Name: integration free derivation of human induced pluripotent stem cells using laminin 521 matrix
Uploaded: 2017-07-07T15:00:00.000+00:00
Duration: 10 min 48 s
Description: Watch this Scientific Journal Video about Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix at JoVE.com

这项工作得到SSF（B13-0074）和VINNOVA（2016-04134）资助者的支持。

患者资料的收集和hiPS细胞的推导得到道德评估委员会，2012年3月28日，斯德哥尔摩，注册号:2012 / 208-31 / 3的批准。细胞培养步骤应在生物安全柜内进行，除非另有说明。在使用细胞时，始终使用无菌处理技术。允许介质，板和试剂在启动前达到室温。在37℃，5％CO 2高湿度条件下孵育细胞。 1.从真皮活检中分离人成纤维细胞<ol><li> 培养基的制备，消化酶，菜肴和解剖工具的涂层。 <ol><li>准备成纤维细胞培养基:44.5mL Iscove&#39;s Modified Dulbecco&#39;s Medium（IMDM），5mL胎牛血清（FBS）和500μL青霉素/链霉素（PEST）（ 见表1 ），储存在4℃。 </li><li>以1mg / mL的工作浓度制备消化酶:wei在分开的15-mL管中加入4mg分散酶和胶原酶I型粉末的gh等分试样并溶于4mL DMEM / F12 + 1％PEST中。通过将其通过0.22μm过滤器来消毒酶溶液。每次准备新鲜的酶溶液。 </li><li>在6孔组织培养板（或35mm组织培养皿）中，每孔用1mL 0.1％明胶在磷酸盐缓冲盐水（DPBS）中切片进行活检，涂一个孔。在室温下孵育30分钟。 </li><li>高压灭菌一次手术剪刀和镊子每次活检进行解剖。 </li></ol></li><li> 活检处理 注意:采集后尽可能快地处理活组织检查。在4℃下在PBS + 1％PEST中存放长达48小时。活检的大小应为2-4毫米，符合道德准证。 <ol><li>将活检转移到35毫米的盘中。将活组织检查移至新的35毫米皿中，并将活检浸没在2毫升70％乙醇中30秒。 </li><li>将活检移至第三位35毫升菜，并用1毫升无菌汉克平衡盐溶液（HBSS）和1％PEST进行洗涤。 </li><li>将活检转移到第四个35毫米皿中，加入1毫升新鲜制备的0.1％分散液。 </li><li>使用手术剪刀和镊子将皮肤活检切成盘中1-2mm 3片，然后将包括分泌酶溶液的活检片转移到15mL管中。加入另外2 mL的分散液。 </li><li>用1 mL分散液冲洗35 mm的培养皿并转移到15 mL管中。 </li><li>在4℃孵育活检过夜。 </li><li>加入4 mL 0.1％胶原酶Ⅰ至37℃孵育4 h。 </li><li>将消化的细胞离心300×g 3分钟，抽出上清液并将消化物重悬于2mL成纤维细胞培养基中。 </li><li>从6孔组织培养板中取出明胶溶液。将包括任何剩余片段的细胞悬浮液转移到6孔组织培养板（或35μm）中m组织培养皿），用DPBS中的0.1％明胶预涂。 </li><li>每三天更换一次媒体。 </li></ol></li><li> 人类成纤维细胞的扩增 注意:当80％汇合时，成纤维细胞可以传代给T25（25cm 2 ）组织培养瓶（ 图2B ）。成纤维细胞通常在7天内达到80％的汇合。然而，所需时间可能会有很大差异，但不得超过4周。 <ol><li>吸出培养基，并用2.5mL DPBS洗涤一次。 </li><li>取出DPBS，加入1 mL胰蛋白酶-EDTA（0.05％），37℃孵育5 min，直至细胞呈圆形边缘，开始分离。 </li><li>加入2 mL成纤维细胞培养基，如有必要，冲洗细胞，确保细胞正确分离。将细胞溶液转移到15-mL管中，并以300xg离心3分钟。 </li><li>弃去上清液，将细胞沉淀重悬于5mL成纤维细胞培养基和平板成纤维细胞中包被细胞组织培养T25烧瓶。当在该步骤之后扩增成纤维细胞时，如上所述解离并种子12×10 3个细胞/ cm 2 。 </li><li>一旦融合的细胞准备冻结，扩大或平板进行重新编程。在任何重新编程开始之前冻结两轮成纤维细胞（参见下一部分的冷冻程序）。下通道成纤维细胞的重编程效率一般较高，第2-4代细胞最佳。然而，使用该方案已经使成纤维细胞成功重编程直到第10代。 </li></ol></li><li> 冻融成纤维细胞 <ol><li>准备新鲜的成纤维细胞冷冻培养基:90％FBS + 10％二甲基亚砜（DMSO）。保持在4°C（ 表1 ）。一旦汇合，T25组织培养瓶可以冷冻成三个冷冻管。每瓶冷冻制备500μL成纤维细胞冷冻培养基。 </li><li>冻结细胞，解离c使用血细胞计数器计数细胞并将3×10 5个细胞的部分转移至15-mL管。旋转管300×g 3分钟。吸出上清。 </li><li>将细胞沉淀重悬于500μL的4℃成纤维细胞冷冻培养基中并转移到冷冻管中。将小瓶放入冷冻容器中，并将细胞在-80℃下孵育24小时，然后将其转移至液氮以长期储存。 </li><li>要解冻细胞，将冰箱的底部浸入37°C的水中。一旦液化，将细胞悬浮液转移到15 mL管中，加入5 mL成纤维细胞培养基。旋转细胞300 xg 3 min。 </li><li>去除上清液并将细胞沉淀重悬于5 mL成纤维细胞培养基中。将细胞转移至未涂覆的T25组织培养瓶中，并在37℃下孵育。 </li></ol></li></ol> 2.成纤维细胞的SeV载体重编程<ol><li> 载体等分试样的制备 </strong&gt; 小心:在生物安全2级遏制下处理SeV。参见制造商协议和本地准则17 。病毒滴度在批次之间变化。 <ol><li>在制备等分试样之前，根据制造商的方案（ 例如 CytoTune 2.0）计算5×10 4个细胞所需的载体量。