板后，细胞应再次以类似的方式传代，以确保所有的iMEFs已被淘汰和细胞已适于馈线 - 自由的条件。 <ol><li> iPSC的汇合前一天准备新涂矩阵板在第1节的讨论。 </li><li>上通道的当天，除去从4℃孵育基质涂层板，并允许所述板到适应室温1小时在组织尽头TURE罩。 </li><li>从吸1井iPS细胞的6孔板（最初是从进纸器矩阵转换）的媒介和500μL细胞分离缓冲液进行更换。 注:此处使用的商业细胞解离缓冲液（见材料表）更适合用相比胶原酶mTeSR1介质使用。 </li><li>在室温下孵育3 - 7分钟，直到的iPSC集落的边缘出现稍微抬起。如前所述，培养时间是线依赖性和可变的。因此，监视在显微镜下的细胞解离，以确定的最佳时间。 </li><li>小心吸出解离缓冲液，并用PBS轻轻洗2次。 </li><li>添加500μL室温mTeSR1完全培养基用10ng / mL的Y-27632 ROCK抑制剂到孔中。 </li><li>代替如步骤2.6的得分菌落，用P200尖端推菌落所需数量到媒体并轻轻磨碎细胞悬液的2倍股份ainst井的一个角落，打破hiPSC殖民地变成〜50团块 - 尺寸200微米。例如，对于一个12孔板接送1 - 2菌落转移到每个1新井。传送从老的细胞悬浮液以及成12孔板的单个新井。 </li><li>摇动平板在几个快，背部的往复，并且边对边动作来均匀地分布所述细胞，并让24小时的板其余在37℃，5％CO 2培养箱。 </li><li>执行与mTeSR1完全培养基每日完整的媒体的变化。如前面提到的，这是没有必要的初始镀覆后添加Y-27632 ROCK抑制剂。 注:来自步骤3.7未使用的iPSC集落可以是在这一点上容易冻存。 </li></ol> 4.制备室载玻片和盖玻片为iPS细胞的免疫细胞化学播种注:以下协议采用4孔塑料玻片，用都是适宜12毫米盖玻片在多井的培养基E卷来支持经济有效的免疫。但是，如果使用较大的井和盖玻片大小的期望能够提高介质的卷。 <ol><li>一天前hiPSC汇合，准备室玻片新涂与细胞外基质的第1节中讨论补充足够的基质溶液（〜300 - 每4井室幻灯片以及500微升），充分大衣每良好，没有任何忧虑蒸发。可替代地，将1清洁和灭菌盖玻片到24孔板的每个孔中，涂层用500微升的细胞外基质，并确保盖玻片不漂浮在溶液的顶部之后。 </li><li>上通道的一天，从4℃除去涂覆室载玻片/盖玻片并允许驯化至室温在组织培养罩1小时。 </li><li>从1井人iPS细胞的12孔板的吸出培养基并用500μL的解离缓冲液替换。 </li><li>孵育在室温坦佩叉涂抹了〜3 - 7分钟，直到的iPSC菌落边缘出现小幅提升。 </li><li>小心吸出解离缓冲液，并用10纳克/毫升Y-27632 ROCK抑制剂添加室温mTeSR1完全培养基的1毫升到孔中。 </li><li>使用放置在组织培养罩内的小相差显微镜，手动选择一个4孔腔式载玻片或1新盖玻片1集落/新井待镀（〜1000 - 1500微米的直径），使用P200的吸管尖通过推它从板表面到培养基中。 </li><li>从新室幻灯片/盖玻片吸矩阵，小心不要去打扰涂层。 </li><li>针对很好地打破了hiPSC殖民地的一个角落2次轻轻磨碎细胞悬液成〜50团块 - 尺寸200微米。转移的细胞悬液250μL的从旧井到4孔腔式载玻片/盖玻片的每个新井。 </li><li>带来每一个新的容量以及高达500μL与mTeSR1续癌宁10毫微克/毫升Y-27632 ROCK抑制剂。 </li><li>放置在孵化室滑动/盖玻片，在37℃，5％ 的 CO 2。 </li><li> 24小时之后，用mTeSR1完全培养基每日完整的媒体的变化。再次如前面提到的，添加Y-27632 ROCK抑制剂的初始镀覆后是没有必要的。 </li></ol> 5.准备为iPS细胞的免疫细胞化学<ol><li>一旦汇合，通过从孔中除去培养基并加入预热小心 4％低聚甲醛保留通过固定的hiPSC菌落（〜300微升/孔的4孔腔式载玻片/盖玻片）。在室温下孵育20分钟。 </li><li>适当地根据机构生物危害和化学安全指引弃多聚甲醛。 </li><li>洗涤细胞3次，每次5分钟，在用PBS台式摇床。 注:在腔室玻片细胞/盖玻片可以在4℃无限期在这个阶段存储有充足的PBS。可替代地，它们可以立即用于IC卡。 </li></ol> 6.免疫染色用多能标记iPS细胞的注意:免疫显iPS细胞具有多能性标准的抗体。作为一个例子，在这里细胞是双染色的抗体针对一种1表面和以顺序方式1的细胞内抗原如下:抗阶段特异性胚胎抗原-4（SSEA4）用抗八聚体结合蛋白4（OCT- 4）和抗TRA-1-60具有抗SRY相关HMG盒基因2（SOX2）（请参阅抗体细节材料表）。 <ol><li>从孔中吸出PBS并在500微升/孔封闭溶液添加不含Triton-X-100。孵育在室温下1小时。 </li><li>准备预先确定的浓度（这里，用1:200）的表面的抗体，无论是SSEA4或TRA-1-60，通过在封闭液不含Triton X-100的稀释。 <ol><li>对于玻片，准备了4孔腔式滑动300微升/孔通过混合封闭液上方和抗SSEA4或TRA-1-60一抗的1.5微升的298.5微升。 </li><li>为盖玻片，通过混合封闭液和抗SSEA4或TRA-1-60一抗的1微升199微升制备40微升/盖玻片。 </li></ol></li><li>吸从玻片封闭液和分配的主要抗体为适当的井。对盖玻片将一块封口膜牢牢培养皿内并分配在上面作为一个液滴的主抗体的40微升。使用弯曲的注射器针头和镊子小心抬起盖玻片出培养的井，并放置电池侧向下到抗体上的封口膜的压降。在4℃孵育过夜。 </li><li>第二天早上，洗细胞3次，5 - 每10分钟，通过从腔室滑动除去抗体并在PBS中加入。对于盖玻片，抬起一次性封口膜，并把它早在24孔板，细胞面朝上的好。小心加入PBS。 </li><li>在封闭液500:1，准备适当的二级抗体。 <ol><li>对于腔室玻片，准备300μL/孔的4孔室载玻片通过混合499微升封闭液和1微升的山羊抗小鼠IgG3的的Alexa Fluor 488（为SSEA4）或1微升的山羊抗小鼠IgM的Alexa Fluor 488（用于TRA-1-60）。 </li><li>为盖玻片，通过混合封闭液499微升制备40微升/盖玻片和1微升的山羊抗小鼠IgG3的的Alexa Fluor 488（为SSEA4）或1山羊μL的抗小鼠IgM的Alexa Fluor 488（为TRA-1-60 ）。 </li></ol></li><li>从室滑动吸出PBS中并分配所述第二抗体为适当的孔中。为盖玻片，下列步骤6.2，翻转的盖玻片上，在室温下放置在封口膜2小时抗体溶液。 注意:如果使用荧光标记的二级抗体，该细胞必须避光保存。放置玻片和盖玻片在黑暗的ENV孵育和洗涤过程中ironment。 </li><li>洗涤细胞3次，每次5 - 10分钟各由从腔室滑动除去抗体并在PBS中加入。为盖玻片，从封口膜小心地每一个和放回一个24孔板，电池侧上的孔中。轻轻洗涤过程中添加PBS。 </li><li>之前探测第二抗原，细胞内的OCT-4或在此情况下SOX2，在阻断用透试剂（的Triton-X-100）在室温下1小时制成溶液再次孵育。 </li><li>免疫染色为OCT-4或SOX2，只要按照在步骤6.1-6.6用于施加第一和第二抗体描述的方法。对于此协议的目的，使用OCT-4和SOX2以1:1的浓度:200和二级抗体的山羊抗兔IgG的Alexa Fluor 594以1:500。 </li><li>上述主要和次要抗体治疗后，洗细胞3次，每次5 - 10分钟各用PBS并用4染液&#39;，6&#39;-二脒基-2--phenylindole，二盐酸盐（DAPI）如果核可视化需要按照标准协议。 </li></ol> 7.准备查看<ol><li>对于腔室玻片，小心地从滑动除去上室，按照制造商的说明进行操作。然后，漂洗在蒸馏水中，添加封固剂和盖玻片启用显微镜。 </li><li>为盖玻片，反转上的安装件中置于玻璃显微镜载玻片一滴盖玻片。 </li><li>标准或共聚焦荧光显微镜下的图像。 </li></ol>

