Name: alternative cultures for human pluripotent stem cell production, maintenance, and genetic analysis
Uploaded: 2014-07-24T15:00:00.000+00:00
Duration: 8 min 27 s
Description: Watch this Scientific Journal Video about Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis at JoVE.com

This work was supported by the Intramural Research Program of the National Institutes of Health (NIH) at the National Institute of Neurological Disorders and Stroke. We would like to thank Dr. Ronald D. McKay for his discussion and comments on this project.

hPSCs的单细胞为主的非殖民地式单层（NCM）的文化。 1，准备工作<ol><li>制成500毫升培养基为小鼠胚胎成纤维细胞（MEFs细胞）培养:补充有10％FBS，2mM L-谷氨酰胺和0.1mM非必需氨基酸（NEAA）的DMEM培养基中。 </li><li>分离小鼠胚胎成纤维细胞的CF1系以下例行协议16和培养的MEF上0.1％明胶包被的6孔细胞培养板中的DMEM培养基中衍生（的MEF）细胞。另外，购买股票的MEF第3代从商业资源。 </li><li>准备基质胶板。 <ol><li>稀释将5ml的hESC合格基质胶股票用5毫升的DMEM/F12培养基（冷藏至〜4℃），并存储为50％等分试样在-20℃下冷冻。解冻冷冻基底膜股票在冰箱中（〜4°C）O / N和进一步稀释基底膜在寒冷的DMEM/F12培养基（以产生2.5％的工作浓度）。 </li><l我&gt;外套6孔细胞培养板用1.5ml的2.5％基质胶每孔，存储涂层板在冰箱中O / N（注:2个星期内使用的基质胶涂层板）。 </li><li>从冰箱中取出基底膜板，使板在室温暖在细胞培养罩为10至30分钟（注:不暖板在细胞培养孵化器），并吸出DMEM培养基之前，电镀hPSCs。 </li></ol></li><li>使500毫升人类胚胎干细胞培养基中:80％DMEM/F12培养基，20％KSR，2mM的L-谷氨酰胺，0.1mM非必需氨基酸，0.1mM的β-巯基乙醇和4纳克FGF-2 /毫升。 </li><li>准备10毫升2X HPSC冷冻介质:60％FBS，20％DMSO和20μM的Y-27632在mT​​eSR1介质，通过过滤灭菌，并在1周内使用介质。 </li></ol> 2，协议1（基本）:成长HPSC殖民地上料机<ol><li>使用MEF通道数为5或6（指定为P5和P6）为HPSC培养，以获得一致的雷苏尔TS。有丝分裂通过用10微克/毫升丝裂霉素C 3小时，在37℃处理细胞灭活的MEF，洗涤细胞3倍用1X Dulbecco氏磷酸盐缓冲盐水（D-PBS），再分离的MEF用0.05％胰蛋白酶在0.53毫EDTA。可选地，照射的MEF为8000拉德的剂量用X射线照射。 </li><li>使用显微镜下的台盼蓝染色排除法计数细胞数。另外，使用自动细胞计数器。 </li><li>在6孔聚苯乙烯板板照射的MEF涂覆有0.1％明胶以每孔1.88×10 5个细胞（ 即 1.96×10 4个细胞/ cm 2）的密度。孵育的细胞在37℃和5％CO 2中放置24小时。 </li><li>吸用巴斯德吸管（:需要在这个时候没有洗涤步骤注）拆下MEF培养基。 </li><li>从以前的文化磨碎HPSC菌落成小团块，检查显微镜下的团块，以确保其大小不等，FROM 50到100微米的直径，和板对MEF饲养层顶HPSC团块于一体的含2ml HPSC介质。 </li><li>通过照片改变人类胚胎干细胞培养基，每日3〜5天，记录菌落生长，标记任何形态的改变还是有区别的殖民地（〜5％），并手动轻轻吸用巴斯德吸管取出标菌落。 </li><li>漂洗在MEFs上剩余的菌落两次（每次用D-PBS 2分钟），并孵育的菌落用2ml的1毫克/毫升胶原酶IV中HPSC介质为10至30分钟），并检查HPSC菌落从脱离MEF涂层表面（注意:用刮刀以帮助该拆卸过程只有在殖民地都紧紧地贴在板上）。 </li><li>加入5 ml HPSC培养基到每个孔中，以最小化的酶反应，转移菌落到15毫升管中，允许HPSC的菌落，以在室温下沉淀3〜5分钟，并确保菌落的沉淀由C的直接可视化细胞l在管的底部（注意:不要在这个步离心管）中。 </li><li>除去含残余的MEF的培养上清和游离的单细胞，重悬菌落用5ml人类胚胎干细胞培养基中，并重复该沉淀步骤两次。 </li><li>除去培养基，磨碎HPSC菌落成小团块，存储在1X HPSC冷冻介质（或CryoStore CS10冷冻介质），用于冷冻保存（1合流每冷冻小瓶孔）或盘上的6孔板的细胞传代。 </li></ol> 3协议2:转换HPSC从殖民地到喂料机NCM <ol><li>冲洗HPSC粒料在D-PBS后，孵育粒料用1ml 1X的Accutase的10分钟，并在显微镜下研究酶促反应，以确保单细胞解离。 </li><li>用含有10μM的Y-27632，然后离心以200×g离心在RT 5分钟轻轻重悬细胞于5ml mTeSR1介质的终止酶促反应（注意，不要使用过多的离心势力，造成细胞损伤）。 </li><li>加入5 ml mTeSR1介质的沉淀，重悬沉淀为单细胞，并过滤通过40微米的细胞过滤（以除去任何残留的细胞聚集体），解离的细胞。 </li><li>种子1.3-2×10 6个hPSCs到一个井一个6孔板中涂有2.5％的hESC合格基质胶（1.4-2.1×10 5个细胞/ cm 2），添加mTeSR1介质可达2.5毫升，并包括10μM Y-27632的培养基中（以便于初始24小时后单池镀覆）。另外，利用下面的小分子，以取代10微米的Y-27632增强单细胞接种:1微米Y型39983（ROCK I抑制剂），1微米phenylbenzodioxane（ROCK II抑制剂），1微米thiazovivin（一种新型的ROCK抑制剂）和2μM的JAK抑制剂1。 </li><li>与在第二天无药物mTeSR1介质（24小时内）更换培养基，从一个额外的解离细胞好，并计数细胞数，以确定单细胞接种效率（注:约50〜90％，可以在这一阶段可以实现单细胞电镀效率）。 </li><li>使细胞用3ml mTeSR1介质的成长作为单细胞形成单层几天。每日更换培养基。 </li><li>凭经验确定用于传代适应NCM取决于细胞密度的时间表。当细胞生长达到汇合在3天或4天，或在细胞 - 细胞边界消失后的4个小时的时间点通过。分离的细胞在1ml的Accutase的，用1至3个分束比（ 例如 ，1汇合的细胞以及接种于3个孔的6孔培养板），用于细胞传代，并以5代稳定适于NCM。 </li><li>细胞冷冻<ol><li>利用一个汇合井（〜5-6×10 6个细胞）一冷冻小瓶:解离的细胞从一个铺满以及用1毫升的Accutase的10分钟。 </li><li>稀瓦特第i个5毫升如上所述mTeSR1中，离心细胞。 </li><li>重悬沉淀中加入500μlmTeSR1培养基中，然后慢慢地在一个冷冻小瓶中加入500μl2X HPSC冷冻培养基（含有20μM的Y-27632）。可替换地，重悬含有2μM的JAK抑制剂1或1X CryoStor CS10介质中任一10微米的Y-27632或2μM的JAK抑制剂1的存在下，细胞在1X HPSC冷冻介质。 </li><li>将冷冻小瓶至冰 - 冷却的低温容器（底部填充isopranol）和低温容器立即转移到-80℃的冰箱中。 </li><li>转移细胞从-80℃冷冻到液氮罐（第二天），用于长期冷冻保存。 </li></ol></li><li>细胞解冻<ol><li>利用1超低温保存的小瓶板1以及一个6孔板中。 </li><li>在37℃水浴中10分钟，解冻细胞在相同的水浴中放置2分钟预温mTeSR1介质逐滴加入细胞到5ml预热mTeSR1介质中含有10μM的Y-27632，并如上所述离心。 </li><li>轻轻重悬细胞沉淀用2ml mTeSR1介质中含有10μM的Y-27632和缓慢的细胞转移到一个良好的预涂布有基质胶（注:避免产生气泡）。 </li><li>每天都在变化中，预计HPSC增长到3天或4达到汇合。 </li></ol></li></ol> 4协议3:转换HPSC殖民地基底膜上以NCM文化<ol><li>从冰箱取出基质胶包被的板，将板在组织培养橱中10〜30分钟，从该板的每个孔中除去培养基，并添加2预热mTeSR1培养基中，以每孔中。 </li><li>转让研磨HPSC团块从供料器培养（基本协议的步骤2.10），以上述的Matrigel板。加mTeSR1介质到每个孔中2.5毫升最终体积。 </li><li>改变介质每日3〜4天S运动，直到菌落达到80〜90％汇合。 </li><li>用于细胞传代:冲洗用D-PBS将菌落两次，治疗的细胞用1ml的2毫克/毫升分散酶在37℃下进行15至20分钟，沉淀物和传代细胞的团块。 </li><li>对于NCM文化:重复步骤3.1至3.9的协议2所述。 </li></ol> 5协议4:在hPSCs的NCM文化LN-521 <ol><li>使用的前一天解冻的重组LN-521溶液，在4℃和解冻的层粘连蛋白与1X的D-PBS（含Ca 2 + / Mg 2 +的）稀释至终浓度为使层粘连蛋白涂层溶液（LCS） 10微克/毫升。 </li><li>加入1 ml濒海战斗舰的一个很好的6孔板（注:避免干出在涂布过程中）。 </li><li>密封涂层板用封口膜，以防止蒸发和板存放在冰箱（4℃）O / N（注:1周内用板）。 </li><li>轻轻地取出用巴斯德吸管濒海战斗舰，但无uching的涂层表面，并添加2毫升mTeSR1介质到一个阱。 </li><li>所有温暖的培养液（含D-PBS），在37℃水浴25分钟。 </li><li>转换HPSC殖民地NCM <ol><li>如在协议2所述游离细胞聚集到单个细胞用Accutase。 </li><li>种子1.3-2×10 6个hPSCs成一个LN-521-包被的孔（1.4-2.1×10 5个细胞/ cm 2）无ROCK抑制剂的存在。 </li><li>改变介质每日3〜4天。 </li><li>分离的细胞在1ml的Accutase在3天或4为用1至3个分束比的下一个传代细胞。 </li></ol></li></ol> 6，协议5:NCM文化的质粒DNA转染<ol><li>适应HPSC殖民地NCM文化作为协议2描述4。 </li><li>板分离在12孔板hPSCs为7.5×10细胞密度5个细胞/孔在mTeSR1介质中的10μM的Y 2763的存在2。 </li><li>电镀后的细胞替换mTeSR1培养基4-8小时之间无药mTeSR1媒体。 </li><li>对于每个转染，稀2.5表达质粒微克（ 例如 ，pmaxGFP）和5μl脂质体2000在独立的Eppendorf管中每个的Opti-MEM的125微升低血清培养基。 </li><li> 5分钟后，混合的稀释试剂，孵育20分钟，在RT（以形成转染复合物）。 </li><li>添加250μl的转染复合物，以1毫升mTeSR1培养基每孔含有hPSCs并在CO 2培养箱中培养24小时，培养的细胞在37℃。 </li><li>检查荧光显微镜和照片为随机的实际转染效率计算下的转染效率。 </li></ol> 7，协议6:NCM文化的小分子RNA的转染<ol><li>通过加入在61毫升的Accutase每孔离解半汇合hPSCs在NCM条件下2天<html>孔板。 </li><li>加入10 ml mTeSR1培养基来稀释的酶反应和离心机在200×g离心5分钟。 </li><li>重悬细胞沉淀用mTeSR1介质，种子细胞以每孔7.5×10 5个细胞在12孔板中含有10μM的Y-27632的密度在mTeSR1介质，并让细胞附着4小时。 </li><li>与Y27632无mTeSR1培养基更换培养基。 </li><li>使用市售的microRNA（ 例如 ，非靶向miRIDIAN的miRNA转染对照标记DY547）。使用提供的试剂滴定浓度为0-160纳米，并按照制造商的指示。 </li><li>通过成像活细胞的DY547荧光显​​微镜在转染后24小时下优化转染效率对于每个浓度在HPSC线。 </li><li>计算基于DY547阳性细胞在所有成像细胞的百分比的转染效率。 </li></ol>。地幔"&gt; 8协议7:NCM文化的慢病毒载体转导<ol><li>重复步骤7.1至协议6 7.4。 </li><li>计算用于转导病毒颗粒的量:使用下面的公式:TU =（MOI x CN）/ VT，而TU表示总的数字变换单元，MOI等于感染的期望的多样性在阱（MOI或TU /细胞），CN指定单元格的每个数好，和VT表明该股的病毒滴度（TU /毫升）。例如，由于库存的病毒滴度为SMART-shRNA表达载体等于1×10 9 TU /毫升（每毫升变换单元），7.5×10 5个每孔的细胞在转导的时间，和20的预期MOI:使用15个微升股票的病毒颗粒为每口井的（注:建议这种情况下的瞬态慢病毒诱导）。 </li><li>预温300微升mTeSR1介质用10毫克/毫升的聚凝胺进行的30分钟的。 </li><li>加入15微升股票病毒颗粒的成包含聚凝胺的预热mTeSR1中，轻轻混匀的解决方案。 </li><li>用300μl预热的培养基中含有的病毒颗粒更换细胞培养基。 </li><li>经过4小时培养，考察turboGFP荧光（一个制造商的shRNA表达），以确定转染效率。 </li><li>加入300微升额外mTeSR1介质来换能顺利当细胞开始表达turboGFP。 </li><li>经过12-16小时培养，重新审视涡轮GFP的表达。 </li><li>与在72小时这些转导的细胞执行所需的后续实验。 </li></ol>