病毒滴度通常为8×10 7 - 1.5×10 8 。解冻和组合载体5:5:3（KOS MOI = 5，hc-myc MOI = 5和hKlf4 = MOI 3）。将5×10 4个细胞的重新编程计算的部分分成高压灭菌的1.5mL管。用成纤维细胞稀释病毒等分试样至250μL的最终体积用于重新编程。 注意:每个小瓶有效重新编程一个具有5×10 4成纤维细胞的24孔组织培养板的一个孔。将病毒等分试样储存在-80°C。 </li></ol></li><li> SeV载体转导 <ol><li>吸气孔切片培养基并用1mL DPBS洗涤。吸入DPBS并加入1mL胰蛋白酶-EDTA（0.05％）。在37℃孵育5分钟或直到细胞分离。 </li><li>加入2mL成纤维细胞培养基，并重悬细胞并转移到15-mL管中。离心300 xg 3 min。 </li><li>吸出上清液并将沉淀重悬于2mL成纤维细胞培养基中。 </li><li>在24孔组织培养板的一个孔中使用血细胞计数器和种子5×10 4细胞计数成纤维细胞。在37℃孵育细胞过夜。 </li><li>吸出细胞培养基，加入250μLSeV溶液，37℃孵育过夜。 </li><li>每天更换培养基至新鲜成纤维细胞培养基。允许转导的细胞生长7天，然后传播到LN-521涂覆的平板。在通过前一天准备LN-521板。涂层程序参见2.3.1。 </li></ol></li><li> 替代转导的成纤维细胞 <ol><li>制备LN-521涂层:DPBS稀释LN-521至0.63μg/ cm 2的终浓度。每60毫米组织培养皿移取2毫升LN-521溶液。使用石蜡膜密封60毫米组织培养皿。在4℃下孵育过夜。 </li><li>准备基本8中等（E8）等分试样以便于处理:48.5mL E8培养基，1mL E8补充物和500μLPEST（ 表1 ）。 </li><li>转导后7天，将细胞通过LN-521涂覆的60mm组织培养皿。加入250μL胰蛋白酶-EDTA（0.05％），37℃孵育5分钟或直到细胞分离。加入2mL成纤维细胞培养基，并以300×g离心细胞3分钟。 </li><li>吸出上清液。将沉淀重悬于2mL成纤维细胞培养基中，并在血细胞计数器中计数细胞。将来自预涂板的吸液LN-521溶液和在LN-521预涂的60mm培养皿中的5ml成纤维细胞培养基中种子4×10 4细胞。添加Rho激酶抑制剂Y-27632（ROCKi）至最终浓度为5μM。在37℃下孵育过夜。 </li><li>重要的是，第二天将介质更换为E8中等。每天更换E8中等hiPS细胞集落将在2-3周内出现。 </li></ol></li></ol> 3.采摘殖民地和hiPS细胞扩增注意:以下步骤是在立体显微镜下在生物安全柜之外完成的。推荐使用发丝网和程序口罩。小心不要污染细胞培养。 <ol><li>在采摘前一天准备LN-521涂层组织培养24孔板。 DPBS稀释LN-521 0.63μg/ cm 2 。将250μL的LN-521溶液移至24孔组织培养板的一个孔中。密封板使用石蜡膜。在4℃下孵育过夜。 注意:当大小达到直径1毫米的大小时，殖民地已准备好挑选。殖民地应显示锋利的边缘，并以均匀的单株生长olayers（ 图2D ）。 </li><li>吸出LN-521溶液，并将500μLE8加入24孔组织培养板的一个孔中。 </li><li>用立体显微镜下的网格像格子的手术刀将殖民地切成小块。机械地从盘中刮下细胞片并使用200μL移液管将片材转移到制备好的孔中（ 图2D-2D ）。选择殖民地分成不同的井。 </li><li>允许细胞在不改变培养基的情况下附着48小时。此后，每天更换E8。 </li><li>当菌落达到&gt; 5mm的大小时，将细胞作为单细胞酶促传代至LN-521涂层板的新孔。 </li><li>从细胞吸出细胞培养基，用250μLDPBS洗涤。 </li><li>吸气DPBS。加入250μL解离试剂，37℃孵育3 min。 </li><li>观察细胞已经开始向上。分离通过用解离试剂冲洗细胞几次，从板上取出细胞。 </li><li>向15 mL管中加入500μLE8培养基。将解离试剂中的细胞转移到管中，并以300×g离心3分钟。 </li><li>从LN-521预涂井抽出LN-521溶液，加入500μL室温E8培养基。 </li><li>吸出上清并将细胞沉淀重悬于500μL的E8培养基中。 </li><li>使用血细胞计数器计数细胞，并在制备的LN-521预包被孔（1.25×10 4 - 2.5×10 4个细胞/ cm 2 ）中种2.5×10 4 -5×10 4个细胞。细胞培养格式可以轻松地放大到适合预期目的。 </li><li>将ROCKi添加到终浓度为10μM。孵育细胞在37°C。 </li><li>每天更换E8中等细胞通常在4-6天后通过。当约90％融合时，将上述细胞作为单细胞进行酶促传代。 注意:重编程载体在hiPS细胞扩增期间逐渐稀释，在第12代后无法检测到。通常重新编程的细胞通常停止在6-10周围增殖或丧失其特征性多能干细胞形态（ 图3B ）。 </li><li> hiPS细胞的冻融 <ol><li>为了冷冻细胞，如上所述用单细胞酶促离解细胞，在血细胞计数器中计数细胞，并将2.5×10 5个细胞转移到含有1mL E8培养基的15-mL管中。以300 xg离心3分钟。吸出上清液。 </li><li>将细胞沉淀重悬于250μL4℃的PSC冷冻细胞中，并转移至低温条件下。将小瓶放入冷冻容器中，并将细胞在-80℃下孵育24小时，然后将其转移至液氮以进行长期储存。 </li><li>为了解冻细胞，准备24孔组织培养物的预涂LN-521孔通过吸取LN-521溶液并加入250μLE8培养基。将冰箱的底部浸入37°C的水中，直到只剩下一个小冰柱。 </li><li>向冷冻机中加入250μLE8培养基，并将细胞悬浮液转移到15 mL试管中，加入1 mL E8培养基。旋转细胞300 xg 3 min。 </li><li>取出上清液，将细胞沉淀重悬于250μL室温E8培养基中。将细胞悬浮液转移到准备好的孔中，加入1％细胞活力补充剂或10μMROCKi。在37℃下孵育组织培养板。 </li></ol></li></ol>