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The authors have nothing to disclose.

这里介绍的系统协议提供了一个节省时间和成本效益的方法，在一个按比例缩小的培养技术的形式，专门通过免疫组化来支持有效的多能性的分析。 是所描述的方法的主要优点如下。需要传统上超过3至4个通道，以过渡从饲养层的iPSC馈线-自由培养条件，以消除使用散装解离技术后剩余的残余的iMEFs和典型单层形态出现12,13。与此相?...

重新编程人成年体细胞到诱导多能干细胞（iPS细胞）提供了一种方法，以获得患者特异性的细胞的潜在无限供给到研究疾病1,2。扼要体外的疾病表型将使得可信检验与疾病相关的细胞和分子机制，增强药物发现和个性化药物3。此外，人iPS细胞（iPS细胞）提供导出其可以用作唯一的资源来替换死亡或功能失调的细胞，并在几个障碍4的范围内，恢复功能的特定细胞类型的可能性。 以使用上述应用程序的iPSC的重要前提是，确保在培养膨胀过程中他们的多能性和分化状态被维持。通常情况下，techniq的UE，如流式细胞术，Western印迹，聚合酶链反应和功能分析，这需要大量的细胞和专用设备，用于IPSC的多能性6，7，8，9，10的详细的分析。然而，iPS细胞"未分化状态的常规评估可能有效地通过这些细胞的有限传播专门为免疫细胞化学（ICC）来实现，因此，涉及减少时间和资源。 最新进展允许的iPSC的所限定的无血清条件下生长的，它是在需要的鼠成纤维细胞饲养层和含血清介质常规培养系统一个显著改善。然而，目前的文献不包括描述如何尽快过渡馈线iPSCs的逐步明确协议层到无饲养系统。 在此背景下，本协议系统详细说明了如何生长在辐照的小鼠胚胎成纤维细胞人iPS细胞（IMEF）饲养层可以是（1），适于在无血清培养基中传播，和（2）在小规模培养的特别支持稳健免疫细胞化学分析。总体而言，这种方法代表了用于传播在无血清条件人类iPS细胞用于确认使用免疫细胞化学在常规基础上它们的多能及时和成本有效的方法。

