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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Countess automated cell counter&#160;&#160;</td>
			<td style="width:220px;">Invitrogen Inc.&#160;</td>
			<td style="width:187px;">C10227</td>
			<td style="width:187px;">Automatic cell counting</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Faxitron Cabinet X-ray System</td>
			<td style="width:220px;">Faxitron X-ray Corporation, Wheeling, IL&#160;</td>
			<td style="width:187px;">Model RX-650</td>
			<td style="width:187px;">X-ray irradiation of MEFs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">MULTIWELL six-well plates&#160;</td>
			<td style="width:220px;">Becton Dickinson Labware</td>
			<td style="width:187px;">353046</td>
			<td style="width:187px;">Polystyrene plates&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">DMEM</td>
			<td style="width:220px;">Invitrogen Inc.</td>
			<td style="width:187px;">11965&#8211;092</td>
			<td style="width:187px;">For MEF medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">mitomycin C</td>
			<td style="width:220px;">Roche&#160;</td>
			<td style="width:187px;">107 409</td>
			<td style="width:187px;">Mitotic inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Trypsin</td>
			<td style="width:220px;">Invitrogen Inc.</td>
			<td style="width:187px;">25300-054</td>
			<td style="width:187px;">For MEF dissociation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">DMEM/F12&#160;</td>
			<td style="width:220px;">Invitrogen Inc.</td>
			<td style="width:187px;">11330&#8211;032</td>
			<td style="width:187px;">For hPSC medium</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:203px;">Opti-MEM I Reduced Serum Medium&#160;</td>
			<td style="width:220px;">Invitrogen Inc.</td>
			<td style="width:187px;">31985-062</td>
			<td style="width:187px;">For hPSC transfection</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Heat-inactivated FBS</td>
			<td style="width:220px;">Invitrogen Inc.</td>
			<td style="width:187px;">16000&#8211;044</td>
			<td style="width:187px;">Component of MEF medium</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Knockout Serum Replacer&#160;</td>
			<td style="width:220px;">Invitrogen Inc.</td>
			<td style="width:187px;">10828&#8211;028</td>
			<td style="width:187px;">KSR, Component of hPSC medium</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:203px;">Dulbecco&#8217;s Phosphate-Buffered Saline</td>
			<td style="width:220px;">Invitrogen Inc.</td>
			<td style="width:187px;">14190-144</td>
			<td style="width:187px;">D-PBS, free of Ca2+/Mg2+</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Non-essential amino acids&#160;</td>
			<td style="width:220px;">Invitrogen&#160;</td>
			<td style="width:187px;">11140&#8211;050</td>
			<td style="width:187px;">NEAA, component of hPSC medium</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">L-Glutamine&#160;</td>
			<td style="width:220px;">Invitrogen&#160;</td>
			<td style="width:187px;">25030&#8211;081</td>
			<td style="width:187px;">Component of hPSC medium</td>
		</tr>
		<tr height="20">
			<td height="41" rowspan="2" style="height:41px;width:203px;">mTeSR1 &#38; Supplements</td>
			<td rowspan="2" style="width:220px;">StemCell Technologies</td>
			<td rowspan="2" style="width:187px;">5850</td>
			<td style="width:187px;">Animal protein-free</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:187px;">medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">TeSR2 &#38; Supplements</td>
			<td style="width:220px;">StemCell Technologies</td>
			<td style="width:187px;">5860</td>
			<td style="width:187px;">Xeno-free medium</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">&#946;-mercaptoethanol&#160;</td>
			<td style="width:220px;">Sigma&#160;</td>
			<td style="width:187px;">7522</td>
			<td style="width:187px;">Component of hPSC medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;"> 
			MEF (CF-1) ATCC</td>
			<td style="width:220px;">American Type Culture Collection (ATCC)&#160;</td>
			<td style="width:187px;">SCRC-1040</td>
			<td style="width:187px;">For feeder culture of hPSCs</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">hESC-qualified Matrigel</td>
			<td style="width:220px;">BD Bioscience</td>
			<td style="width:187px;">354277</td>
			<td style="width:187px;">For feeder-free culture of hPSCs</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Laminin-521</td>
			<td style="width:220px;">BioLamina</td>
			<td style="width:187px;">LN521-02</td>
			<td style="width:187px;">Human recombinant protein</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">FGF-2 (recombinant FGF, basic)</td>
			<td style="width:220px;">R&#38;D Systems, MN</td>
			<td style="width:187px;">223-FB</td>
			<td style="width:187px;">Growth factor in hPSC medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">CryoStor CA10&#160;</td>
			<td style="width:220px;">StemCell Technologies</td>
			<td style="width:187px;">7930</td>
			<td style="width:187px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Accutase</td>
			<td style="width:220px;">Innovative Cell Technologies</td>
			<td style="width:187px;">AT-104</td>
			<td style="width:187px;">1X mixed enzymatic solution</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">JAK inhibitor I</td>
			<td style="width:220px;">EMD4 Biosciences</td>
			<td style="width:187px;">420099</td>
			<td style="width:187px;">An inhibitor of Janus kinase</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Y-27632</td>
			<td style="width:220px;">EMD4 Biosciences</td>
			<td style="width:187px;">688000</td>
			<td style="width:187px;">ROCK inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Y-27632</td>
			<td style="width:220px;">Stemgent</td>
			<td style="width:187px;">04-0012</td>
			<td style="width:187px;">ROCK inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Y-39983</td>
			<td style="width:220px;">Stemgent</td>
			<td style="width:187px;">04-0029</td>
			<td style="width:187px;">ROCK I inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Phenylbenzodioxane&#160;</td>
			<td style="width:220px;">Stemgent</td>
