所有小鼠实验按照与当地机构动物护理委员会（Landesamt附耳NATUR，UMWELT UND Verbraucherschutz，北莱茵 - 威斯特法伦州[批准ID号批准同意保护动物和德国法律规定进行:AZ 84-02.04.2013 .A428]）。 1.媒体准备<ol><li>制备293T培养基:Dulbecco改良的Eagle培养基（DMEM），10％（V / V）FBS，L-谷氨酰胺（2毫摩尔），丙酮酸钠（1毫米），青霉素/链霉素（1×）。 </li><li>制备MEF培养基:DMEM，10％（V / V）FBS，L-谷氨酰胺（2毫摩尔），1×非必需氨基酸，青霉素/链霉素（1×）。 </li><li>制备的TS培养基（W / O型FGF4 /肝素）:罗斯韦尔园区纪念研究所培养基（RPMI）1640 20％（V / V）FBS的（干细胞培养级），青霉素/链霉素（1×），L-谷氨酰胺（2mM的），丙酮酸钠（1毫摩尔），2-巯基乙醇（0.1毫摩尔）。 </li><li>制备的TS培养基FGF4 /肝素（TS + F4H）:RPMI 1640，20％（V / V）FBS的（干细胞培养级），青霉素/链霉素（1×），L-谷氨酰胺（2毫摩尔），丙酮酸钠（1毫摩尔），2-巯基乙醇（0.1毫摩尔），25纳克/毫升FGF4，1微克/毫升肝素。 注意:FGF4和肝素被直接添加到媒体即将使用前。 </li><li>制备的TS-CM与FGF4 /肝素（TS-CM + F4H）:70％MEF条件的TS培养基+ 30％新制备的TS培养基+ 25毫微克/毫升FGF4，1微克/毫升肝素。 注意:FGF4和肝素被直接添加到媒体即将使用前。 </li><li>制备转染培养基:高级DMEM，2％（V / V）FBS的（干细胞培养级），L-谷氨酰胺（2mM的），青霉素/链霉素（1×）。 </li><li>制备病毒生产培养基:高级DMEM，5％（V / V）FBS的（干细胞培养级），L-谷氨酰胺（2mM的），青霉素/链霉素（1×）。 </li></ol> 2.小​​鼠胚胎成纤维细胞的推导<ol><li> （B6.Cg-GT（ROSA）26Sor TM1 rtTA;设置定时交配使用重量129S2SV女性和男性的纯合子R26 :: rtTA小鼠（品系名称* M2）宰/ J 7）。上通过颈椎脱位E13.5牺牲小鼠和隔离根据标准方案8初级小鼠胚胎成纤维细胞（MEF中）。此后，板细胞一个胚胎在MEF媒体150毫米的菜。 </li><li>一旦主要的MEF培养150 mm培养皿中达到汇合，洗涤细胞两次用10毫升的PBS，并添加4毫升0.05％胰蛋白酶/ EDTA溶液。于37℃在培养箱中4分钟 - 孵育3菜肴。 </li><li>孵育检查后，如果使用倒置显微镜（使用10X透镜）从培养皿分离的细胞。添加5毫升MEF培养基以停止胰蛋白酶消化，并收集细胞悬浮到50ml锥形管中。 </li><li>从几盘池细胞悬浮液，并收集到50ml锥形管中。离心细胞悬浮液，在200 xg离心3分钟，10℃。 </li><li>丢弃在含有10％（体积/体积）二甲基亚砜的FBS上清，重悬细胞沉淀。冻结等分进一步使用MEF中。 注:另外，用野生典型Ë主要的MEF;然而，需要表达rtTA额外慢病毒载体也可以与其它慢病毒载体一起转导（例如，使用由Maherali 等人 9所述的FUdeltaGW-rtTA质粒）。 </li><li> MEF条件TS培养基的制备 注:在转换实验，MEF条件（CM）TS媒体已经做好准备，这是需要单独ITSC线（在第6条所述）的亚文化。 <ol><li>板1×10 7每150毫米的菜有丝分裂灭活的MEF在菜肴所需数量MEF培养基。通过失活γ射线的MEF用剂量9戈瑞。 注:每150毫米的菜产量约为60毫升CM的。 </li><li>后镀，吸白天和丢弃MEF培养基，并添加新鲜制备的TS培养基（无FGF4 /肝素）20毫升到有丝分裂失活的MEF和地点的菜肴的每个培养皿放回37℃的培养箱。 </li><li>孵化后对有丝分裂灭活的MEF3天TS中，收集在50毫升锥形管网上平台。