<table><tbody><tr><td>DMEM-F12/HEPES</td><td>Life Technologies</td><td>11330032</td><td></td></tr><tr><td>Knockout Serum Replacement</td><td>Life Technologies</td><td>10828028</td><td></td></tr><tr><td>L-Glutamine</td><td>Life Technologies</td><td>25030081</td><td></td></tr><tr><td>MEM-NEAA</td><td>Life Technologies</td><td>11140050</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Life Technologies</td><td>21985023</td><td></td></tr><tr><td>Recombinant Human FGF-Basic</td><td>Cell Sciences</td><td>CRF001B</td><td></td></tr><tr><td>Y-27632 ROCK Inhibitor</td><td>R&amp;amp;D</td><td>1254</td><td></td></tr><tr><td>Collagenase Type IV</td><td>Life Technologies</td><td>17104019</td><td></td></tr><tr><td>Matrigel hESC-qualified Matrix</td><td>Corning</td><td>354277</td><td></td></tr><tr><td>mTeSR1 Basal Medium</td><td>StemCell Technologies</td><td>05850</td><td></td></tr><tr><td>mTeSR1 5x Supplement</td><td>StemCell Technologies</td><td>05850</td><td></td></tr><tr><td>Gentle Cell Dissociation Buffer</td><td>StemCell Technologies</td><td>07174</td><td></td></tr><tr><td>0.1 M PO&lt;sub&gt;4&lt;/sub&gt; Buffer</td><td>In-House</td><td>n/a</td><td></td></tr><tr><td>Paraformaldehyde, prill</td><td>Electron Microscopy Sciences</td><td>19202</td><td></td></tr><tr><td>1x Phosphate Buffered Saline</td><td>n/a</td><td>n/a</td><td></td></tr><tr><td>Normal Goat Serum</td><td>Life Technologies</td><td>16210072</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A2153</td><td></td></tr><tr><td>Triton-X-100</td><td>Sigma-Aldrich</td><td>X100</td><td></td></tr><tr><td>Oct-4A (C30A3) Rabbit mAb,&lt;br/&gt; Sox2 (D6D9) Rabbit mAb,&lt;br/&gt; SSEA4 (MC813) Mouse mAb,&lt;br/&gt; TRA-1-60(S) (TRA-1-60(S)) Mouse mAb</td><td>Cell Signaling&lt;br/&gt; Cell Signaling&lt;br/&gt; Cell Signaling&lt;br/&gt; Cell Signaling</td><td>2840&lt;br/&gt; 3579&lt;br/&gt; 4755&lt;br/&gt; 4746</td><td>Alternatively, a combination of 6 pluripotency primary antibodies can be purchased together as a kit in Catalog #9656</td></tr><tr><td>Goat anti-Ms IgM Alexa Fluor 488</td><td>Life Technologies</td><td>A21042</td><td></td></tr><tr><td>Goat anti-Ms IgG3 Alexa Fluor 488</td><td>Life Technologies</td><td>A21151</td><td></td></tr><tr><td>Goat anti-Rb IgG Alexa Fluor 594</td><td>Life Technologies</td><td>A11037</td><td></td></tr><tr><td>Multiwell Cell Culture Plates</td><td>Fisher Scientific&amp;nbsp;</td><td>0720080/0720081</td><td>Available in 6, 12, 24, 48, 96 well sizes</td></tr><tr><td>Chamber Slides</td><td>Fisher Scientific&amp;nbsp;</td><td>12 565 21</td><td>Available in Glass or Permanox Plastic in 1, 2, 4, 8, 16 well sizes</td></tr><tr><td>Coverglass for growth</td><td>Fisher Scientific&amp;nbsp;</td><td>12 545 82</td><td>Available in 12, 15, 18, 22 and 25 mm sizes</td></tr></tbody></table>

small-scale propagation of human ipscs in serum-free conditions for routine immunocytochemical characterization

There is great interest in utilizing human induced pluripotent stem cells (hiPSCs) for disease modeling and cell therapeutics due to their patient specificity and characteristic stemness. However, the pluripotency of iPSCs, which is essential to their functionality, must be confirmed before these cells can be used in such applications. While a rigorous characterization of iPSCs, through different cellular and functional assays is necessary to establish their pluripotency, routine assessment of pluripotency maintenance can be achieved more simply and effectively through immunocytochemical techniques. Here, we present a systematic protocol for culturing hiPSCs, in a scaled-down manner, to particularly facilitate the verification of their pluripotent state using immunocytochemistry. More specifically, this methodology encompasses an efficient and cost-effective means of growing iPSCs in serum-free conditions and plating them on small chamber slides or glass coverslips ideal for immunocytochemistry.

这个协议提供的iPSCs的人类如何可以从饲养层转移至无饲养条件下，并随后传播以有限的方式来具体实现，用于确认多能性维持成本效益的免疫细胞化学的逐步描述。 图1显示了该协议的示意图。 图2A示出hiPSC菌落在6孔板的iMEFs生长。这些殖民地表现出有确定的边界和密相明亮的中心典型形态。 如图2B所示，在12孔板的iPSC移交后馈线-自...

Watch this Scientific Journal Video about Small-scale Propagation of Human iPSCs in Serum-free Conditions for Routine Immunocytochemical Characterization at JoVE.com

Small-scale Propagation of Human iPSCs in Serum-free Conditions for Routine Immunocytochemical Characterization

Regular characterization of induced pluripotent stem cells (iPSCs), to ascertain maintenance of their pluripotent state, is an important step before these cells are used for other applications. Here we describe a method for the small-scale propagation of human iPSCs specifically designed to enable their easy and routine characterization via immunocytochemistry.

在常规免疫细胞化学鉴定无血清条件下人类iPS细胞的小规模的传播

There is great interest in utilizing human induced pluripotent stem cells (hiPSCs) for disease modeling and cell therapeutics due to their patient ...

small-scale-propagation-human-ipscs-serum-free-conditions-for-routine

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

6.9K 次观看.  The University of Arizona. Regular characterization of induced pluripotent stem cells (iPSCs), to ascertain maintenance of their pluripotent state, is an important step before these cells are used for other applications. Here we describe a method for the small-scale propagation of human iPSCs specifically designed to enable their easy and routine characterization via immunocytochemistry.