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+pluripotent+stem+cell+culture:+considerations+for+maintenance,+expansion,+and+therapeutics.">Human pluripotent stem cell culture: considerations for maintenance, expansion, and therapeutics.</a> Cell Stem Cell. 14 (1), 13-26 (2014).</li><li>Jacobs, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Higher-Density+Culture+in+Human+Embryonic+Stem+Cells+Results+in+DNA+Damage+and+Genome+Instability.">Higher-Density Culture in Human Embryonic Stem Cells Results in DNA Damage and Genome Instability.</a> Stem Cell Reports. 6 (3), 330-341 (2016).</li><li>Schlaeger, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+non-integrating+reprogramming+methods.">A comparison of non-integrating reprogramming methods.</a> Nat Biotech. 33 (1), 58-63 (2015).</li><li>. Ideal Xeno-Free Culture Conditions For Human Fibroblasts That Facilitate Efficient Reprogramming Available from: <a target="_blank" href="https://tools.thermofisher.com/content/sfs/posters/xeno-free-culture-conditions-fibroblasts-poster.pdf">https://tools.thermofisher.com/content/sfs/posters/xeno-free-culture-conditions-fibroblasts-poster.pdf</a> (2016)</li><li>Trokovic, R., Weltner, J., Noisa, P., Raivio, T., Otonkoski, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+negative+effect+of+donor+age+and+time+in+culture+on+the+reprogramming+efficiency+into+induced+pluripotent+stem+cells.">Combined negative effect of donor age and time in culture on the reprogramming efficiency into induced pluripotent stem cells.</a> Stem Cell Res. 15 (1), 254-262 (2015).</li></ol>