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The authors declare that they have no competing financial interests.

有两种主要的方法来培养hPSCs 体外 :常规菌落型培养物（细胞上馈线或细胞外基质）和hPSCs作为骨料未经进料器6的悬浮培养物。这两个殖民地式和悬浮培养方法的局限性包括累积的异质性和可继承的表观遗传变化。 NCM培养的基础上，无论是单细胞传代和高密度的细胞接种，代表了一种新的培养方法对HPSC生长6,18。尽管各单细胞传代的方法已被记载在文献中，但他们都不是用于常规的传播。例如，Wu和同事采用了一种单细胞传代的方法来研究各种小分子鸡尾酒HPSC增长19的影响。然而，他们最终文化产品的菌落。这样，低密度的基于单细胞传代方法仍属于集落类型的文化。关于NCM培养，高窝点性电镀变换该培养到一个新的方法，由于最终细胞产物是均匀的单层。近日，德沃夏克和他的同事使用了类似的方法，全面分析这种增长的条件下，18 HPSC属性。他们的研究结果也支持我们的主要结论。很显然，现在NCM文化是具有几个新的属性一个新兴的方法，并可以进一步修改为多能干细胞生物学的许多潜在的应用。 从NCM文化hPSCs的基本性能 可以想象的是，hPSCs下NCM文化共享许多特征具有相同类型的细胞生长成的菌落9,18。人类胚胎干细胞的标记物在hPSCs，包括Oct-4的，NANOG，SSEA-3/4，TRA-1-60，Tra（山茶）-1-81和SSEA-1，表达水平是相似的两个NCM和菌落型培养。 NCM适应细胞也保持类似的全球基因表达模式，以种植M上HPSC殖民地EF饲养层9。显然，NCM适应细胞维持多能性状态由畸胎瘤实验9,18决定。然而，不同于HPSC菌落，NCM条件下的细胞具有的特征是一致的生长曲线，细胞周期，细胞数9可预测的增长率。由于高密度的单细胞电镀，NCM条件下hPSCs呈现天2和4（ 图1B）之间呈指数增长，从而增加细胞数目由4倍带生长在MEF中HPSC菌落进行比较。长期的文化不增加电池的生产，而是增加细胞凋亡和分化压力9。 NCM还实现了最佳冷冻保存，从而在电镀解冻的细胞（协议1）使细胞快速恢复。此外，NCM方便HPSC文化的适应各种无异物协议9。在一般情况下，NCM支持hPSCs的生长，维持多能性状态，并维持该电位TiAl金属hPSCs分化成三个胚层的成体组织中。 NCM下hPSCs染色体的稳定性 在染色体稳定性方面，存在从不同的培养方法的细胞之间没有直接的比较。我们大部分的NCM培养条件下hPSCs有正常核型和基因拷贝数由G显带，基于阵列的比较基因组杂交（ACGH）和荧光原位杂交（FISH）9确定。两行（〜13％）表现出异常核型，其中一条线（ 即 WA09）表现出升高的多倍体和另一个（ES01）有细胞的20号染色体三体9 14％。目前还不清楚这些异常核型是否来自选择已经存在的前NCM文化或在NCM适应诱导突变的细胞。重要的是起始HPSC菌落或菌落的子集用于NCM培养的sh乌尔德是良好的特点在他们的同质性和染色体稳定性方面在当时的NCM适应。值得注意的是，NCM条件下异常核型的速度比报道在最近的队列分析，在此笔者透露主要聚居区式培养条件20岁以下的34％核型异常低得多。因此，我们的研究表明，我们可以成长分离的单细胞和NCM条件下保持其染色体的稳定性，不过，我们还需要使用更灵敏的方法来检查hPSCs的染色体异常在未来几年。特别是，我们需要使用更高的分辨率探头（ 即 &lt;50 KB）扫描染色体1，12，17，和20，因为一些小的病变（ 如 20q11.21扩增子）在这些经常改变染色体不能按常规进行检测核型分析和FISH 20-22。此外，不同的NCM协议的发展使我们能够识别ROBUST和安全的方法，为未来的应用。 根据不同的修改NCM文化 使用ROCK抑制剂Y-27632，已经打开了门为单细胞基础的分析。多样化的ROCK抑制剂的可用性将提供NCM-based扩展hPSCs和冷冻保存（ 图2）更多的选择。从理论上讲，任何小分子，可以显著提高单池镀覆效率，可用于促进NCM培养。 JAK抑制剂1，它的功能不同于那些ROCK抑制剂，代表这样一个例子9。利用单分子或组合的方法可以提供我们最佳的细胞生长，细胞分析，并hPSCs冷冻保存。然而，在限定的细胞外基质蛋白，如层粘连蛋白同种型LN-521，通常表示在胚胎干细胞23-25 ​​hPSCs替代NCM培养可消除小的干扰分子（ 图3）。 LN-521可能会提高分离，HPSC生存，并通过α6β1-PI3K/AKT通道24的激活维持多能性。传代hPSCs与LN-521的简单降低了细胞，使用完全确定的培养基（方案4）的各种无饲养和无异物的细胞培养系统兼容的异质性。它还提供了高通量检测的附加模块，而小分子的影响。虽然根据NCM培养于LN-521的iPSCs的保留HPSC标记，这些细胞用畸胎瘤测定和胚状体介导的多向分化的进一步表征一个面板的表达可能是必要的，以确认这些细胞的多能性状态。值得注意的是，人iPS细胞培养的层粘连蛋白亚型LN-521据报道细胞遗传学稳定（http://biolamina.com/）。但是，我们也需要应用更高分辨率和敏感的方法（如上所述）到examine在这些细胞中的染色体的稳定性。 多功能NCM文化的基因工程 NCM为基础的方法代表了简单，可靠，经济的系统，可能是特别有用的hPSCs（协议5-7）的遗传操作。它是已知的人类胚胎干细胞集落难以转染或转导，具有不同的实验室26-28之间的转染/转导的效率很大的可变性。例如，在人胚胎干细胞的转染效率为3〜菌落培养条件26下的35％。在集落的条件下BG01细胞的慢病毒转导的信号被认为是非常低9。然而，转染效率通过NCM介导率大于75％和9的转导方法的修改可以增加慢病毒介导的转导效率高达90％28。一个严密的比较研究显示，它是个ê多细胞组织中人类胚胎干细胞克隆，有助于对转染效率低9。此外，我们最近修改了小分子RNA转染协议，能够实现高转染效率（〜91％），而无需使用慢病毒（协议6和图4）。这个协议是易于使用，并且用于在3瞬时转染实验中特别有用 - 至5天的时间帧。正如我们在划定上述协议，多种因素应该被考虑的考虑，当我们优化转染/转导效率hPSCs。这些因素包括细胞的密度，浓度的质粒，慢病毒滴度，感染复数（MOI），转染/转导的持续时间，所用试剂和细胞毒作用，并用于监测转染/转导效率的方法。 我们制定并延长NCM方法对基质胶和上定义的细胞外基质文化hPSCs。这种培养方法是为了消除HPSC菌落并聚集培养通常发现的异质性的有效方法。根据NCM生长条件人多能干细胞是多能干细胞与染色体稳定。这种新的文化系统是简单和通用的HPSC维护，大规模的扩张，以及遗传操作。