视频: Small-scale Propagation of Human iPSCs in Serum-free Conditions for Routine Immunocytochemical Characterization

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

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

从人体外周血单个核细胞融合，无诱导多能干细胞的生成方法游离型载体

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

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 ...

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 ...

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted for research in regenerative medicine and are currently in clinical trials for eye diseases, diabetes, heart diseases, and other disorders. The potential to differentiate into specialized cell types coupled with the recent advances in genome editing technologies including the CRISPR/Cas system have provided additional opportunities for tailoring the genome of iPSC for varied applications including disease modeling, gene therapy, and biasing pathways of differentiation, to name a few. Among the available editing technologies, the CRISPR/Cas9 from Streptococcus pyogenes has emerged as a tool of choice for site-specific editing of the eukaryotic genome. The CRISPRs are easily accessible, inexpensive, and highly efficient in engineering targeted edits. The system requires a Cas9 nuclease and a guide sequence (20-mer) specific to the genomic target abutting a 3-nucleotide &#34;NGG&#34; protospacer-adjacent-motif (PAM) for targeting Cas9 to the desired genomic locus, alongside a universal Cas9 binding tracer RNA (together called single guide RNA or sgRNA). Here we present a step-by-step protocol for efficient generation of feeder-independent and footprint-free iPSC and describe methodologies for genome editing of iPSC using the Cas9 ribonucleoprotein (RNP) complexes. The genome editing protocol is effective and can be easily multiplexed by pre-complexing sgRNAs for more than one target with the Cas9 protein and simultaneously delivering into the cells. Finally, we describe a simplified approach for identification and characterization of iPSCs with desired edits. Taken together, the outlined strategies are expected to streamline generation and editing of iPSC for manifold applications.

This protocol describes in detail the generation of footprint-free induced pluripotent stem cells (iPSCs) from human pancreatic cells in feeder-free conditions, followed by editing using CRISPR/Cas9 ribonucleoproteins and characterization of the modified single-cell clones.

利用 CRISPR-Cas9 系统高效生成和编辑人胰细胞无饲养干细胞

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted ...

生物工程

Developing HiPSC Derived Serum Free Embryoid Bodies for the Interrogation of 3-D Stem Cell Cultures Using Physiologically Relevant Assays

Although a number of in vitro disease models have been developed using hiPSCs, one limitation is that these two-dimensional (2-D) systems may not represent the underlying cytoarchitectural and functional complexity of the affected individuals carrying suspected disease variants. Conventional 2-D models remain incomplete representations of in vivo-like structures and do not adequately capture the complexity of the brain. Thus, there is an emerging need for more 3-D hiPSC-based models that can better recapitulate the cellular interactions and functions seen in an in vivo system.
Here we report a protocol to develop a 3-D system from undifferentiated hiPSCs based on the serum free embryoid body (SFEB). This 3-D model mirrors aspects of a developing ventralized neocortex and allows for studies into functions integral to living neural cells and intact tissue such as migration, connectivity, communication, and maturation. Specifically, we demonstrate that the SFEBs using our protocol can be interrogated using physiologically relevant and high-content cell based assays such as calcium imaging, and multi-electrode array (MEA) recordings without cryosectioning. In the case of MEA recordings, we demonstrate that SFEBs increase both spike activity and network-level bursting activity during long-term culturing. This SFEB protocol provides a robust and scalable system for the study of developing network formation in a 3-D model that captures aspects of early cortical development.

Here we report a protocol to develop a three-dimensional (3-D) system from human induced-pluripotent stem cells (hiPSCs) called the serum free embryoid body (SFEB). This 3-D model can be used like an organotypic slice culture to model human cortical development and for the physiological interrogation of developing neural circuits.

开发HiPSC衍生的无血清胚胎体用于使用生理相关测定的3-D干细胞培养物的询问

Although a number of in vitro disease models have been developed using hiPSCs, one limitation is that these two-dimensional (2-D) systems may not ...

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 ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.

This protocol describes a dynamic culture system to produce controlled size aggregates of human pluripotent stem cells and further stimulate differentiation in cerebellar organoids under chemically-defined and feeder-free conditions using a single-use bioreactor.

人类多能干细胞和免疫损伤的成熟小脑有机体可伸缩生成

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

人类诱导多能干细胞衍生心肌细胞中的单细胞光学作用潜在测量

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...