作者没有报告利益冲突。

该协议的预期结果是成功生成几个克隆导出的hiPS细胞系。重要的是，这里描述的用于维持和扩增所建立的hiPS细胞的方法是可靠的，并且可以以少量干细胞培养的经验进行。已知使用ROCKi与LN-521基因进行单酶细胞传代，将细胞维持在核型正常，多能性和易于分化，同时避免诱导的异质性，基于集落的传代可刺激10,19,20。在LN-521上培养的hiPS细胞可以作为单细胞传代，而不添加ROCKi 10 ...

由于Takahashi 等人首先推导出hiPS细胞系1,2 ，hiPS细胞为疾病建模，药物发现和用于在再生医学中产生细胞疗法的源材料提供了有用的工具3 。长期以来，HiPS细胞培养依赖于与成纤维细胞饲养细胞4,5或基质胶6和含有胎牛血清（FBS）的培养基配制物的共培养。批次差异是这些文化条件未定义性质的常见后果，导致不可预测的变化，这是这些协议不可靠性的主要原因7 。定义的培养基如基本8（E8） 8和定义的细胞培养基质例如LN-521 9的开发允许s用于建立高度可重复的方案，并有助于稳健的产生和维持均质的hiPS细胞7,8,9,10。 一体化免费重编程技术的发展已经跨越式发展。最初，重编程依赖于随机整合到基因组中的逆转录病毒载体，对基因组完整性具有破坏性影响11 。重编程方法的进展包括开发基于RNA的载体。 RNA载体相对于基于DNA的重编程方法具有优势，因为通过基因组重组的非预期整合是不可能的12 。 SeV载体通过没有DNA相11的单链RNA提供高和短暂的外源因子表达。由SeV交付的重编程载体在整个细胞扩增过程中被稀释，最终从培养物中脱落，提供了免打印方式的重编程。此后，多能性的维持依赖于多能性基因的内源性表达2 。 作为开创性的基于hiPS细胞的疗法正在开始进入临床试验，标准化批次，再现性和安全性的要求是解决13的重要问题。因此，应避免动物产地。例如，异种产品的使用与非人类病原体污染的风险相关。此外，在动物衍生的培养物组分存在下培养的细胞已经显示出将非人类硅酸掺入可能导致衍生细胞免疫原性的细胞膜中14 。因此，消除异种产品的需要对于任何未来的临床追求都是必要的。此协议适用于xe免疫和定义的培养在保持hiPS细胞移动细胞更接近临床依从性。 该协议描述了从成纤维细胞产生标准化的hiPS细胞的一致，高度可重现和易于使用的方法。它还提供了一种用户友好的文化系统，用于维护已建立的hiPS细胞。该协议已被用于在Karolinska机构的瑞典国家人类iPS核心设施中获得超过300个hiPS细胞系，其中一些线路之前已被描述为15,16 。