hPSCs的分化走向多向成体组织的能力开辟了新的途径来治疗从谁，涉及心血管，肝，胰腺和神经系统，严重的1-4患有疾病的患者。从hPSCs派生的各种细胞类型也将提供强大的移动平台，疾病模型，基因工程，药物筛选和毒理试验1,4。确保其未来的临床药理及应用的关键问题是大量的临床级hPSCs通过体外细胞培养的一代。然而，目前的文化系统是不足或固有可变的，涉及hPSCs为菌落5,6各馈线和无饲养层培养。 殖民地式增长的hPSCs股的内细胞团哺乳动物早期胚胎（ICM）的许多结构特征。将ICM容易分化成三个胚层因为在异构信号梯度的存在，多细胞环境。因此，收购异质性在胚胎发育早期被认为是分化的必要过程，但HPSC文化的不必要的功能。在HPSC文化的异质性往往被过度凋亡信号和自发分化，由于不理想的生长条件引起的。因此，在集落类型的文化，异质细胞通常在7,8菌落的周围观察到。它也已表明，细胞在人胚胎干细胞（hESC细胞）菌落表现出不同反应到的信号分子，如BMP-4 9。此外，集落培养方法由于不可控的增长率及凋亡信号通路6,9产生低电池产量以及极低的电池回收率从冷冻保存。在最近几年，各种悬浮培养物已被开发用于培养hPSCs，particul阿尔利扩张的馈线和无基质条件6,10-13大量hPSCs的。显然，不同的文化系统都有自己的优点和缺点。在一般情况下，hPSCs的异质性表示的主要缺点在菌落型的和聚集的培养方法，这是不理想的用于递送DNA和RNA的物料进入hPSCs遗传工程6中的一个。 显然，有一个迫切需要开发出规避目前的文化方法的一些不足之处的新系统。小分子抑制剂（如ROCK抑制剂Y-27632和JAK抑制剂1）提高单细胞存活的发现铺平了道路分离，HPSC文化14,15。通过使用这些小分子，我们最近开发出一种培养方法基于非集群式分离- hPSCs 9（NCM）的增长。这种新颖的培养方法结合了单细胞传代和高密度电镀方法，使我们能够在一致的增长周期产生大量同质hPSCs无重大染色体异常9。或者，NCM培养可能与不同的小分子和定义矩阵（如层粘连蛋白），以优化的培养方法在广泛的应用中实现。在这里，我们提出几个具体的协议的基础上NCM文化和划定详细程序，基因工程。为了证明NCM协议的通用性，我们还测试了NCM的文化与不同的ROCK抑制剂和单层粘连蛋白亚型521（ 即 ，LN-521）。