通过0.2微米的过滤器过滤介质和2.6.4进行。用20ml新鲜的TS培养基的制备另一批次的CM的替换CM。 注意:有丝分裂失活的MEF可用于CM制备高达三倍。 </li><li>等分试样35毫升过滤的CM到50ml锥形管中并储存在-20℃直到需要。解冻后，以获得70％的TS-CM和储存于4℃最多一个月增加15毫升新制备的TS培养基。 </li></ol></li></ol> 3.慢病毒载体生产的293T细胞注意:所有步骤都是在授权给生物安全2级实验室空间进行。 <ol><li>在此之前转换实验，准备多西环素（DOX）依赖过度表达TFAP2C，GATA3，EOMES，ETS2和mCherry的慢病毒载体。对于每一个慢病毒，共转染的293T细胞用各自的慢病毒转移连同包装质粒和VSV-G包膜表达质粒（质粒信息，请参阅材料表）载体。 注意:有用的信息有关病毒的产生是指GAVRILESCU 等[10]。虽然它描述逆转录病毒生产，关于293T细胞和质粒DNA的质量一般评论是慢病毒的生产也是如此。 小心:适当的安全措施需要与慢病毒载体和工作时进行拍摄，具有在生物安全等级2设施执行。 </li><li>外套个100毫米培养皿用聚-L-赖氨酸（100微克/毫升）通过覆盖菜用5ml溶液并轻轻摇动以均匀分布的涂层溶液溶液。放置碗碟放回培养箱中于37℃30分钟。 30分钟后吸出涂布溶液，并用PBS漂洗碗碟一次​​。 </li><li>板7×10 6 293T细胞培养皿每10毫升293T细胞媒体，SWIRL菜在37℃下均匀地分布的细胞和地方菜肴放回培养箱中。 </li><li>第二天早上，用5ml转染培养基替换生长培养基。添加6微升1000倍的股票氯溶液（25毫米），地方菜的回37℃培养箱。 </li><li> 同时，准备转染混合物: <ol><li>对于每一个慢病毒载体准备两个5毫升的反应管中，标记为A和B. </li><li>添加600μl的2×HEPES缓冲盐水（HBS）到管A. </li><li>管B中混合61.5微升的CaCl 2溶液（2M），用18.5微克慢病毒DNA和9.25微克psPAX2和9.25微克pMD2.G.的音量调整到600微升无菌文化品位H 2 O. </li><li>从加入B管滴来管的解决方案，而涡旋。孵育转染混合物在室温15分钟。 </li></ol></li><li>添加转染混合物非常小心滴明智的准备293T细胞上100毫米的菜。一个压脚提升5至6小时除去培养基并用10毫升的病毒生产培养基替换。放置碗碟放回培养箱中于37℃。 注:拆卸和更换介质，因为293T细胞很容易地从盘分离时要小心。更换培养基在转染后5-6小时是至关重要的，因为长时间暴露于氯喹是对细胞有毒。 </li><li>第二天早晨，小心地取出并抛弃中，用7ml病毒生产培养基替换。 </li><li>第二天早上收获了第一批含病毒的培养基。吸中，收集15毫升锥形管。再次，添加7毫升病毒生产培养基到细胞上。含病毒介质在冰箱商店直到第二批收获第二天。 </li><li>第二天早上吸第二批含有病毒的介质其加入到15ml含有第一批锥形管的。 293T细胞现在可以丢弃。含病毒介质通过0.45μm河畔滤波器免费factant - 醋酸纤维素-membrane过滤器。等分试样过滤，介质和存储在-80℃用于进一步使用含病毒。 注:病毒生产也可以在6孔皿进行。因此缩小体积和手机号码。 </li></ol> 4.成纤维细胞转导有4个因素与mCherry矢量控制注意:所有步骤在生物安全等级2设施进行。 <ol><li>为实验概要的示意图见图1。解冻初级rtTA-MEF中的一个等分试样和板每6孔培养皿的孔3.33×10 5个细胞在2毫升的MEF培养基的总体积。至少准备3井和4个因素（4F），mCherry和控制贴上标签。 注意:可替换地，野生型MEFs中可以使用，当步骤4.2中一个rtTA表达慢病毒载体被添加到慢病毒鸡尾酒。 </li><li>细胞后第二天从cryopre完全恢复servation，更换三六井传导介质为0.6毫升（4F），0.9毫升（mCherry）1毫升（控制）MEF媒体分别。解冻PLV-TETO-TFAP2C，-Gata3，-Eomes，-ets和-mCherry病毒之一等分在室温，加入100微升各含病毒的上清液（TFAP2C，GATA3，EOMES，ETS2）到4F好。 注意:在野生型的MEF的情况下，由100微升减少预电镀媒体的体积和另加100μl包含上清到4F混合FUdeltaGW-rtTA病毒。 </li><li>加入100微升mCherry含有病毒的媒体与mCherry井标记。加入聚凝胺至8微克/毫升到每个孔中的最终浓度。放置培养皿放回培养箱中于37℃孵育的MEF O / N与含有病毒的上清液。 注意:在野生型的MEF的情况下，由100微升减少预电镀媒体的体积和另加100微升FUdeltaGW-rtTA含病毒上清。 </li><li>第二天早晨，小心地取出含有病毒的介质和洗涤三次用2ml PBS中。最后，加2ml TS媒体（不FGF4 /肝素）。 注意:转导的MEF既可以直接用于ITSC感应实验或冷冻用于进一步使用。 </li></ol> 5.成纤维细胞转化ITSC 注意:所有步骤都是在授权给生物安全2级实验室空间进行。 <ol><li> 4分钟 - 用2毫升PBS和加入0.5ml胰蛋白酶/ EDTA溶液孵化它们在孵化器3洗净后转的MEF的两倍。检查细胞的合适脱离倒置显微镜下。 <ol><li>通过加入1ml的TS介质灭活胰蛋白酶。细胞悬液转移到15毫升锥形管中并离心，在200×g下3分钟。弃去上清，重悬在2ml的TS培养基中的细胞。使用未转染对照样品计数细胞。 </li><li>板4F-的MEF，mCherry-的MEF和未转导的MEF控制各以2×10 5的密度每100mm细胞培养皿细胞在10毫升的TS培养基含有2微克/毫升DOX（TS培养基+ FGF4 /肝素+ DOX）。 </li><li>另外，在较小的菜肴进行转。板上所需盘大小，缩放量相应地每平方厘米3.6×10 3个 。 </li></ol></li><li>第二天，用落射荧光显微镜（使用10X透镜和适合于红色荧光激发过滤器）监视器，以估计转导效率mCherry蛋白质的表达。 注意:转导效率应接近100％。如果增加的细胞死亡是转基因诱导后显而易见。这意味着病毒滴度过高。转导应具有减少的含病毒介质的量被重复。 </li><li>从现在开始，改变媒体隔日，直到电镀后的10天所有的菜。此后，与饲料中的TS细胞无DOX（TS培养基+ FGF4 /肝素）。 </li><li>使用倒置显微镜监测的外观"转分化区"（参照图2B和C）上4F盘长达3周。这样的菌落不应该在两个控制盘（mCherry-MEF中和未转的MEF）可见。 </li></ol> 6.隔离个别ITSC线路注意:对于步骤6.3不需要组织培养罩。建议擦拭用70％乙醇在倒置显微镜，以减少细菌污染的机会。个别ITSC菌落天21和转分化28天之间进行分离。 <ol><li>加入40微升0.05％胰蛋白酶/ EDTA溶液到96 U型底孔盘的12口井，并置于冰上的菜。 </li><li>菌落分离，在4F-MEF转碟吸介质之前。用10ml的PBS洗涤细胞一次。再次，加10毫升的PBS和地点培养皿倒置显微镜下。 </li><li>使用100微升移液器设置为40微升抬单个菌落，用C枪头ircling他们吸浮殖民地。一个菌落转移细胞每到准备好的96孔盘的一个孔中。采摘后12菌落置于在37℃下孵化5分钟的96孔培养皿中。 </li><li>通过上下吹打数次和转移细胞悬液与制备500μl的TS-CM培养基（30％的新鲜的TS培养基+ 70％的CM + FGF4 /肝素）的24孔培养皿的一个孔中解离的细胞。 </li><li>第二天用新鲜的TS-CM培养基更换培养基去除死细胞。更换培养基隔日1次。 </li><li>监视器明亮的界限上皮菌落外观（参见图2）。一旦出现菌落，培养至70％汇合或长达一个星期，并逐步扩大更大的菜肴。从现在开始，培养细胞作为真正的TSC，根据在含血清培养基或无血清，确定的培养基-3,5,11-标准协议。 </li></ol>

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The authors have nothing to disclose.

这里介绍的4因子（TFAP2C，GATA3，EOMES，ETS2）的转协议提供了一种可靠的方法来生成忠实地转换从小鼠胚胎成纤维细胞iTSCs。此外，该方法也适用于产后尾成纤维细胞，虽然在效率的降低相比胚胎成纤维细胞3。在一般情况下，初级成纤维细胞的质量是转结果和护理的一个关键因素，应采取使用早期传代细胞（传代两到三个）。 该协议的优点是使用强力霉素诱导的载体，...

最近，比较小鼠胚胎干细胞的几种方法来滋养层干细胞转化的一项研究显示，在所有的分析系统，沿袭转换仍然是不完整的。代替诱导滋养层干细胞（iTSCs）所谓的滋养层干的已产生细胞样细胞保持原点1的细胞命运的存储器中。在这里，我们跟着ITSC一代不同的方法。类似于来自小鼠胚胎成纤维细胞（MEF中）2多能干细胞的直接感应，iTSCs已直接从分化的体细胞组织转化。首先，我们确定了MEF中过表达时诱导TSC命运12个候选的因素。稍后，因素TFAP2C，GATA3，EOMES和ETS2已经确定是必要的和足够的ITSC感应3。同时，另一组独立地发现TFAP2C，GATA3和EOMES足以MEF中转换成iTSCs。然而，在研究中，对转基因表达所需要的时间相当长相比，我们的研究中，表明不同的转化动力学，当ETS2从转鸡尾酒4不存在。 含有诱导和囊胚衍生滋养层干细胞的培养常规胎牛血清（FBS）依赖于由生长灭活的MEF 5,6-分泌的因子的存在。在ITSC感应过程中，由MEF中，缺乏转基因的充分结合，并且不接受转提供了这些因素。然而，一旦个人ITSC菌落亚培养，它们需要媒体，已预处理由生长灭活的MEF。从那里，iTSCs可以培养并根据标准协议，如胚泡衍生的TSC处理。值得注意的是，在对比田中等人的5，我们经常培养的TSC和iTSCs无凝胶化的细胞培养皿。