<table><tbody><tr><td>Dispase</td><td>Life Technologies</td><td>171105-041</td><td>Biopsy digestion</td></tr><tr><td>Collagenase</td><td>Sigma</td><td>C0130</td><td>Biopsy digestion</td></tr><tr><td>Gelatin</td><td>Sigma</td><td>G1393</td><td>Fibroblast matrix</td></tr><tr><td>IMDM</td><td>Life Technologies</td><td>21980002-032</td><td>Fibroblast medium</td></tr><tr><td>FBS</td><td>Invitrogen</td><td>10270106</td><td>Fibroblast medium</td></tr><tr><td>Penicillin/Streptomycin</td><td>Life Technologies</td><td>15070-063</td><td>Antibiotic</td></tr><tr><td>Essential 8 media</td><td>ThermFisher Scientific</td><td>A1517001</td><td>iPS cell culture media</td></tr><tr><td>LN-521</td><td>Biolamina</td><td>LN521-03</td><td>iPS cell culture matrix</td></tr><tr><td>TrypLE select 1X</td><td>ThermoFisher Scientific</td><td>12563011</td><td>Dissociation reagent</td></tr><tr><td>Rho-kinase inhibitor Y27632</td><td>Millipore</td><td>SCM075</td><td>Rho-kinase inhibitor (ROCKi)</td></tr><tr><td>CytoTune &amp;ndash; iPS 2.0 reprogramming kit</td><td>Life Technologies</td><td>A1377801</td><td>Sendai virus reprogramming vector</td></tr><tr><td>35 mm tissue culture dish</td><td>Sarstedt</td><td>83.3900.300</td><td>Cell culture</td></tr><tr><td>60 mm tissue culture dishes</td><td>Sarstedt</td><td>83.3901.300</td><td>Cell culture</td></tr><tr><td>24 well tissue culture plates</td><td>Sarstedt</td><td>83.3922.300</td><td>Cell culture</td></tr><tr><td>T25 tissue culture flasks</td><td>VWR</td><td>734-2311</td><td>Cell culture</td></tr><tr><td>15 mL tubes</td><td>Corning</td><td>430791</td><td>Centrifuge tubes</td></tr><tr><td>1.5 mL tube</td><td>Eppendorf</td><td>0030123.301</td><td>1.5 mL tube</td></tr><tr><td>CoolCell cell LX</td><td>Biocision</td><td>BCS-405</td><td>Freezing container</td></tr><tr><td>DMSO</td><td>Sigma</td><td>D2650-100ml</td><td>Fibroblast freezing</td></tr><tr><td>Cryovials 1.8 mL</td><td>VWR</td><td>479-6847</td><td>Cryovials</td></tr><tr><td>PSC Cryopreservation Kit</td><td>Gibco</td><td>A2644601</td><td>PSC CryomediumRevitaCell supplement</td></tr><tr><td>Trypsin-EDTA (0.05%)</td><td>ThermoFisherScientific</td><td>25300054</td><td>Fibroblast dissociation enzyme</td></tr><tr><td>DMEM/F12</td><td>Life Technologies</td><td>31331-028</td><td>Digestive enzymes dilutant</td></tr><tr><td>DPBS</td><td>Life Technologies</td><td>14190-094</td><td>PBS</td></tr><tr><td>HBSS</td><td>ThermoFIsher Scientific</td><td>14025-050</td><td>Biopsy preparation</td></tr><tr><td>Haemocytometer</td><td>Sigma</td><td>BRAND, 718920</td><td>Cell counting</td></tr><tr><td>Parafilm</td><td>VWR</td><td>291-1213</td><td>Sealing plates for storage</td></tr></tbody></table>

integration free derivation of human induced pluripotent stem cells using laminin 521 matrix

无性和完全定义的条件是稳定和可重复产生均质人诱导多能干细胞（hiPS）细胞的关键参数。饲养细胞或未定义基质上的hiPS细胞的维持易受批次差异，致病性污染和免疫原性风险的影响。使用定义的重组人层粘连蛋白521（LN-521）基质与无异种素和限定的培养基制剂组合减少变异性并允许hiPS细胞的一致产生。仙台病毒（SeV）载体是一种非整合的基于RNA的系统，因此规避了与基因组完整性整合载体可能具有的潜在破坏性影响相关的问题。此外，这些载体在真皮成纤维细胞的重新编程中已经表现出相对较高的效率。此外，细胞的酶促单细胞传代促进了hiPS细胞的均匀维持，而没有实质的茎干经验文化。在这里，我们描述了一个经过广泛测试和开发的方案，重点是重现性和易用性，为从成纤维细胞中产生定义和不含异种的人类hiPS细胞提供了一种可靠和实用的方法。

从活检到hiPS细胞从活检到建立的hiPS细胞，清除重编程载体并准备表征的整个过程大约需要16周（ 图1 ）。 图2A中指定了更详细的时间表。需要大约4周来建立和扩大成纤维细胞培养。第一个hiPS细胞集落开始在仙台载体转导后约三周出现。首先通过机械采集菌落，然后以单细胞酶促传代...