			<td style="width:187px;">04-0030</td>
			<td style="width:187px;">ROCK II inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Thiazovivin</td>
			<td style="width:220px;">Stemgent</td>
			<td style="width:187px;">04-0017</td>
			<td style="width:187px;">A novel ROCK inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">BD Falcon Cell Strainer&#160;</td>
			<td style="width:220px;">BD Bioscience</td>
			<td style="width:187px;">352340</td>
			<td style="width:187px;">40-&#181;m cell strainer</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Nalgene 5100-0001 Cryo 1&#176;C</td>
			<td style="width:220px;">Thermo Scientific&#160;</td>
			<td style="width:187px;">C6516F-1</td>
			<td style="width:187px;">&#8220;Mr. Frosty&#8221; Freezing Container</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Lipofectamine 2000&#160;</td>
			<td style="width:220px;">Invitrogen Inc.</td>
			<td style="width:187px;">11668-027</td>
			<td style="width:187px;">Transfection reagents</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">DharmaFECT Duo&#160;</td>
			<td style="width:220px;">Thermo Scientific</td>
			<td style="width:187px;">T-2010-02</td>
			<td style="width:187px;">Transfection reagent</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:203px;">Non-targeting miRIDIAN miRNA Transfection Control</td>
			<td style="width:220px;">Thermo Scientific</td>
			<td style="width:187px;">IP-004500-01-05</td>
			<td style="width:187px;">Labeled with Dy547, to monitor the delivery of microRNAs&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">SMART-shRNA</td>
			<td style="width:220px;">Thermo Scientific&#160;</td>
			<td style="width:187px;">To be determined</td>
			<td style="width:187px;">Lentiviral vector</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">pmaxGFP</td>
			<td>amaxa Inc (Lonza)</td>
			<td>Included in every transfection kit</td>
			<td style="width:187px;">Expression plasmid for transfection control</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">4-Oct</td>
			<td style="width:220px;">Santa Cruz Biotechnology</td>
			<td style="width:187px;">sc-5279</td>
			<td style="width:187px;">Mouse IgG2b, pluripotent marker</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">SSEA-1</td>
			<td>Santa Cruz Biotechnology</td>
			<td style="width:187px;">sc-21702</td>
			<td style="width:187px;">Mouse IgM, differentiation marker</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">SSEA-4</td>
			<td style="width:220px;">Santa Cruz Biotechnology</td>
			<td style="width:187px;">sc-21704</td>
			<td style="width:187px;">Mouse IgG3, pluripotent marker</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Tra-1-60</td>
			<td style="width:220px;">Santa Cruz Biotechnology</td>
			<td style="width:187px;">sc-21705&#160;</td>
			<td style="width:187px;">Mouse IgM, pluripotent marker</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Tra-1-81</td>
			<td style="width:220px;">Santa Cruz Biotechnology</td>
			<td style="width:187px;">sc-21706</td>
			<td style="width:187px;">Mouse IgM, pluripotent marker</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">CK8 (C51)</td>
			<td style="width:220px;">Santa Cruz Biotechnology</td>
			<td style="width:187px;">sc-8020</td>
			<td style="width:187px;">Mouse IgG1, against cytokeratin 8</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">&#945;-fetoprotein</td>
			<td style="width:220px;">Santa Cruz Biotechnology</td>
			<td style="width:187px;">sc-8399</td>
			<td style="width:187px;">AFP, mouse IgG2a</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">HNF-3&#946; (P-19)</td>
			<td style="width:220px;">Santa Cruz Biotechnology</td>
			<td style="width:187px;">sc-9187</td>
			<td style="width:187px;">FOXA2, goat polyclonal antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Troponin T (Av-1)</td>
			<td style="width:220px;">Thermo Scientific</td>
			<td style="width:187px;">MS-295-P0</td>
			<td style="width:187px;">Mouse IgG1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Desmin&#160;</td>
			<td style="width:220px;">Thermo Scientific</td>
			<td style="width:187px;">RB-9014-P1</td>
			<td style="width:187px;">Rabbit IgG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Anti-NANOG</td>
			<td style="width:220px;">ReproCELL Inc, Japan</td>
			<td style="width:187px;">RCAB0004P-F</td>
			<td style="width:187px;">Polyclonal antibody&#160;</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Rat anti-GFAP</td>
			<td style="width:220px;">Zymed</td>
			<td style="width:187px;">13-0300</td>
			<td style="width:187px;">Glial fibrillary acidic protein</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Albumin (clone HSA1/25.1.3)</td>
			<td style="width:220px;">Cedarlane Laboratories Ltd. (</td>
			<td style="width:187px;">CL2513A</td>
			<td style="width:187px;">Mouse IgG1,</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Smooth muscle actin (clone 1A4)</td>
			<td style="width:220px;">DakoCytomation Inc</td>
			<td style="width:187px;">IR611/IS611</td>
			<td style="width:187px;">Mouse IgG2a</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Nestin</td>
			<td style="width:220px;">Chemicon International</td>
			<td style="width:187px;">MAB5326</td>
			<td style="width:187px;">Rabbit polyclonal antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">TUBB3</td>
			<td style="width:220px;">Convance Inc</td>
			<td style="width:187px;">MMS-435P</td>
			<td style="width:187px;">Tuj1, mouse IgG2a</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">HNF4&#945; (C11F12)</td>
			<td style="width:220px;">Cell Signaling Technologies</td>
			<td style="width:187px;">3113</td>
			<td style="width:187px;">Rabbit monoclonal antibody</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Paraformaldehyde (solution)</td>
			<td style="width:220px;">Electron Microscopy Sciences</td>
			<td style="width:187px;">15710</td>
			<td style="width:187px;">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/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/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/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 ...

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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

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

小鼠表皮神经嵴干细胞（EPI的非公务员合约雇员）文化

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

科研

生物学

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 ...

Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for mesenchymal stromal cells (MSCs), fibroblasts and endothelial colony-forming cells with platelets activated by thrombin3-5 or lysed by freeze/thaw cycles6-14 before the platelet releasate is added to the cell culture medium. The trophic effect of platelet derived growth factors has already been tested in several trials for tissue engineering and regenerative therapy.1,15-17 Varying efficiency is considered to be at least in part due to individually divergent concentrations of growth factors18,19 and a current lack of standardized protocols for platelet preparation.15,16 This protocol presents a practicable procedure to generate a pool of human platelet lysate (pHPL) derived from routinely produced platelet rich plasma (PRP) of forty to fifty single blood donations. By several freeze/thaw cycles the platelet membranes are damaged and growth factors are efficiently released into the plasma. Finally, the platelet fragments are removed by centrifugation to avoid extensive aggregate formation and deplete potential antigens. The implementation of pHPL into standard culture protocols represents a promising tool for further development of cell therapeutics propagated in an animal protein-free system.

Preparation of Pooled Human Platelet Lysate pHPL as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Human platelet lysate is a rich source of growth factors and a potent supplement in cell culture. This protocol presents the process of preparing a large pool of human platelet lysate by starting from platelet rich plasma, performing several freeze-thaw cycles and depleting the platelet fragments.

汇集人类血小板裂解液作为一种有效的补充（pHPL）的动物血清自由人干细胞培养制备

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

视频人类胚胎干细胞克隆生长的生物信息学分析

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

人类诱导多能干细胞的转染和Nucleofecting

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

人类心肌细胞的生成:从馈线人类诱导多能干细胞的分化协议

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

从人类体细胞与仙台病毒高效的新一代人类诱导多能干细胞

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

A Multi-hole Cryovial Eliminates Freezing Artifacts when Muscle Tissues are Directly Immersed in Liquid Nitrogen

Studies on skeletal muscle physiology face the technical challenge of appropriately processing the specimens to obtain sections with clearly visible cytoplasmic compartments. Another hurdle is the tight apposition of myofibers to the surrounding tissues. Because the process of tissue fixation and paraffin embedding leads to the shrinkage of muscle fibers, freezing is an optimal means of hardening muscle tissue for sectioning. However, a commonly encountered issue, the formation of ice crystals, occurs during the preparation of frozen sections because of the high water content of muscle. The protocol presented here first describes a simple and efficient method for properly freezing muscle tissues by immersing them in liquid nitrogen. The problem with using liquid nitrogen alone is that it causes the formation of a nitrogen gas barrier next to the tissue, which acts as an insulator and inhibits the cooling of the tissues. To avoid this "vapor blanket" effect, a new cryovial was designed to increase the speed of liquid flow around the tissue surface. This was achieved by punching a total of 14 inlet holes in the wall of the vial. According to bubble dynamics, a higher rate of liquid flow results in smaller bubbles and fewer chances to form a gas barrier. When liquid nitrogen flows into the cryovial through the inlet holes, the flow velocity around the tissue is fast enough to eliminate the gas barrier. Compared to the method of freezing muscle tissues using pre-chilled isopentane, this protocol is simpler and more efficient and can be used to freeze muscle in a throughput manner. Furthermore, this method is optimal for institutions that do not have access to isopentane, which is extremely flammable at room temperature.

This protocol describes a procedure to freeze muscle tissues by plunging them directly into liquid nitrogen. This protocol also highlights a new cryovial that can avoid the &#34;blanket effect&#34; of nitrogen gas when liquid nitrogen contacts the tissue surface of a specimen.

的多孔冷冻管省去冻结工件时肌肉组织在液氮中直接浸入

Studies on skeletal muscle physiology face the technical challenge of appropriately processing the specimens to obtain sections with clearly visible ...

Automated Production of Human Induced Pluripotent Stem Cell-Derived Cortical and Dopaminergic Neurons with Integrated Live-Cell Monitoring

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are prone to spontaneous differentiation into unwanted cell types. The methods are labor-intensive and are not easily amenable to large-scale experiments. To overcome these limitations, we developed an automated cell culture system coupled to a high-throughput imaging system and implemented protocols for maintaining multiple hiPSC lines in parallel and neuronal differentiation. We describe the automation of a short-term differentiation protocol using Neurogenin-2 (NGN2) over-expression to produce hiPSC-derived cortical neurons within 6&#8210;8 days, and the implementation of a long-term differentiation protocol to generate hiPSC-derived midbrain dopaminergic (mDA) neurons within 65 days. Also, we applied the NGN2 approach to a small molecule-derived neural precursor cells (smNPC)&#160;transduced with GFP lentivirus and established a live-cell automated neurite outgrowth assay. We present an automated system with protocols suitable for routine hiPSC culture and differentiation into cortical and dopaminergic neurons. Our platform is suitable for long term hands-free culture and high-content/high-throughput hiPSC-based compound, RNAi and CRISPR/Cas9 screenings to identify novel disease mechanisms and drug targets.

We show the automation of human induced pluripotent stem cell (hiPSC) cultures and neuronal differentiations compatible with automated imaging and analysis.

通过集成活细胞监测，自动生产人类诱导多能干细胞衍生皮质神经元和多巴胺神经元

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are ...

A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe a new strategy that combines the use of chemical epigenetic erasing with mechanosensing-related cues, to generate mammalian pluripotent cells. Two main steps are required. In the first step, adult mature (terminally differentiated) cells are exposed to the epigenetic eraser 5-aza-cytidine to drive them into a pluripotent state. This part of the protocol was developed, based on the increasing understanding of the epigenetic mechanisms controlling cell fate and differentiation, and involves the use of the epigenetic modifier to erase cell differentiated state and then drive into a transient high plasticity window.
In the second step, erased cells are encapsulated in polytetrafluoroethylene (PTFE) micro-bioreactors, also known as Liquid Marbles, to promote 3D cell rearrangement to extend and stably maintain the acquired high plasticity. PTFE is a non-reactive hydrophobic synthetic compound and its use permits the creation of a cellular microenvironment, which cannot be achieved in traditional 2D culture systems. This system encourages and boosts the maintenance of pluripotency though bio-mechanosensing-related cues.
The technical procedures described here are simple strategies to allow for the induction and maintenance of a high plasticity state in adult somatic cells. The protocol allowed the derivation of high plasticity cells in all mammalian species tested. Since it does not involve the use of gene transfection and is free of viral vectors, it may represent a notable technological advance for translational medicine applications. Furthermore, the micro-bioreactor system provides a notable advancement in stem cell organoid technology by in vitro re-creating a specific micro-environment that allows for the long-term culture of high plasticity cells, namely as ESCs, iPSCs, epigenetically erased cells and MSCs.

We here present a method that combines the use of chemical epigenetic erasing with mechanosensing-related cues to efficiently generate mammalian pluripotent cells, without the need of gene transfection or retroviral vectors. This strategy is, therefore, promising for translational medicine and represents a notable advancement in stem cell organoid technology.

结合表观遗传修饰和生物力学线索的两步策略，以产生哺乳动物多能细胞

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe ...