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 border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="84">
			<td height="84" style="height:84px;width:199px;">Doxycycline hyclate</td>
			<td style="width:93px;">Sigma-Aldrich</td>
			<td style="width:101px;">D9891-10G</td>
			<td style="width:180px;">Dissolve in sterile PBS, prepare 2 mg/ml stock solution. Store aliquots at -20&#176;C</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:199px;">Chloroquine diphosphate salt&#160;</td>
			<td style="width:93px;">Sigma-Aldrich</td>
			<td>C6628-25G</td>
			<td style="width:180px;">Prepare 25 mM stock solution in sterile cell culture grade water. Store aliquots at -20&#176;C</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:199px;">Polybrene (Hexadimethrine bromide)</td>
			<td style="width:93px;">Sigma-Aldrich</td>
			<td>107689-10G</td>
			<td style="width:180px;">Prepare 8 mg/ml stock solution in sterile cell culture grade water. Store aliquots at -20&#176;C</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:199px;">human recombinant Fibroblast Growth Factor 4</td>
			<td style="width:93px;">ReliaTech</td>
			<td>300-131L</td>
			<td style="width:180px;">Dissolve in sterile 0.1% BSA in PBS, prepare 25 &#181;g/ml stock solution. Store aliquots at -80&#176;C</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:199px;">Heparin sodium salt</td>
			<td style="width:93px;">Sigma-Aldrich</td>
			<td>H3149-10KU</td>
			<td style="width:180px;">Prepare 1 mg/ml stock solution by dissolving in sterile PBS. Store aliquots at -80&#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:199px;">ProFection Mammalian Transfection System</td>
			<td style="width:93px;">Promega</td>
			<td>E1200</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:199px;">PBS, no calcium, no magnesium</td>
			<td style="width:93px;">ThermoFisher Scientific</td>
			<td>1419-094</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:199px;">DMEM (1x) + GlutaMAX</td>
			<td style="width:93px;">ThermoFisher Scientific</td>
			<td>31966-021</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:199px;">RPMI 1640, no glutamine</td>
			<td style="width:93px;">ThermoFisher Scientific</td>
			<td>31870-025</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:199px;">Advanced DMEM (1x)</td>
			<td style="width:93px;">ThermoFisher Scientific</td>
			<td>12491-015</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:199px;">Trypsin-EDTA (0.05%), phenol red</td>
			<td style="width:93px;">ThermoFisher Scientific</td>
			<td>25300-054</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:199px;">Poly-L-lysine</td>
			<td style="width:93px;">Sigma-Aldrich</td>
			<td>P4707</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">Fetal bovine serum</td>
			<td style="width:93px;">Hyclone</td>
			<td>SH30071.03IR</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:199px;">Surfactant-free cellulose acetate-membrane filter</td>
			<td style="width:93px;">Corning</td>
			<td>431220</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:199px;">Non-essential amino acids</td>
			<td style="width:93px;">ThermoFisher Scientific</td>
			<td>11140-035</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:199px;">Penicillin Streptomycin</td>
			<td style="width:93px;">ThermoFisher Scientific</td>
			<td>15140-122</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:199px;">Sodium Pyruvate&#160;</td>
			<td style="width:93px;">ThermoFisher Scientific</td>
			<td>11360-039</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:199px;">L-glutamine&#160;</td>
			<td style="width:93px;">ThermoFisher Scientific</td>
			<td>25030-24</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:199px;">2-Mercaptoethanol</td>
			<td style="width:93px;">ThermoFisher Scientific</td>
			<td>31350-010</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:199px;">Dimethyl sulfoxide (DMSO), cell culture grade</td>
			<td style="width:93px;">Panreac AppliChem GmbH</td>
			<td>A3672,0100</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">pLV-tetO-Tfap2c</td>
			<td style="width:93px;">Addgene</td>
			<td>70269</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">pLV-tetO-Gata3</td>
			<td style="width:93px;">Addgene</td>
			<td>70270</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">pLV-tetO-Eomes</td>
			<td style="width:93px;">Addgene</td>
			<td>70271</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">pLV-tetO-Ets2</td>
			<td style="width:93px;">Addgene</td>
			<td>70272</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">pLV-tetO-mCherry</td>
			<td style="width:93px;">Addgene</td>
			<td>70273</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">psPAX2</td>
			<td style="width:93px;">Addgene</td>
			<td>12260</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">pMD2.G</td>
			<td style="width:93px;">Addgene</td>
			<td>12259</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">FUdeltaGW-rtTA&#160;</td>
			<td style="width:93px;">Addgene</td>
			<td>19780</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Mice B6.Cg-Gt(ROSA)26Sortm1(rtTA*M2)Jae/J 7</td>
			<td style="width:93px;">JAX Mice</td>
			<td>6965</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:199px;">129S2SV</td>
			<td style="width:93px;">Charles River</td>
			<td>129</td>
			<td style="width:180px;"></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 ...

protocol-for-direct-conversion-murine-embryonic-fibroblasts-into

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Developmental Biology

7.5K 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

Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such cells are not readily available, they can be obtained using different approaches that include, among the many, reprogramming and trans-differentiation, with advantages and limitations that are specific of the different techniques. Here a new strategy for the conversion of an adult mature fibroblast into an insulin-secreting cell, arbitrarily designated as epigenetic converted cells (EpiCC), is described. The method has been developed, based on the increasing understanding of the mechanisms controlling epigenetic regulation of cell fate and differentiation. In particular, the first step uses an epigenetic modifier, namely 5-aza-cytidine, to drive adult cells into a "highly permissive" state. It then takes advantage of this brief and reversible window of epigenetic plasticity, to re-address cells toward a different lineage. The approach is designated "epigenetic cell conversion". It is a simple and robust way to obtain an efficient, controlled and stable cellular inter-lineage switch. Since the protocol does not involve the use of any gene transfection, it is free of viral vectors and does not involve a stable pluripotent state, it is highly promising for translational medicine applications.

Here, a new method that allows the conversion of adult skin fibroblasts into insulin-secreting cells is presented. This technique is based on epigenetic conversion, does not involve the use of retroviral vectors nor the acquisition of a stable pluripotent state. It is therefore highly promising for translational medicine applications.

后生转换为安全和简单的方法来获得成人皮肤成纤维细胞胰岛素分泌细胞

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such ...

科研

发育生物学

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

小鼠胚胎Hemogenic内皮细胞的分离

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

从人多能干细胞使用高度可重复的小分子为基础的分化协议心肌细胞规模化生产

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

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 ...

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.

Human induced pluripotent stem cells (hiPSCs) are considered a powerful tool for drug and chemical screening and for the development of new in vitro models for toxicity testing, including neurotoxicity. Here, a detailed protocol for the differentiation of hiPSCs into neurons and glia is described.

将人诱导的多能干细胞分化为神经元和神经胶质的混合培养物进行神经毒性测试的方案

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

水平整体安装:厚三维组织横截面皮肤的新型加工和成像方案

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

人胚胎或诱导多能干细胞对视网膜色素上皮细胞的快速定向分化

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

活完好斑马鱼的单细胞 Photoconversion

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative therapeutic requires a safe, robust, and expedient reprogramming protocol. Here, we present a clinically relevant, step-by-step protocol for the extremely high-efficiency reprogramming of human dermal fibroblasts into iPSCs using a non-integrating approach. The core of the protocol consists of expressing pluripotency factors (SOX2, KLF4, cMYC, LIN28A, NANOG, OCT4-MyoD fusion) from in vitro transcribed messenger RNAs synthesized with modified nucleotides (modified mRNAs). The reprogramming modified mRNAs are transfected into primary fibroblasts every 48 h together with mature embryonic stem cell-specific microRNA-367/302 mimics for two weeks. The resulting iPSC colonies can then be isolated and directly expanded in feeder-free conditions. To maximize efficiency and consistency of our reprogramming protocol across fibroblast samples, we have optimized various parameters including the RNA transfection regimen, timing of transfections, culture conditions, and seeding densities. Importantly, our method generates high-quality iPSCs from most fibroblast sources, including difficult-to-reprogram diseased, aged, and/or senescent samples.

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

基于 rna 的人原代成纤维细胞重组成诱导多能干细胞

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...