Watch this Scientific Journal Video about Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix at JoVE.com

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

通过使用非整合仙台病毒（SeV）载体介导的真皮成纤维细胞的重编程，实现人诱导的多能干细胞（hiPS）细胞的稳健衍生。使用重组人层粘连蛋白521（LN-521）基质和Essential E8（E8）培养基，使用无异种素和化学定义的培养条件进行hiPS细胞维持和克隆扩增。

使用层粘连蛋白521矩阵整合人诱导多能干细胞的自由衍生

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...

integration-free-derivation-human-induced-pluripotent-stem-cells

Research

JoVE Journal

Developmental Biology

8.5K 次观看.  Karolinska Institutet. 通过使用非整合仙台病毒（SeV）载体介导的真皮成纤维细胞的重编程，实现人诱导的多能干细胞（hiPS）细胞的稳健衍生。使用重组人层粘连蛋白521（LN-521）基质和Essential E8（E8）培养基，使用无异种素和化学定义的培养条件进行hiPS细胞维持和克隆扩增。 

视频: Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

集成的无人类诱导多能干细胞的生成方法头发衍生角质形成细胞

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

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 ...

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

人类神经祖细胞和神经元小分子诱导人类胚胎干细胞的多能干高效的推导

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

神经科学

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines&#160;promising advances in cell-based modelling and therapies.

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

界定和由人多能干细胞的肝细胞样细胞的可扩展代

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

高效的推导人类心脏的前体和从多能干的小分子诱导人类胚胎干细胞的心肌细胞

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

生物学

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 ...

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 ...

生物工程

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

另类文化的人多能干细胞的生产，维护和遗传分析

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions

Human induced pluripotent stem cells (hiPSCs) can be generated with lentiviral-based reprogramming methodologies. However, traces of potentially oncogenic genes remaining in actively transcribed regions of the genome, limit their potential for use in human therapeutic applications1. Additionally, non-human antigens derived from stem cell reprogramming or differentiation into therapeutically relevant derivatives preclude these hiPSCs from being used in a human clinical context2. In this video, we present a procedure for reprogramming and analyzing factor-free hiPSCs free of exogenous transgenes. These hiPSCs then can be analyzed for gene expression abnormalities in the specific intron containing the lentivirus. This analysis may be conducted using sensitive quantitative polymerase chain reaction (PCR), which has an advantage over less sensitive techniques previously used to detect gene expression differences3. Full conversion into clinical-grade good manufacturing practice (GMP) conditions, allows human clinical relevance. Our protocol offers another methodology&#8212;provided that current safe-harbor criteria will expand and include factor-free characterized hiPSC-based derivatives for human therapeutic applications&#8212;for deriving GMP-grade hiPSCs, which should eliminate any immunogenicity risk due to non-human antigens. This protocol is broadly applicable to lentiviral reprogrammed cells of any type and provides a reproducible method for converting reprogrammed cells into GMP-grade conditions.

We describe a protocol for deriving lentiviral-based reprogrammed and characterized factor-free human induced pluripotent stem cells and conversion into putative clinical-grade conditions.

推导与表征的转基因无人类诱导多能干细胞系，并转换成定义的临床级条件

Human induced pluripotent stem cells (hiPSCs) can be generated with lentiviral-based reprogramming methodologies. However, traces of potentially oncogenic ...

Measuring the Confluence of iPSCs Using an Automated Imaging System

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence in real time has been established without the need to count cells in single cell suspension to avoid any growth perturbation. A high-content image analysis system was used to assess iPCS confluence on 4 different ECMs over time in an automated manner. Different analysis settings were used to assess cell confluence of adherent iPSCs and only a slight difference (at 24 and 48 hours with laminin) has been observed whether a 60, 80 or 100% mask was applied. We also show that laminin lead to the best confluence compared to Matrigel, vitronectin and fibronectin.

The goal of the protocol is to compare different extracellular matrix (ECM) coating conditions to assess how differential coating affects the growth rate of induced pluripotent stem cells (iPSCs). In particular, we aim to set up conditions to obtain optimal growth of iPSC cultures.

使用自动成像系统测量 iPSC 的汇合度。

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence ...