This work was supported by CARIPARO Foundation Grant number 13/04 and Fondazione Istituto di Ricerca Pediatrica Citt&#224; della Speranza Grant number 10/02. Martina Piccoli, Chiara Franzin and Michela Pozzobon are funded by Fondazione Istituto di Ricerca Pediatrica Citt&#224; della Speranza. Enrica Bertin is funded by CARIPARO Foundation Grant number 13/04. Paolo De Coppi is funded by Great Ormond Street Hospital Children's Charity.

所有的程序都是按照意大利法律。鼠AF样品来自孕鼠13.5天交配后（DPC）从C57BL / 6-的Tg（UBC-GFP）30Scha / J小鼠被称为GFP收获。 1.转座子生产注意:使用标准克隆程序生成座子表达载体。使用商业试剂盒制备用于小鼠的AF细胞转染的质粒DNA。 <ol><li>混合的质粒DNA的10毫微克，在1.5ml微量离心管中加入50μl的DH5α细菌。在冰上孵育的微量离心管20分钟。 </li><li>在42℃下进行40秒的热休克。 </li><li>放置微量离心管在冰上2分钟。 </li><li>加入250微升预热（37°C）LB培养基（LB）肉汤。摇动（300转），在37℃30分钟。 </li><li>蔓延30微升各变换到LB平板上用0.1mg / ml氨苄青霉素。允许板干燥并在37℃OV孵育反转ernight。 </li><li>第二天，下午，挑3个菌落，用无菌200微升提示，从每个转化和转移到0.1毫克/毫升的LB放大器。 </li><li>摇动，在37℃过夜（转速300rpm）的细菌。 </li><li>对于质粒纯化使用商业试剂盒。股票的质粒在-20℃。 </li></ol> 2.小​​鼠胚胎成纤维细胞（MEF）文化<ol><li> 在6孔板的涂层用0.1％明胶 <ol><li>罩加入1毫升无菌0.1％明胶的每个六个孔的下涂层板。让在培养箱中（37℃）明胶聚合1小时。 </li><li>丢弃过量的，并干燥板1小时。 </li><li>用封口膜在室温下以密封和存储板。 </li></ol></li><li> MEF的灭活丝裂霉素 <ol><li>解冻使用37℃浴4×10 6 MEF细胞的小瓶。 </li><li>在培养皿（150毫米）MEF培养基罩种子MEF。 </li><li>培养细胞在37℃和5％的CO 2。 </li><li> （注意）加入丝裂霉素C（5毫克/毫升），当细胞融合。 </li><li>地方细胞进入培养箱（37℃和5％CO 2）3小时。 </li><li>用丝裂霉素C.洗涤细胞除去培养基用1×磷酸盐缓冲盐水（PBS）的三倍。 </li><li>加2ml商业胰蛋白酶和地点细胞进入37℃培养箱中培养5分钟。后，加入18毫升隔断介质停止胰蛋白酶的反应。 </li><li>算使用根据制造商的协议的burker室中的细胞。 </li><li>离心细胞，在145×g离心5分钟。弃去上清液。 </li><li>种子60,000个细胞/上0.1％明胶包被的板灭活的MEF的厘米2。 </li><li>培养细胞在37℃下24小时和5％的CO 2，播种AF和/或的iPS-AF细胞前。 </li></ol></li></ol> 3.鼠标AF细胞分离<ol><li>设置定时交配，检查阴道PLU合房24小时后GS。 </li><li>观察到13.5天，通过颈椎脱臼牺牲怀孕水坝。 </li><li>清洁用70％的乙醇的动物的腹壁。 </li><li>使用剪刀，执行中线剖腹切开腹壁的长度来访问进入腹腔。 </li><li>揭露使用镊子子宫。除去用剪刀和转让子宫的100毫米的培养皿充满无菌1×PBS中置于冰上。 </li><li>使用立体显微镜收集AF在10倍的放大倍率。 </li><li>握住钳子子宫和用剪刀取出子宫壁。要小心，以避免胎膜和随之而来的羊水泄漏任何损害。 </li><li>收集胎儿的100毫米的培养皿充满无菌1×PBS中，并放置在冰上。 </li><li>抓住一个胎儿用细镊子点和地点的胎盘在干净的100毫米的培养皿。 </li><li>取出卵黄囊用细镊子点。 </li><l我&gt;用细镊子点扰乱羊膜，并收集30微升的自动对焦使用胰岛素注射器到一个15毫升的小瓶锥形每个胎儿。 </li><li> AF收集之后，洗净用无菌1×PBS中的每个胎儿补充有1％胎牛血清（FBS），并收集到15ml含有自动对焦锥形管中。 </li><li>离心机对焦在145×g离心5分钟。引擎盖下去除上清，种子沉淀的AF细胞。 </li></ol> 4.鼠标AF细胞培养<ol><li> 鼠标AF细胞接种 <ol><li>播种前一天，准备与丝裂霉素C处理的MEF 0.1％明胶包被的6孔板中。 </li><li> AF收集之后，使用AF培养基种子从羊膜穿刺术获得的细胞（来自6胎儿得到约1×10 7个细胞）到有丝分裂失活的MEF。 </li><li>培养细胞在37℃和5％的CO 2，更换培养基每隔一天。 </li><li>经过7天的文化，跨FECT AF细胞按照以下所述的步骤。 </li></ol></li></ol> 5.转染，IPS-AF细胞的产生与文化注:鼠标AF细胞与PB-tetO2-IRES-OKMS座子的质粒转染，在转座表达质粒（mPBase）结合，并与反四环素反式（PB-CAG-rtTA）转座子的质粒。所有质粒通过安德拉什纳吉33教授提供。 <ol><li>在1.5ml微量离心管:混合PB-tetO2-IRES-OKMS的1微克用mPBase 0.5微克和0.5 PB-CAG-rtTA的微克（2微克总DNA）。 </li><li>稀释该DNA到100微升无血清培养基（Dulbecco改良的Eagle培养基 - DMEM）中。 </li><li>添加8微升转染试剂（ 例如 ，FUGENE）（在质粒DNA的比率:2微克（DNA）至8微升转染试剂转染试剂）放入含有DNA 1.5毫升离心管中。 </li><li>孵育转染试剂/ DNA混合物在室温下15分钟。 </li><li>逐滴加入100微升每孔的转染试剂/ DNA混合物的与小鼠AF细胞的6孔平板，并通过温和涡旋分布，用2mL的最终体积/孔。离开24小时。 </li><li>一天后，通过用补充有1.5微克/毫升的强力霉素（2毫升的媒体的每个孔）新鲜的iPS-AF介质供给细胞诱导的山中的因素（Oct4的，Klf4的，cMYC的，Sox2的）的表达。 </li><li>每天给细胞，而不通道，用新鲜的含DOX培养基（保持辅以强力霉素，直到点5.13介质细胞）。 </li><li>观察为mCherry表达在24小时内，并每日为集落形成（集落通常在转染后20到30天之间出现）细胞。 </li><li>殖民地采摘前一天，准备用丝裂霉素C处理的MEF 96孔培养板。 </li><li>在全光照量50微升接单菌落ガ200μL的吸移管，并依次传送，使它们成为单独的孔96孔板中。 </li><li>一天后，通过加入50μl商业胰蛋白酶的解离每一菌落成单个细胞，并在37℃下孵育5分钟。 </li><li>移液器上下分列的殖民地。 </li><li>转让50载涂96孔板0.1％的明胶分离的殖民地与灭活MEF新井的胰蛋白酶微升充满100微升辅以强力霉素新鲜的iPS-AF媒介。 </li><li>当细胞达到60-70％汇合时，分割成一个24孔板的孔中。 </li><li>当细胞达到60-70％汇合，拆1:2两口井，一用强力霉素和其他没有强力霉素，以验证是否殖民地也都出现在条件无强力霉素。 </li><li>继续保持的iPS-AF克隆，多西环素独立，灭活MEF在IPS-AF网上平台。 </li><li>培养在37℃和5％的CO 2中，细胞通道每天anging网上平台。 </li></ol> 6.碱性磷酸酶染色<ol><li>按照制造商的协议来执行碱性磷酸酶染色每当有强力霉素独立的iPS-AF细胞的外观。 </li></ol> 7.免疫<ol><li>每当有强力霉素独立的iPS-AF细胞的出现，种子与灭活的MEF 24孔板的孔150,000个细胞/厘米2，并在37℃和5％的CO 2培养细胞48小时。 </li><li> （注意）加入每4％低聚甲醛（PFA）的井300微升，在室温下15分钟固定细胞。 </li><li>添加300微升0.1％的NP-40通透细胞的。 </li><li>冲洗细胞与300微升0.1％PBS-吐温20。 </li><li>加入300微升封闭缓冲液（在1×PBS中的10％马血清）中在室温下1小时。 </li><li>使用以下主要抗体:Oct4的（1:80），Sox2的（1:80），Klf4的（1:80），Nanog的（1:100）和SSEA1（1:80）。稀释SSEA1用1％牛血清白蛋白（BSA），10％正常山羊血清，0.3M甘氨酸在0.1％PBS-Tween-20的。稀释在1×PBS中的10％马血清的所有其它抗体。 </li><li>孵育抗体过夜，在4℃。 </li><li>用300μl0.1％的PBS-Tween-20中的冲洗。 </li><li>使用在1X PBS中的10％马血清稀释的二级抗体:Alexa594缀合的山羊抗小鼠IgG（1:200），Alexa594缀合的鸡抗山羊IgG（1:200），Alexa594缀合的鸡抗兔IgG （1:200），Alexa568缀合的山羊抗小鼠IgM（1:200）。孵育在室温下2小时。 </li><li>用1X PBS冲洗。 </li><li>染色核用Hoechst溶液（1:10,000）在1×PBS中。 </li></ol> 8. 在体外细胞分化<ol><li>为形成悬滴胚状体（EBS），培养单滴（2,000个细胞/ 20微升）到培养皿盖子，用分化培养基。 </li><li>孵化的菜在37℃和5％的CO 2 4天。 </li><li>转移到EB中100毫米细菌级的菜肴在悬浮培养在分化培养基3天。 </li><li>板胚涂有基底膜基质（DMEM中1:10稀释）24孔板另外14天。 </li><li>对于免疫荧光分析，使用第一抗体:T（1:100），αfp（1:200）和TUBB3（1:500）37。 </li></ol> 9. 在体内形成畸胎瘤<ol><li>注入100μl的1×PBS中含有1×10 6的iPS-AF细胞进入的Rag2的后肢的肌肉- / - γC - / -小鼠。 </li><li>通过细胞注射6周后颈椎脱位牺牲小鼠。 </li><li>取出肿块，其大小区分，从小鼠用于以下分析的后肢。 </li><li>使用切低温恒温器异戊烷冷冻肿瘤的7-10微米厚的横截面。 </li><li> 对于苏木的ð曙红（HE）染色: <ol><li>用HE染色，以评估肿瘤组织组合物，按照制造商的协议。 </li></ol></li><li> 对于免疫过氧化物酶染色: <ol><li>固定畸胎瘤切片，用4％PFA中于室温下15分钟。 </li><li>用0.1％的Triton X-100透化。 </li></ol></li><li>用以下抗体孵育:TUBB3（1:100），αfp（1:50）和αSMA（1:100），在1X PBS中的1％BSA稀释。孵育在37℃（对TUBB3）1小时或过夜，在4℃下（对于其它抗体）。 </li><li>用100％甲醇20分钟块过氧化物酶。 </li><li>孵育在37℃下用45分钟的第二抗体抗小鼠HRP缀合（1:150）。 </li><li>用1X PBS冲洗。 </li><li>与过氧化物酶（HRP）底物（见材料表 ）5分钟孵育。 </li><li>用自来水冲洗5分钟。 </li><li>与苏木QS染色，持续9秒。 </li><li>用自来水冲洗。 </li></OL&gt; 从电池10 RNA提取<ol><li>根据制造商建议的协议使用商业的RNA提取试剂盒。 </li></ol>从畸胎瘤11 RNA提取<ol><li>均质使用杵和研钵和液氮，以避免解冻样品的畸胎瘤。 </li><li>称取25毫克的组织。 </li><li>添加1毫升的商业试剂提取RNA和氯仿的200微升。 </li><li>在室温下孵育5分钟。 </li><li>离心机以13,000 xg离心4℃15分钟。 </li><li>转移澄清的上清液至新管。 </li><li>以纯化的RNA，根据制造商的方案使用商业的RNA提取试剂盒。 </li></ol> 12.反转录聚合酶链反应<ol><li>合成使用反转录（RT）-polymerase链反应（PCR）和寡聚（dT）雅逆转录酶第一链cDNA叮叮制造商的协议，获得50纳克/ cDNA的微升。在10毫微克/微升的浓度与无RNA酶的水稀释的cDNA。 </li><li>利用RT-PCR的DNA聚合酶根据制造商的协议，使用1纳克执行/ cDNA的微升。 </li></ol>

作者什么都没有透露。

选择以获得多能性的诱导的方法是相关的细胞的临床安全相对于长期的移植。如今，有适合的重编程的几种方法。间的非整合方法中，仙台病毒（SeV载体）载体是能产生大量的蛋白的未经结合进感染的细胞40的核，可以是获得iPS细胞的策略的RNA病毒。 SeV载体可能是平移级iPS细胞的产生有吸引力的候选者，但它提出了一些缺点。病毒复制酶是转基因序列的性质敏感。由于SeV载体组?...

产前诊断是一个重要的临床工具，遗传性疾病（ 如染色体异常，单基因或多基因/多因素疾病）和先天性畸形（ 如先天性膈疝，囊性肺部病变，exomphalos，腹）评价。羊水（AF）细胞是简单怀孕的第二个三个月期间定期调度的程序，以获取（ 即羊膜穿刺术和amnioreduction）或剖腹产1，2。从产前或新生儿患者AF细胞的可用性提供了使用这种源再生医学的可能性，一些研究人员调查处理利用AF 3，4，5，6分离出来的干细胞群不同的组织损伤或疾病的可能性，7，8，9，10，11，12。容易地从患病的患者获得的AF细胞，其中的疾病是经常固定的时间窗口的可能性，开辟了道路使用用于重编程的目的本细胞来源的想法。的确，从AF细胞的诱导的多能干（iPS细胞）可以在用于体外药物测试或用于组织工程的方法感兴趣的细胞分化，为了分娩前准备适当的患者特异性治疗。许多研究已经证实的AF细胞的能力进行重新编程并分化成范围广泛的细胞类型13，14，15，16，17 &lt;的/ SUP&gt;，18，19，20，21，22，23，24，25，26，27。 由于通过四个转录因子（Oct4的，SOX2，cMYC的和Klf4）强制表达的发现高桥和重新编程体细胞的山中伸弥28日 ，取得了长足的重新编程领域取得。考虑到不同的方法，我们可以病毒和非病毒方法之间区别。所述第一预计的病毒载体（逆转录病毒和慢病毒），它们具有高效率，但在反转录病毒转基因的通常不完全沉默，具有部分重编程的细胞系的两种后果和风险的使用插入诱变29，30，31。的非病毒方法使用不同的策略: 即质粒，载体，基因，蛋白质，转座子。自由的转基因序列的iPS细胞的推导的目的，以规避渗漏的转基因表达和插入突变的潜在有害影响。在所有的上述的非病毒策略，所述piggyBac转（PB）转座子/转座系统只需要在反向末端重复侧翼的转基因和转座酶的瞬时表达来催化插入或切除事件32。在使用转座子上的其它方法iPS细胞产生的优点是获得免费矢量-iPS细胞与非病毒载体的方法，其中显示的逆转录病毒载体的相同的效率的可能性。这可以通过对reprogr集成座子编码的痕量少切除amming以下在iPS细胞33转座的新的瞬时表达的因素。鉴于PB为在不同的细胞类型34，35，36，37有效，是更适合于临床方法相对于病毒载体，并允许生产无异物-iPS细胞的违背使用的生物外源性电流病毒产生的协议条件下，该系统用于从鼠的AF获得iPS细胞。 在这里我们建议按照已经发表的作品展示从小鼠AF细胞（iPS-AF细胞）38生产多能干的iPS克隆的详细的协议。

<table><tbody><tr><td>100 mm Bacterial-grade Petri Dishes&amp;nbsp;</td><td>BD Falcon</td><td>351029</td><td>For &lt;em&gt;in vitro&lt;/em&gt; differentiation</td></tr><tr><td>2-mercaptoethanol&amp;nbsp;</td><td>Sigma</td><td>M6250</td><td>For mouse AF, iPS-AF cells and differentiation medium</td></tr><tr><td>Alexa568-conjugated goat anti-mouse IgM&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>A21043</td><td>Secondary antibody (immunofluorescence)</td></tr><tr><td>Alexa594-conjugated chicken anti-goat IgG&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>A21468</td><td>Secondary antibody (immunofluorescence)</td></tr><tr><td>Alexa594-conjugated chicken anti-rabbit IgG&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>A21442</td><td>Secondary antibody (immunofluorescence)</td></tr><tr><td>Alexa594-conjugated goat anti-mouse IgG&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>A11005</td><td>Secondary antibody (immunofluorescence)</td></tr><tr><td>Alkaline Phosphatase kit&amp;nbsp;</td><td>Sigma</td><td>85L1</td><td>Alkaline Phosphatase&amp;nbsp; staining</td></tr><tr><td>Ampicillin</td><td>Sigma</td><td>A0166</td><td>For bacterial selection</td></tr><tr><td>Bovine Serum Albumin&amp;nbsp;</td><td>Sigma</td><td>A7906</td><td>BSA, for blocking solution. Diluted in PBS 1x</td></tr><tr><td>Chloroform</td><td>Sigma</td><td>C2432</td><td>For RNA extraction</td></tr><tr><td>DH5&amp;alpha; cells</td><td>Thermo Fisher Scientific</td><td>18265-017</td><td>Bacteria for cloning procedure</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Thermo Fisher Scientific</td><td>41965039</td><td>For MEF, mouse AF, iPS-AF cells and differentiation medium</td></tr><tr><td>Doxycycline&amp;nbsp;</td><td>Sigma</td><td>D9891</td><td>For exogenous factors expression</td></tr><tr><td>Microcentrifuge tubes (1.5 ml)&amp;nbsp;</td><td>Sarstedt&amp;nbsp;</td><td>72.706</td><td>For PB production&amp;nbsp;</td></tr><tr><td>ES FBS&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>10439024</td><td>For mouse AF, iPS-AF cells and differentiation medium</td></tr><tr><td>FBS&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>10270106</td><td>For MEF medium</td></tr><tr><td>Fine point forceps</td><td>F.S.T</td><td>Dumont #5&amp;nbsp;</td><td>AF isolation</td></tr><tr><td>Gelatin</td><td>J.T.Baker</td><td>131</td><td>Used 0.1%, diluted in PBS 1x</td></tr><tr><td>Glycine</td><td>Bio-Rad</td><td>161-0718</td><td>For blocking solution. Diluted in PBS 1x</td></tr><tr><td>Haematoxylin QS</td><td>Vector Laboratories</td><td>H3404</td><td>Nuclei detection</td></tr><tr><td>HE&amp;nbsp;</td><td>Bio-Optica</td><td>04-061010</td><td>Histological analysis of teratoma</td></tr><tr><td>Hoechst&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>H3570</td><td>Nuclei detection</td></tr><tr><td>Horse Serum&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>16050-122</td><td>For blocking solution</td></tr><tr><td>HRP-conjugated goat anti-mouse IgG</td><td>SantaCruz</td><td>sc2005</td><td>Secondary antibody (immunoperoxidase)</td></tr><tr><td>ImmPACT NovaRED&amp;nbsp;</td><td>Vector Laboratories</td><td>SK4805</td><td>Peroxidase substrate</td></tr><tr><td>Insulin syringe with needle (25 G)</td><td>Terumo</td><td>SS+01H25161</td><td>Amniocentesis procedure</td></tr><tr><td>Klf4&amp;nbsp;</td><td>SantaCruz</td><td>sc-20691</td><td>Rabbit polyclonal IgG</td></tr><tr><td>L-glutamine&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>25030</td><td>For mouse AF, iPS-AF cells and differentiation medium</td></tr><tr><td>LB broth (Lennox)</td><td>Sigma</td><td>L3022</td><td>For bacterial growth</td></tr><tr><td>LIF&amp;nbsp;</td><td>Sigma</td><td>L5158</td><td>For mouse AF and iPS-AF cells medium</td></tr><tr><td>Matrigel&amp;nbsp;</td><td>BD</td><td>354234</td><td>For &lt;em&gt;in vitro&lt;/em&gt; differentiation. Diluted 1:10 in DMEM</td></tr><tr><td>Methanol</td><td>Sigma</td><td>32213</td><td>Peroxidase blocking</td></tr><tr><td>MULTIWELL 24 well plate</td><td>BD Falcon</td><td>353047</td><td>For &lt;em&gt;in vitro&lt;/em&gt; differentiation</td></tr><tr><td>MULTIWELL 6 well plate</td><td>BD Falcon</td><td>353046</td><td>For MEF, mouse AF and iPS-AF cells culture</td></tr><tr><td>Nanog&amp;nbsp;</td><td>ReproCELL</td><td>RCAB0002P-F</td><td>Rabbit polyclonal IgG</td></tr><tr><td>Non-essential amino acids&amp;nbsp;</td><td>Sigma</td><td>M7145</td><td>For mouse AF, iPS-AF cells and differentiation medium</td></tr><tr><td>Normal Goat Serum</td><td>Vector Laboratories</td><td>S2000</td><td>For blocking solution. Diluted in PBS 1x</td></tr><tr><td>NP-40</td><td>Sigma</td><td>12087-87-0</td><td>For cell permeabilization. Diluted in PBS 1x</td></tr><tr><td>Oct4</td><td>SantaCruz</td><td>sc-5279</td><td>Mouse monoclonal IgG2b</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Oligo (dT)&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>18418012</td><td>For RT-PCR</td></tr><tr><td>Paraformaldehyde (solution)</td><td>Sigma</td><td>441244</td><td>PFA, fixative, diluted in PBS</td></tr><tr><td>PBS 10x</td><td>Thermo Fisher Scientific</td><td>14200-067</td><td>D-PBS, free of Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;. Diluted with sterile water to obtain PBS 1x</td></tr><tr><td>Penicillin - Streptomycin&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>15070063</td><td>For MEF, mouse AF, iPS-AF cells and differentiation medium</td></tr><tr><td>Petri Dish (150 mm)</td><td>BD Falcon</td><td>353025</td><td>For MEF culture, tissue culture</td></tr><tr><td>PiggyBac transposase expression plasmid&amp;nbsp;</td><td>Provided by professor Andras Nagy laboratory</td><td>-</td><td>mPBase</td></tr><tr><td>PiggyBac-tetO2-IRES-OKMS transposon plasmid</td><td>Provided by professor Andras Nagy laboratory</td><td>-</td><td>PB-tetO2-IRES-OKMS</td></tr><tr><td>QIAprep Spin Maxiprep Kit</td><td>Qiagen</td><td>12663</td><td>For plasmids purification</td></tr><tr><td>QIAprep Spin Miniprep Kit</td><td>Qiagen</td><td>27106</td><td>For plasmids purification</td></tr><tr><td>Reverse tetracycline transactivator transposon plasmid&amp;nbsp;</td><td>Provided by professor Andras Nagy laboratory</td><td>-</td><td>rtTA</td></tr><tr><td>RNeasy Mini Kit&amp;nbsp;</td><td>Qiagen</td><td>74134</td><td>For RNA extraction</td></tr><tr><td>Sox2&amp;nbsp;</td><td>SantaCruz</td><td>sc-17320</td><td>Goat polyclonal IgG</td></tr><tr><td>SSEA1&amp;nbsp;</td><td>Abcam</td><td>ab16285</td><td>Mouse monoclonal IgM</td></tr><tr><td>SuperScript II Reverse Transcriptase&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>18064-014</td><td>For RT-PCR</td></tr><tr><td>T&amp;nbsp;</td><td>Abcam</td><td>ab20680</td><td>Rabbit polyclonal IgG</td></tr><tr><td>Taq DNA Polymerase</td><td>Thermo Fisher Scientific</td><td>10342020</td><td>PCR</td></tr><tr><td>Trypsin&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>25300-054</td><td>Cell culture passaging</td></tr><tr><td>Triton X-100</td><td>Bio-Rad</td><td>161-047</td><td>For cell permeabilization, diluted in PBS 1x</td></tr><tr><td>TRIzol Reagent</td><td>Thermo Fisher Scientific</td><td>15596-026</td><td>For RNA extraction</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Tubb3&amp;nbsp;&amp;nbsp;</td><td>Promega&amp;nbsp;</td><td>G712A</td><td>Mouse monoclonal IgG1</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>TWEEN-20</td><td>Sigma</td><td>P1379</td><td>For cell permeabilization, diluted in PBS 1x</td></tr><tr><td>&amp;alpha;fp&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>R&amp;amp;D Systems</td><td>MAB1368</td><td>Mouse Monoclonal IgG1</td></tr><tr><td>&amp;alpha;SMA&amp;nbsp;</td><td>Abcam</td><td>ab7817</td><td>Mouse Monoclonal IgG2a</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Transfection Reagent (FuGENE HD)</td><td>Promega&amp;nbsp;</td><td>E2311</td><td>For AF cells transfection</td></tr><tr><td>Stereomicroscope</td><td>Nikon</td><td>SM2645</td><td>To perform amniocentesis&amp;nbsp;</td></tr><tr><td>200 &amp;mu;l tips</td><td>Sarstedt&amp;nbsp;</td><td>70.760012</td><td>To pick bacteria colonies</td></tr><tr><td>Scissor</td><td>F.S.T</td><td>14094-11 stainless 25U</td><td>To perform amniocentesis&amp;nbsp;</td></tr><tr><td>Ethanol</td><td>Sigma</td><td>2860</td><td>To clean the abdominal wall of the pregnant dam</td></tr><tr><td>Tissue culture petri dish (150 mm)&amp;nbsp;</td><td>BD Falcon</td><td>353025</td><td>For MEF expansion</td></tr><tr><td>Mitomycin C</td><td>Sigma</td><td>M4287-2MG</td><td>For MEF inactivation</td></tr><tr><td>MULTIWELL 96 well plate</td><td>BD Falcon</td><td>353071</td><td>For iPS-AF culture</td></tr></tbody></table>

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.

为了评估重新编程的能力，小鼠的AF细胞从GFP小鼠的胎儿收集。细胞用圆形座子的质粒PB-tetO2-IRES-OKMS，它表示连接到mCherry荧光蛋白在多西环素诱导的方式山因子（Oct4的，SOX2，cMYC的和Klf4）转染，和反向四环素式激活（PB- CAG-rtTA）与转座表达质粒（mPBase）一起质粒。小鼠的AF细胞1周超过MEF饲养层体外膨胀后的Oct4，Klf4的，cMYC的和Sox2染。对于外源因子的表达，强力霉?...

Watch this Scientific Journal Video about The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System at JoVE.com

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

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 ...

the-production-pluripotent-stem-cells-from-mouse-amniotic-fluid-cells

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

6.9K 次观看.  Fondazione Istituto di Ricerca Pediatrica Citta della Speranza. In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system. 

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

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

使用非整合附加型质粒从冷冻血沉棕黄层诱导多能干细胞的产生

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

科研

发育生物学

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

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining the ability to self-renew and to differentiate into all subtypes of the trophoblast lineage, equivalent to the in vivo stem cell population giving rise to the fetal portion of the placenta. Therefore, TSCs offer a unique model to study placental development and embryonic versus extra-embryonic cell fate decision in vitro. From the blastocyst stage onwards, a distinct epigenetic barrier consisting of DNA methylation and histone modifications tightly separates both lineages. Here, we describe a protocol to fully overcome this lineage barrier by transient over-expression of trophoblast key regulators Tfap2c, Gata3, Eomes and Ets2 in murine embryonic fibroblasts. The induced trophoblast stem cells are able to self-renew and are almost identical to blastocyst derived trophoblast stem cells in terms of morphology, marker gene expression and methylation pattern. Functional in vitro and in vivo assays confirm that these cells are able to differentiate along the trophoblast lineage generating polyploid trophoblast giant cells and chimerizing the placenta when injected into blastocysts. The induction of trophoblast stem cells from somatic tissue opens new avenues to study genetic and epigenetic characteristics of this extra-embryonic lineage and offers the possibility to generate trophoblast stem cell lines without destroying the respective embryo.

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 ...

Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006; Wernig, et al., 2007; Okita, et al., 2007; Maherali, et al., 2007). We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors (Brambrink et al., 2008). Using these inducible constructs, we can derive induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs). In this video, we demonstrate the procedure for the generation of inducible lentiviruses that express the four transcription factors and show how to infect MEFs with these viruses in order to produce iPS cells. By using inducible lentiviruses, the expression of the four factors in controlled by the addition of doxycyline to the culture medium. The advantage of this system over the traditional retroviral infection is the ability to turn the genes on and off so that the kinetics of reprogramming and gene expression requirements can be analyzed in detail.

This video shows the procedure for generating induced pluripotent stem cells using inducible lentivirus that express Oct4, Sox2, c-Myc and Klf4.

从MEFS iPS细胞生成通过强制SOX - 2的表达，10月4，c - Myc基因和Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006;  Wernig, et al., ...

生物学

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

从人体外周血单个核细胞融合，无诱导多能干细胞的生成方法游离型载体

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Reprogramming Primary Amniotic Fluid and Membrane Cells to Pluripotency in Xeno-free Conditions

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic fluid and membrane mesenchymal stem cells, represent a unique type of undifferentiated cells with promise in tissue engineering and for reprogramming into iPSC for future pediatric interventions and stem cell banking. The protocol presented here describes an optimized procedure for extracting and culturing primary amniotic fluid and membrane mesenchymal stem cells and generating episomal induced pluripotent stem cells from these cells in fully chemically defined culture conditions utilizing human recombinant vitronectin and the E8 medium. Characterization of the new lines by applying stringent methods &#8211; flow cytometry, confocal imaging, teratoma formation and transcriptional profiling &#8211; is also described. The newly generated lines express markers of embryonic stem cells &#8211; Oct3/4A, Nanog, Sox2, TRA-1-60, TRA-1-81, SSEA-4 &#8211; while being negative for the SSEA-1 marker. The stem cell lines form teratomas in scid-beige mice in 6-8 weeks and the teratomas contain tissues representative of all three germ layers. Transcriptional profiling of the lines by submitting global expression microarray data to a bioinformatic pluripotency assessment algorithm deemed all lines pluripotent and therefore, this approach is an attractive alternative to animal testing. The new iPSC lines can readily be used in downstream experiments involving the optimization of differentiation and tissue engineering.

This protocol describes the reprogramming of primary amniotic fluid and membrane mesenchymal stem cells into induced pluripotent stem cells using a non-integrating episomal approach in fully chemically defined conditions. Procedures of extraction, culture, reprogramming, and characterization of the resulting induced pluripotent stem cells by stringent methods are detailed.

无异条件下多原代羊水和膜细胞重新编程

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic ...

Introducing Point Mutations into Human Pluripotent Stem Cells Using Seamless Genome Editing

Custom designed endonucleases, such as RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, enable efficient genome editing in mammalian cells. Here we describe detailed procedures to seamlessly genome edit the hepatocyte nuclear factor 4 alpha (HNF4&#945;) locus as an example in human pluripotent stem cells. Combining a piggyBac-based donor plasmid and the CRISPR-Cas9 nickase mutant in a two-step genetic selection, we demonstrate correct and efficient targeting of the HNF4&#945; locus.

Here, we describe a detailed method for seamless gene editing in human pluripotent stem cells using a piggyBac-based donor plasmid and the Cas9 nickase mutant. Two point mutations were introduced into exon 8 of the hepatocyte nuclear factor 4 alpha (HNF4&#945;) locus in human embryonic stem cells (hESCs).

使用无缝基因组编辑将点突变引入人类多能干细胞

Custom designed endonucleases, such as RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, enable efficient genome editing ...

遗传学

Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc, and Klf4 is capable of inducing pluripotency in mouse fibroblasts.1 Subsequent reports have demonstrated the utility of the doxycycline (DOX) inducible lentiviral delivery system for the generation of both primary and secondary iPS cells from a variety of other adult mouse somatic cell types.2,3
Induced pluripotent stem (iPS) cells are similar to embryonic stem (ES) cells in morphology, proliferation and ability to induce teratoma formation. Both types of cell can be used as the pluripotent starting material for the generation of differentiated cells or tissues in regenerative medicine.4-6 iPS cells also have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction.
Here we demonstrate the protocol for reprogramming mouse embryonic fibroblast (MEF) cells with the Stemgent DOX Inducible Mouse TF Lentivirus Set. We also demonstrate that the Stemgent DOX Inducible Mouse TF Lentivirus Set is capable of expressing each of the four transcription factors upon transduction into MEFs thereby inducing a pluripotent stem cell state that displays the pluripotency markers characteristic of ES cells.

The Stemgent Dox Inducible Mouse TF Lentivirus Set can reprogram mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. Here we demonstrate the protocol for DOX-inducible expression of mouse reprogramming transcription factors Oct4, Sox2, Klf4 and c-Myc to generate iPS colonies that express common mES pluripotency markers.

重新用四个转录因子的小鼠胚胎成纤维细胞诱导多能干细胞的产生，强力霉素诱导慢病毒转导系统

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)1-4. Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies5.
We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors6. These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs7-9. In one study, a Tet inducible version of the STEMCCA vector was employed9, which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies.
Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described10,11 in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

代人类诱导多能干细胞从外周血中使用STEMCCA慢病毒载体

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...

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 ...