<table><tbody><tr><td>Countess automated cell counter</td><td>Invitrogen Inc.</td><td>C10227</td><td>Automatic cell counting</td></tr><tr><td>Faxitron Cabinet X-ray System</td><td>Faxitron X-ray Corporation, Wheeling, IL&amp;nbsp;</td><td>Model RX-650</td><td>X-ray irradiation of MEFs</td></tr><tr><td>MULTIWELL 6-well plates</td><td>Becton Dickinson Labware</td><td>353046</td><td>Polystyrene plates</td></tr><tr><td>DMEM</td><td>Invitrogen Inc.</td><td>11965&amp;ndash;092</td><td>For MEF medium</td></tr><tr><td>Mitomycin C</td><td>Roche</td><td>107409</td><td>Mitotic inhibitor</td></tr><tr><td>Trypsin</td><td>Invitrogen Inc.</td><td>25300-054</td><td>For MEF dissociation</td></tr><tr><td>DMEM/F12</td><td>Invitrogen Inc.</td><td>11330&amp;ndash;032</td><td>For hPSC medium</td></tr><tr><td>Opti-MEM I Reduced Serum Medium&amp;nbsp;</td><td>Invitrogen Inc.</td><td>31985-062</td><td>For hPSC transfection</td></tr><tr><td>Heat-inactivated FBS</td><td>Invitrogen Inc.</td><td>16000&amp;ndash;044</td><td>Component of MEF medium</td></tr><tr><td>Knockout Serum Replacement</td><td>Invitrogen Inc.</td><td>10828&amp;ndash;028</td><td>KSR, Component of hPSC medium</td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate-Buffered Saline</td><td>Invitrogen Inc.</td><td>14190-144</td><td>D-PBS, free of Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;</td></tr><tr><td>Non-essential amino acids</td><td>Invitrogen</td><td>11140&amp;ndash;050</td><td>NEAA, component of hPSC medium</td></tr><tr><td>L-Glutamine</td><td>Invitrogen&amp;nbsp;</td><td>25030&amp;ndash;081</td><td>Component of hPSC medium</td></tr><tr><td>mTeSR1 &amp;amp; Supplements</td><td>StemCell Technologies</td><td>5850</td><td>Animal protein-free</td></tr><tr><td>TeSR2 &amp;amp; Supplements</td><td>StemCell Technologies</td><td>5860</td><td>Xeno-free medium</td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Sigma&amp;nbsp;</td><td>M7522</td><td>Component of hPSC medium</td></tr><tr><td>&lt;br/&gt; MEF (CF-1) ATCC</td><td>American Type Culture Collection (ATCC)&amp;nbsp;</td><td>SCRC-1040</td><td>For feeder culture of hPSCs</td></tr><tr><td>hESC-qualified Matrigel</td><td>BD Bioscience</td><td>354277</td><td>For feeder-free culture of hPSCs</td></tr><tr><td>Laminin-521</td><td>BioLamina</td><td>LN521-02</td><td>Human recombinant protein</td></tr><tr><td>FGF-2 (recombinant FGF, basic)</td><td>R&amp;amp;D Systems, MN</td><td>223-FB</td><td>Growth factor in hPSC medium</td></tr><tr><td>CryoStor CS10</td><td>StemCell Technologies</td><td>7930</td><td></td></tr><tr><td>Accutase</td><td>Innovative Cell Technologies</td><td>AT-104</td><td>1X mixed enzymatic solution</td></tr><tr><td>JAK inhibitor I</td><td>EMD4 Biosciences</td><td>420099</td><td>An inhibitor of Janus kinase</td></tr><tr><td>Y-27632</td><td>EMD4 Biosciences</td><td>688000</td><td>ROCK inhibitor</td></tr><tr><td>Y-27632</td><td>Stemgent</td><td>04-0012</td><td>ROCK inhibitor</td></tr><tr><td>Y-39983</td><td>Stemgent</td><td>04-0029</td><td>ROCK I inhibitor</td></tr><tr><td>Phenylbenzodioxane</td><td>Stemgent</td><td>04-0030</td><td>ROCK II inhibitor</td></tr><tr><td>Thiazovivin</td><td>Stemgent</td><td>04-0017</td><td>A novel ROCK inhibitor</td></tr><tr><td>BD Falcon Cell Strainer</td><td>BD Bioscience</td><td>352340</td><td>40 &amp;micro;m cell strainer</td></tr><tr><td>Nalgene 5100-0001 Cryo 1 &amp;deg;C</td><td>Thermo Scientific&amp;nbsp;</td><td>C6516F-1</td><td>&amp;ldquo;Mr. Frosty&amp;rdquo; Freezing Container</td></tr><tr><td>Lipofectamine 2000</td><td>Invitrogen Inc.</td><td>11668-027</td><td>Transfection reagents</td></tr><tr><td>DharmaFECT Duo</td><td>Thermo Scientific</td><td>T-2010-02</td><td>Transfection reagent</td></tr><tr><td>Non-targeting miRIDIAN miRNA Transfection Control</td><td>Thermo Scientific</td><td>IP-004500-01-05</td><td>Labeled with Dy547, to monitor the delivery of microRNAs&amp;nbsp;</td></tr><tr><td>SMART-shRNA</td><td>Thermo Scientific&amp;nbsp;</td><td>To be determined</td><td>Lentiviral vector</td></tr><tr><td>pmaxGFP</td><td>amaxa Inc (Lonza)</td><td>Included in every transfection kit</td><td>Expression plasmid for transfection control</td></tr><tr><td>Oct-4</td><td>Santa Cruz Biotechnology</td><td>sc-5279</td><td>Mouse IgG2b, pluripotent marker</td></tr><tr><td>SSEA-1</td><td>Santa Cruz Biotechnology</td><td>sc-21702</td><td>Mouse IgM, differentiation marker</td></tr><tr><td>SSEA-4</td><td>Santa Cruz Biotechnology</td><td>sc-21704</td><td>Mouse IgG3, pluripotent marker</td></tr><tr><td>Tra-1-60</td><td>Santa Cruz Biotechnology</td><td>sc-21705&amp;nbsp;</td><td>Mouse IgM, pluripotent marker</td></tr><tr><td>Tra-1-81</td><td>Santa Cruz Biotechnology</td><td>sc-21706</td><td>Mouse IgM, pluripotent marker</td></tr><tr><td>CK8 (C51)</td><td>Santa Cruz Biotechnology</td><td>sc-8020</td><td>Mouse IgG1, against cytokeratin 8</td></tr><tr><td>&amp;alpha;-fetoprotein</td><td>Santa Cruz Biotechnology</td><td>sc-8399</td><td>AFP, mouse IgG2a</td></tr><tr><td>HNF-3&amp;beta; (P-19)</td><td>Santa Cruz Biotechnology</td><td>sc-9187</td><td>FOXA2, goat polyclonal antibody</td></tr><tr><td>Troponin T (Av-1)</td><td>Thermo Scientific</td><td>MS-295-P0</td><td>Mouse IgG1</td></tr><tr><td>Desmin</td><td>Thermo Scientific</td><td>RB-9014-P1</td><td>Rabbit IgG</td></tr><tr><td>Anti-NANOG</td><td>ReproCELL Inc, Japan</td><td>RCAB0004P-F</td><td>Polyclonal antibody&amp;nbsp;</td></tr><tr><td>Rat anti-GFAP</td><td>Zymed</td><td>13-0300</td><td>Glial fibrillary acidic protein</td></tr><tr><td>Albumin (clone HSA1/25.1.3)</td><td>Cedarlane Laboratories Ltd.</td><td>CL2513A</td><td>Mouse IgG1</td></tr><tr><td>Smooth muscle actin (clone 1A4)</td><td>DakoCytomation Inc</td><td>IR611/IS611</td><td>Mouse IgG2a</td></tr><tr><td>Nestin</td><td>Chemicon International</td><td>MAB5326</td><td>Rabbit polyclonal antibody</td></tr><tr><td>TUBB3</td><td>Convance Inc</td><td>MMS-435P</td><td>Tuj1, mouse IgG2a</td></tr><tr><td>HNF4&amp;alpha; (C11F12)</td><td>Cell Signaling Technologies</td><td>3113</td><td>Rabbit monoclonal antibody</td></tr><tr><td>Paraformaldehyde (solution)</td><td>Electron Microscopy Sciences</td><td>15710</td><td>PFA, fixative, diluted in D-PBS</td></tr></tbody></table>

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.

NCM文化的一般模式 图1显示出高密度的单细胞接种在ROCK抑制剂Y-27632的存在后hPSCs的动态变化典型的NCM的文化模式。这些形态变化包括电镀，细胞簇的形成，并呈指数生长的细胞，然后进行细胞缩合（ 图1A）之后，细胞间的连接。代表性实验表明WA01（H1）的胚胎干细胞，镀成单细胞在1.9×10 5个细胞/ cm 2在10μMY-27632在第1天的存在下的密度（ 图1B，左面板），进一步传播而不在2天的集落的形成（ 图1B，中间板），并冷凝为一种均匀的单分子层，是适合需要的实验或用于细胞传代在第3天（ 图1B，右图）。 &lt;STRONG&gt;各种ROCK抑制剂支持NCM文化 96孔微孔板检测被用于证明了概念高通量药物筛选的。它也被设计来优化使用各种ROCK抑制剂的支持NCM文化。约，31000解离SCU-i10的细胞，人的诱导多能干细胞（人iPS细胞），17，铺在1基质胶涂覆在不同浓度的ROCK抑制剂的存在下良好。 24小时后，对细胞进行的CCK-8基于存活试验，以确定在这些条件下的细胞存活。之前我们已经表明，NCM方法需要使用的ROCK抑制剂Y-27632，在10微米以提高单细胞电镀。在这份报告中，我们证实，10微米Y-27632显著增加24小时的单细胞镀人iPS细胞的效率性（P &lt;0.05）（ 图2）。我们还发现，Y型39983（ROCK I抑制剂），phenylbenzodioxane（ROCK II抑制剂器），和thiazovivin（一种新颖的ROCK抑制剂）显著调制单细胞接种效率和促进生长的NCM于1μM时与对照组（P &lt;0.05）（ 图2）相比较。此外，3 ROCK抑制剂（在1μM）对单细胞电镀效率的影响，则相当于是Y-27632在10μM（P&gt; 0.05）（ 图2）。值得注意的是，ROCK I抑制剂（5μM）似乎显示明显的细胞毒性的药物在1μM（P &lt;0.05），提示比其它分子的更具体的相互作用。因此，可用于在将来支持NCM培养各种ROCK抑制剂。然而，将需要为将来使用两种胚胎干细胞和人iPS细胞NCM这些新抑制剂的下一个完整的特征。 LN-521支持NCM文化，而 ​​无需使用ROCK抑制剂 要确定吨在支持人类胚胎干细胞的增长，他一个特定的层粘连蛋白亚型的作用，我们在培养的异种培养基TeSR2 LN-521-涂层板SCU-I30人iPS细胞。有趣的是，LN-521单独使用，无需ROCK抑制剂的存在下，支持单细胞镀和随后的NCM生长15代在此条件下（ 图3）。 SCU-i30的细胞与抗Nanog的多克隆抗体的免疫染色表明，在此条件下的细胞具有在细胞核高NANOG的表达（ 图3A）。流式细胞仪分析表明，人类胚胎干细胞标志物表达谱相似，使用ROCK抑制剂Y-27632（ 图3B）成长为NCM细胞。 在不使用的慢病毒颗粒的小分子RNA传递效率高 转染DY547标记寡核苷酸微RNA进行中WA01（H1）细胞NCM条件下进行。 WA01人类胚胎干细胞生长为NCM上<html> 2.5％基质胶中mTeSR1为2（ 图4A）及18（ 图4B）分别通路。这些细胞显示出高的转染效率24小时转染后（ 图4A和4B）。一般来说，我们可以在转染后24小时获得的转染效率高达91％的人类胚胎干细胞。 <img alt="图1" fo:content-width="6.5in" src="/files/ftp_upload/51519/51519fig1highres.jpg" width="600" /> 图1（A）架构NCM文化。线图描绘的多细胞组织中NCM条件下，一个典型的为期3天的文化的动态变化。（二）在mTeSR1介质中的NCM条件下繁殖的2.5％基质胶16代WA01（H1）人类胚胎干细胞代表相位图像（指定作为WA01，mcp16）。下面板是在上面板的放大图。刻度条表示100μm左右。EF ="htt​​ps://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51519/51519fig1highres.jpg"目标="_blank"&gt;请点击这里查看该图的放大版本。 <img alt="图2" fo:content-width="7in" src="/files/ftp_upload/51519/51519fig2highres.jpg" width="600" /> 图2使用96孔格式的单细胞存活测定法。约，31000 SCU-i10的人iPS细胞17人在指定浓度与Rho相关激酶（ROCK）的途径的各种小分子抑制剂的存在下接种于2.5％基质胶。 24小时后，将细胞通过测量450nm处的吸光度（A450）进行的CCK-8存活测定法。学生t-检验来测定各种ROCK抑制剂之间的差异在单池镀覆效率是否是统计显着性。奇异星号标记（*）表示抑制剂（P＆＃之间没有显著差异62; 0.05），而两个星号标志（**）表示观察到的差异是统计学显着性（P &lt;0.05）。列在直方图指定一式四份决定因素和酒吧的平均值表示标准偏差。 <img alt="图3" fo:content-width="6in" src="/files/ftp_upload/51519/51519fig3highres.jpg" width="600" /> 没有ROCK抑制剂的人iPS细胞存在于层粘连蛋白-521（LN-521）图3。NCM文化的 SCU-I30 hiPSC行成立于美国国立卫生研究院干细胞单位。 SCU-i30的细胞生长在LN-521包被的6孔板中的异种培养基TeSR2 15代，而无需使用小分子抑制剂，例如ROCK抑制剂。SCU-i30的细胞（A）的免疫染色用抗-NANOG多克隆抗体并用Hoechst 33342（Hoechst公司）。值得注意的是，某些细胞具有负NANOG染色是第在有丝分裂E细胞（B）中成长为NCM在2.5％基质胶与LN-521采用10微米Y-27632（上图）或NCM没有Y-27632 SCU-I30 HPSC细胞标志物表达的流式细胞仪分析（下图）。该程序为免疫染色和流式细胞仪分析之前5进行了描述。比例尺描绘100微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51519/51519fig3highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" fo:content-width="7in" src="/files/ftp_upload/51519/51519fig4highres.jpg" width="600" /> 图4。hPSCs的小分子RNA转染的NCM文化。WA01人类胚胎干细胞生长为NCM在2.5％基质胶中mTeSR1 2（a）和18（B）的通道，分别为。 WA01细胞转染了镝（Dy）的代表相位和荧光图像547标记的小分子RNA控制监测转染效率在24小时转染后。标尺代表100微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51519/51519fig4highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis at JoVE.com

人类多能干细胞生产、维持和遗传分析的替代培养物|生物学 |视频

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

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 ...

alternative-cultures-for-human-pluripotent-stem-cell-production

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SCIENTISTS IN ACTION

12.3K 次观看.  National Institutes of Health. 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.

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

Scalable 96-well Plate Based iPSC Culture and Production Using a Robotic Liquid Handling System

Continued advancement in pluripotent stem cell culture is closing the gap between bench and bedside for using these cells in regenerative medicine, drug discovery and safety testing. In order to produce stem cell derived biopharmaceutics and cells for tissue engineering and transplantation, a cost-effective cell-manufacturing technology is essential. Maintenance of pluripotency and stable performance of cells in downstream applications (e.g., cell differentiation) over time is paramount to large scale cell production. Yet that can be difficult to achieve especially if cells are cultured manually where the operator can introduce significant variability as well as be prohibitively expensive to scale-up. To enable high-throughput, large-scale stem cell production and remove operator influence novel stem cell culture protocols using a bench-top multi-channel liquid handling robot were developed that require minimal technician involvement or experience. With these protocols human induced pluripotent stem cells (iPSCs) were cultured in feeder-free conditions directly from a frozen stock and maintained in 96-well plates. Depending on cell line and desired scale-up rate, the operator can easily determine when to passage based on a series of images showing the optimal colony densities for splitting. Then the necessary reagents are prepared to perform a colony split to new plates without a centrifugation step. After 20 passages (~3 months), two iPSC lines maintained stable karyotypes, expressed stem cell markers, and differentiated into cardiomyocytes with high efficiency. The system can perform subsequent high-throughput screening of new differentiation protocols or genetic manipulation designed for 96-well plates. This technology will reduce the labor and technical burden to produce large numbers of identical stem cells for a myriad of applications.

The protocols described allow laboratories to perform scalable, adherent stem cell culture in high throughput with minimal labor, experience and equipment investment cost using a programmable liquid handling robot and 96-well plates. iPSCs passaged more than 20 times on this system maintained pluripotency, normal karyotypes and differentiated into cardiomyocytes.

可扩展的96孔板基于IPSC的文化和生产用机器人液体处理系统

Continued advancement in pluripotent stem cell culture is closing the gap between bench and bedside for using these cells in regenerative medicine, drug ...

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发育生物学

A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of interest. Nevertheless, an ideal humanized ex vivo feeder-free propagation method for hPSCs has not been developed; currently, additional exogenous substrates including basic fibroblast growth factor (bFGF), a master hPSC-sustaining factor, is added to all of culture media and synthetic substrata such as Matrigel or laminin are used in all feeder-free cultures. Recently, our group developed a simple and efficient protocol for the propagation of hPSCs using only conditioned media derived from the human placenta on a gelatin-coated dish without additional exogenous supplementation or synthetic substrata specific to hPSCs. This protocol has not been reported previously and might enable researchers to propagate hPSCs efficiently in humanized culture conditions. Additionally, this model obviates hPSC contamination risks by animal products such as viruses or unknown proteins. Furthermore, this system facilitates easy mass production of hPSCs using the gelatin coating, which is simple to handle, dramatically decreases the overall costs of ex vivo hPSC maintenance.

This protocol provides a simple and efficient way to propagate human pluripotent stem cells (hPSCs) using only conditioned media derived from the human placenta in a gelatin-coated dish without additional exogenous supplementation or hPSC-specific synthetic substrata.

一种新的文化模式的人多能干明胶胎盘条件培养基细胞增殖

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of ...

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

培养人多能性和神经干细胞的基本和临床前研究一个封闭的细胞培养系统

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

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.

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 ...

Chemical Reversion of Conventional Human Pluripotent Stem Cells to a Na&#239;ve-like State with Improved Multilineage Differentiation Potency

Na&#239;ve human pluripotent stem cells (N-hPSC) with improved functionality may have a wide impact in regenerative medicine. The goal of this protocol is to efficiently revert lineage-primed, conventional human pluripotent stem cells (hPSC) maintained on either feeder-free or feeder-dependent conditions to a na&#239;ve-like pluripotency with improved functionality. This chemical na&#239;ve reversion method employs the classical leukemia inhibitory factor (LIF), GSK3&#946;, and MEK/ERK inhibition cocktail (LIF-2i), supplemented with only a tankyrase inhibitor XAV939 (LIF-3i). LIF-3i reverts conventional hPSC to a stable pluripotent state adopting biochemical, transcriptional, and epigenetic features of the human pre-implantation epiblast. This LIF-3i method requires minimal cell culture manipulation and is highly reproducible in a broad repertoire of human embryonic stem cell (hESC) and transgene-free human induced pluripotent stem cell (hiPSC) lines. The LIF-3i method does not require a re-priming step prior to the differentiation; N-hPSC can be differentiated directly with extremely high efficiencies and maintain karyotypic and epigenomic stabilities (including at imprinted loci). To increase the universality of the method, conventional hPSC are first cultured in the LIF-3i cocktail supplemented with two additional small molecules that potentiate protein kinase A (forskolin) and sonic hedgehog (sHH) (purmorphamine) signaling (LIF-5i). This brief LIF-5i adaptation step significantly enhances the initial clonal expansion of conventional hPSC and permits them to be subsequently na&#239;ve-reverted with LIF-3i alone in bulk quantities, thus obviating the need for picking/subcloning rare N-hPSC colonies later. LIF-5i-stabilized hPSCs are subsequently maintained in LIF-3i alone without the need of anti-apoptotic molecules. Most importantly, LIF-3i reversion markedly improves the functional pluripotency of a broad repertoire of conventional hPSC by decreasing their lineage-primed gene expression and erasing the interline variability of directed differentiation commonly observed amongst independent hPSC lines. Representative characterizations of LIF-3i-reverted N-hPSC are provided, and experimental strategies for functional comparisons of isogenic hPSC in lineage-primed vs. na&#239;ve-like states are outlined.

We present a protocol for efficient, bulk, and rapid chemical reversion of conventional lineage-primed human pluripotent stem cells (hPSC) into an epigenomically-stable na&#239;ve preimplantation epiblast-like pluripotent state. This method results in decreased lineage-primed gene expression and marked improvement in directed multilineage differentiation across a broad repertoire of conventional hPSC lines.

改良多向分化效力的常规人多能干细胞的化学逆转与幼稚样态

Na&#239;ve human pluripotent stem cells (N-hPSC) with improved functionality may have a wide impact in regenerative medicine. The goal of this protocol is ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

文化和维护人类胚胎干细胞

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

生物学

Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS cell cultures to feeder-free while still maintaining your use of Knockout SR.

The following protocol provides instruction for adapting human induced Pluripotent Stem (iPS) Cells to feeder-free culture using complete KnockOut Serum Replacement Feeder-Free medium (KSR-FF). Once adapted, instructions for continual maintenance are also provided.

免费接驳适应，文化和传代使用完整的基因敲除血清替代馈线中的人类iPS细胞

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem ...

使用自动化培养系统诱导多能干细胞分化

An Automated Culture System for Maintaining and Differentiating Human-Induced Pluripotent Stem Cells

Human induced pluripotent stem cells (hiPSCs) with infinite self-proliferating ability have been expected to have applications in numerous fields, including the elucidation of rare disease pathologies, the development of new medicines, and regenerative medicine aiming to restore damaged organs. Despite this, the social implementation of hiPSCs is still limited. This is partly because of the difficulty of reproducing differentiation in culture, even with advanced knowledge and sophisticated technical skills, due to the high sensitivity of iPSCs to minute environmental changes. The application of an automated culture system can solve this issue. Experiments with high reproducibility independent of a researcher's skill can be expected according to a shared procedure across various institutes. Although several automated culture systems that can maintain iPSC cultures and induce differentiation have been developed previously, these systems are heavy, large, and costly because they make use of humanized, multi-articulated robotic arms. To improve on the above issues, we developed a new system using a simple x-y-z axis slide rail system, allowing it to be more compact, lighter, and cheaper. Furthermore, the user can easily modify parameters in the new system to develop new handling tasks. Once a task is established, all the user needs to do is prepare the iPSC, supply the reagents and consumables needed for the desired task in advance, select the task number, and specify the time. We confirmed that the system could maintain iPSCs in an undifferentiated state through several passages without feeder cells and differentiate into various cell types, including cardiomyocytes, hepatocytes, neural progenitors, and keratinocytes. The system will enable highly reproducible experiments across institutions without the need for skilled researchers and will facilitate the social implementation of hiPSCs in a wider range of research fields by diminishing the obstacles for new entries.

作者聚焦:自动化 iPSC 培养以提高可重复性 

Here, we present a protocol for an automated cell culture system. This automated culture system reduces labor and benefits the users, including researchers unfamiliar with handling induced pluripotent stem (iPS) cells, from the maintenance of iPS cells to differentiation into various types of cells.

另类文化的人多能干细胞的生产，维护和遗传分析

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可扩展的96孔板基于IPSC的文化和生产用机器人液体处理系统

一种新的文化模式的人多能干明胶胎盘条件培养基细胞增殖

培养人多能性和神经干细胞的基本和临床前研究一个封闭的细胞培养系统

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

改良多向分化效力的常规人多能干细胞的化学逆转与幼稚样态

文化和维护人类胚胎干细胞

免费接驳适应，文化和传代使用完整的基因敲除血清替代馈线中的人类iPS细胞

用于维持和分化人诱导的多能干细胞的自动化培养系统

用于维持和分化人诱导的多能干细胞的自动化培养系统

用于维持和分化人诱导的多能干细胞的自动化培养系统