<table><tbody><tr><td>Doxycycline hyclate</td><td>Sigma-Aldrich</td><td>D9891-10G</td><td>Dissolve in sterile PBS, prepare 2 mg/ml stock solution. Store aliquots at -20 &amp;deg;C</td></tr><tr><td>Chloroquine diphosphate salt&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C6628-25G</td><td>Prepare 25 mM stock solution in sterile cell culture grade water. Store aliquots at -20 &amp;deg;C</td></tr><tr><td>Polybrene (Hexadimethrine bromide)</td><td>Sigma-Aldrich</td><td>107689-10G</td><td>Prepare 8 mg/ml stock solution in sterile cell culture grade water. Store aliquots at -20 &amp;deg;C</td></tr><tr><td>human recombinant Fibroblast Growth Factor 4</td><td>ReliaTech</td><td>300-131L</td><td>Dissolve in sterile 0.1% BSA in PBS, prepare 25 &amp;micro;g/ml stock solution. Store aliquots at -80 &amp;deg;C</td></tr><tr><td>Heparin sodium salt</td><td>Sigma-Aldrich</td><td>H3149-10KU</td><td>Prepare 1 mg/ml stock solution by dissolving in sterile PBS. Store aliquots at -80&amp;deg;C</td></tr><tr><td>ProFection Mammalian Transfection System</td><td>Promega</td><td>E1200</td><td></td></tr><tr><td>PBS, no calcium, no magnesium</td><td>ThermoFisher Scientific</td><td>1419-094</td><td></td></tr><tr><td>DMEM (1x) + GlutaMAX</td><td>ThermoFisher Scientific</td><td>31966-021</td><td></td></tr><tr><td>RPMI 1640, no glutamine</td><td>ThermoFisher Scientific</td><td>31870-025</td><td></td></tr><tr><td>Advanced DMEM (1x)</td><td>ThermoFisher Scientific</td><td>12491-015</td><td></td></tr><tr><td>Trypsin-EDTA (0.05%), phenol red</td><td>ThermoFisher Scientific</td><td>25300-054</td><td></td></tr><tr><td>Poly-L-lysine</td><td>Sigma-Aldrich</td><td>P4707</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Hyclone</td><td>SH30071.03IR</td><td></td></tr><tr><td>Surfactant-free cellulose acetate-membrane filter</td><td>Corning</td><td>431220</td><td></td></tr><tr><td>Non-essential amino acids</td><td>ThermoFisher Scientific</td><td>11140-035</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>ThermoFisher Scientific</td><td>15140-122</td><td></td></tr><tr><td>Sodium Pyruvate&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>11360-039</td><td></td></tr><tr><td>L-glutamine&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>25030-24</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>ThermoFisher Scientific</td><td>31350-010</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO), cell culture grade</td><td>Panreac AppliChem GmbH</td><td>A3672,0100</td><td></td></tr><tr><td>pLV-tetO-Tfap2c</td><td>Addgene</td><td>70269</td><td></td></tr><tr><td>pLV-tetO-Gata3</td><td>Addgene</td><td>70270</td><td></td></tr><tr><td>pLV-tetO-Eomes</td><td>Addgene</td><td>70271</td><td></td></tr><tr><td>pLV-tetO-Ets2</td><td>Addgene</td><td>70272</td><td></td></tr><tr><td>pLV-tetO-mCherry</td><td>Addgene</td><td>70273</td><td></td></tr><tr><td>psPAX2</td><td>Addgene</td><td>12260</td><td></td></tr><tr><td>pMD2.G</td><td>Addgene</td><td>12259</td><td></td></tr><tr><td>FUdeltaGW-rtTA&amp;nbsp;</td><td>Addgene</td><td>19780</td><td></td></tr><tr><td>Mice B6.Cg-Gt(ROSA)26Sor&lt;sup&gt;tm1(rtTA*M2)Jae/J 7&lt;/sup&gt;</td><td>JAX Mice</td><td>6965</td><td></td></tr><tr><td>129S2SV</td><td>Charles River</td><td>129</td><td></td></tr></tbody></table>

protocol for the direct conversion of murine embryonic fibroblasts into trophoblast stem cells

滋养层干细胞（TSC的）出现在哺乳动物发育第一细胞命运决定的结果。它们可以在体外进行培养，保持能力自我更新和分化为滋养层谱系的所有子类型，等同于体内干细胞群从而引发胎盘的胎儿部。因此，的TSC提供了一个独特的模型来研究胎盘发育和胚胎与体外胚外细胞命运决定。从开始的囊胚阶段，包括DNA甲基化和组蛋白修饰明显的后生屏障分隔紧密谱系两者。在这里，我们描述了一个协议，通过瞬态过表达的滋养重点监管TFAP2C，GATA3，EOMES和ETS2在小鼠胚胎成纤维细胞完全克服这一障碍的血统。诱导滋养层干细胞是能自我更新和几乎相同的囊胚滋养衍生的干细胞我形态，标记基因的表达和甲基化模式的n项。功能的体外和体内测定确认这些细胞能够沿滋养层谱系产生多倍体滋养层巨细胞和当注入胚泡嵌合化胎盘区分。从体细胞组织滋养层干细胞的诱导打开了新的途径来研究这个胚外谱系遗传和表观遗传特性，并提供给产生滋养层干细胞系，而不破坏各自的胚胎的可能性。

上，其中4F的转基因表达被激活的菜，细胞迅速改变形态（比较图2A和B）。围绕天14 - 21不同转分化区域出现（两个例子在图2B和C给出）。这些主要的菌落缺乏典型的TSC形态;然而，一旦他们的子培养，特色上皮形态与紧边和明亮的界限高度回忆善意的TSC的出现（ 图2D）。 4F-iTSCs染色阳性的滋养层转录因子CDX2和TFAP2C（ 图2E）。</stro...

Watch this Scientific Journal Video about Protocol for the Direct Conversion of Murine Embryonic Fibroblasts into Trophoblast Stem Cells at JoVE.com

Protocol for the Direct Conversion of Murine Embryonic Fibroblasts into Trophoblast Stem Cells

Here we present a protocol for the direct conversion of murine embryonic fibroblasts into fully functional and stable trophoblast stem cells by ten day over-expression of Tfap2c, Gata3, Eomes and Ets2.

协议小鼠胚胎成纤维细胞直接转化为滋养层干细胞

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining ...

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JoVE Journal

Developmental Biology

7.6K 次观看.  Institute of Pathology, University of Bonn Medical School. Here we present a protocol for the direct conversion of murine embryonic fibroblasts into fully functional and stable trophoblast stem cells by ten day over-expression of Tfap2c, Gata3, Eomes and Ets2.

视频: Protocol for the Direct Conversion of Murine Embryonic Fibroblasts into Trophoblast Stem Cells

Kinetic Measurement and Real Time Visualization of Somatic Reprogramming

Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease phenotypes. Recent advances have identified more efficient and safe methods for introduction of reprogramming factors. However, there are few tools to monitor and track the progression of reprogramming. Current methods for monitoring reprogramming rely on the qualitative inspection of morphology or staining with stem cell-specific dyes and antibodies. Tools to dissect the progression of iPSC generation can help better understand the process under different conditions from diverse cell sources. 
This study presents key approaches for kinetic measurement of reprogramming progression using flow cytometry as well as real-time monitoring via imaging. To measure the kinetics of reprogramming, flow analysis was performed at discrete time points using antibodies against positive and negative pluripotent stem cell markers. The combination of real-time visualization and flow analysis enables the quantitative study of reprogramming at different stages and provides a more accurate comparison of different systems and methods. Real-time, image-based analysis was used for the continuous monitoring of fibroblasts as they are reprogrammed in a feeder-free medium system. The kinetics of colony formation was measured based on confluence in the phase contrast or fluorescence channels after staining with live alkaline phosphatase dye or antibodies against SSEA4 or TRA-1-60. The results indicated that measurement of confluence provides semi-quantitative metrics to monitor the progression of reprogramming.

The protocol presented in this study describes methods for the real-time monitoring of reprogramming progression via the kinetic measurement of positive and negative pluripotent stem cell markers using flow cytometry analysis. The protocol also includes the imaging-based assessment of morphology, and marker or reporter expression during iPSC generation.

体细胞重编程的动力学测量和实时可视化

Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease ...

科研

发育生物学

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

重新编程的小鼠胚胎成纤维细胞转录因子诱导Hemogenic计划

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

从小鼠羊水细胞多能干细胞使用转座子系统的生产

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

在人多能的体外干细胞向滋养层细胞

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage specification. In particular, the conversion of fibroblasts into induced cardiomyocyte-like myocytes (iCLMs) by Gata4, Hand2, Mef2c, and Tbx5 (GHMT) represents an important avenue for generating de novo cardiac myocytes in vitro and in vivo. Recent evidence suggests that GHMT generates a greater diversity of cardiac subtypes than previously appreciated, thus underscoring the need for a systematic approach to conducting additional studies. Before direct reprogramming can be used as a therapeutic strategy, however, the mechanistic underpinnings of lineage conversion must be understood in detail to generate specific cardiac subtypes. Here we present a detailed protocol for generating iCLMs by GHMT-mediated reprogramming of mouse embryonic fibroblasts (MEFs). 
We outline methods for MEF isolation, retroviral production, and MEF infection to accomplish efficient reprogramming. To determine the subtype identity of reprogrammed cells, we detail a step-by-step approach for performing immunocytochemistry on iCLMs using a defined set of compatible antibodies. Methods for confocal microscopy, identification, and quantification of iCLMs and individual atrial (iAM), ventricular (iVM), and pacemaker (iPM) subtypes are also presented. Finally, we discuss representative results of prototypical direct reprogramming experiments and highlight important technical aspects of our protocol to ensure efficient lineage conversion. Taken together, our optimized protocol should provide a stepwise approach for investigators to conduct meaningful cardiac reprogramming experiments that require identification of individual CM subtypes.

This manuscript describes a step-by-step protocol for the generation and quantification of diverse reprogrammed cardiac subtypes using a retrovirus-mediated delivery of Gata4, Hand2, Mef2c, and Tbx5.

评估心肌亚型以下鼠标胚胎成纤维细胞的转录因子介导的重编程

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage ...

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines retain the ability to either self-renew or differentiate under specific conditions. Due to these properties, mESC are a useful tool in regenerative medicine, disease modeling, and tissue engineering studies. This article describes a simple protocol to obtain mESC lines with high derivation efficiencies (60-80%) by culturing blastocysts from permissive mouse strains on feeder cells in defined medium supplemented with leukemia inhibitory factor. The protocol can also be applied to efficiently derive mESC lines from non-permissive mouse strains, by the simple addition of a cocktail of two small-molecule inhibitors to the derivation medium (2i medium). Detailed procedures on the preparation and culture of feeder cells, collection and culture of mouse embryos, and derivation and culture of mESC lines are provided. This protocol does not require specialized equipment and can be carried out in any laboratory with basic mammalian cell culture expertise.

This article describes a protocol to efficiently derive and culture pluripotent stem cell lines from mouse embryos at the blastocyst stage.

从小鼠胚胎植入前胚胎干细胞系的推导

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines ...

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. 
While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

Here we present our protocol for producing induced erythroid progenitors (iEPs) from mouse adult fibroblasts using transcription factor-driven direct lineage reprogramming (DLR).

成人小鼠成纤维细胞对红细胞生成子细胞的直接线重编程

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

鼠标滋养层干细胞从胚泡的推导

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...

生物学

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

适用于培养人类胚胎和诱导多能干细胞的小鼠胚胎成纤维细胞的制备

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

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 ...