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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 mm Bacterial-grade Petri Dishes&#160;</td>
			<td>BD Falcon</td>
			<td>351029</td>
			<td>For in vitro differentiation</td>
		</tr>
		<tr>
			<td>2-mercaptoethanol&#160;</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&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>A21043</td>
			<td>Secondary antibody (immunofluorescence)</td>
		</tr>
		<tr>
			<td>Alexa594-conjugated chicken anti-goat IgG&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>A21468</td>
			<td>Secondary antibody (immunofluorescence)</td>
		</tr>
		<tr>
			<td>Alexa594-conjugated chicken anti-rabbit IgG&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>A21442</td>
			<td>Secondary antibody (immunofluorescence)</td>
		</tr>
		<tr>
			<td>Alexa594-conjugated goat anti-mouse IgG&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11005</td>
			<td>Secondary antibody (immunofluorescence)</td>
		</tr>
		<tr>
			<td>Alkaline Phosphatase kit&#160;</td>
			<td>Sigma</td>
			<td>85L1</td>
			<td>Alkaline Phosphatase&#160; staining</td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Sigma</td>
			<td>A0166</td>
			<td>For bacterial selection</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin&#160;</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 rowspan="2">DH5&#945; cells</td>
			<td rowspan="2">Thermo Fisher Scientific</td>
			<td rowspan="2">18265-017</td>
			<td rowspan="2">Bacteria for cloning procedure</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>Dulbecco&#39;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&#160;</td>
			<td>Sigma</td>
			<td>D9891</td>
			<td>For exogenous factors expression</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes (1.5 mL)&#160;</td>
			<td>Sarstedt&#160;</td>
			<td>72.706</td>
			<td>For PB production&#160;</td>
		</tr>
		<tr>
			<td>ES FBS&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>10439024</td>
			<td>For mouse AF, iPS-AF cells and differentiation medium</td>
		</tr>
		<tr>
			<td>FBS&#160;</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&#160;</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&#160;</td>
			<td>Bio-Optica</td>
			<td>04-061010</td>
			<td>Histological analysis of teratoma</td>
		</tr>
		<tr>
			<td>Hoechst&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>H3570</td>
			<td>Nuclei detection</td>
		</tr>
		<tr>
			<td>Horse Serum&#160;</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&#160;</td>
			<td>Vector Laboratories</td>
			<td>SK4805</td>
			<td>Peroxidase substrate</td>
		</tr>
		<tr>
			<td>Insulin syringe with needle (25G)</td>
			<td>Terumo</td>
			<td>SS+01H25161</td>
			<td>Amniocentesis procedure</td>
		</tr>
		<tr>
			<td>Klf4&#160;</td>
			<td>SantaCruz</td>
			<td>sc-20691</td>
			<td>Rabbit polyclonal IgG</td>
		</tr>
		<tr>
			<td>L-glutamine&#160;</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&#160;</td>
			<td>Sigma</td>
			<td>L5158</td>
			<td>For mouse AF and iPS-AF cells medium</td>
		</tr>
		<tr>
			<td>Matrigel&#160;</td>
			<td>BD</td>
			<td>354234</td>
			<td>For in vitro 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 in vitro 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&#160;</td>
			<td>ReproCELL</td>
			<td>RCAB0002P-F</td>
			<td>Rabbit polyclonal IgG</td>
		</tr>
		<tr>
			<td>Non-essential amino acids&#160;</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 rowspan="2">Oct4</td>
			<td rowspan="2">SantaCruz</td>
			<td rowspan="2">sc-5279</td>
			<td rowspan="2">Mouse monoclonal IgG2b</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>Oligo (dT)&#160;</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 Ca2+/Mg2+. Diluted with sterile water to obtain PBS 1X</td>
		</tr>
		<tr>
			<td>Penicillin - Streptomycin&#160;</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 (150mm)</td>
			<td>BD Falcon</td>
			<td>353025</td>
			<td>For MEF culture, tissue culture</td>
		</tr>
		<tr>
			<td>PiggyBac transposase expression plasmid&#160;</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&#160;</td>
			<td>Provided by professor Andras Nagy laboratory</td>
			<td>-</td>
			<td>rtTA</td>
		</tr>
		<tr>
			<td>RNeasy Mini Kit&#160;</td>
			<td>Qiagen</td>
			<td>74134</td>
			<td>For RNA extraction</td>
		</tr>
		<tr>
			<td>Sox2&#160;</td>
			<td>SantaCruz</td>
			<td>sc-17320</td>
			<td>Goat polyclonal IgG</td>
		</tr>
		<tr>
			<td>SSEA1&#160;</td>
			<td>Abcam</td>
			<td>ab16285</td>
			<td>Mouse monoclonal IgM</td>
		</tr>
		<tr>
			<td>SuperScript II Reverse Transcriptase&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>18064-014</td>
			<td>For RT-PCR</td>
		</tr>
		<tr>
			<td>T&#160;</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&#160;</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 rowspan="2">TRIzol Reagent</td>
			<td rowspan="2">Thermo Fisher Scientific</td>
			<td rowspan="2">15596-026</td>
			<td rowspan="2">For RNA extraction</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td rowspan="2">Tubb3&#160;&#160;</td>
			<td rowspan="2">Promega&#160;</td>
			<td rowspan="2">G712A</td>
			<td rowspan="2">Mouse monoclonal IgG1</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>TWEEN-20</td>
			<td>Sigma</td>
			<td>P1379</td>
			<td>For cell permeabilization, diluted in PBS 1X</td>
		</tr>
		<tr>
			<td>&#945;fp&#160;&#160;&#160;</td>
			<td>R&#38;D Systems</td>
			<td>MAB1368</td>
			<td>Mouse Monoclonal IgG1</td>
		</tr>
		<tr>
			<td rowspan="2">&#945;SMA&#160;</td>
			<td rowspan="2">Abcam</td>
			<td rowspan="2">ab7817</td>
			<td rowspan="2">Mouse Monoclonal IgG2a</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>Transfection Reagent (FuGENE HD)</td>
			<td>Promega&#160;</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&#160;</td>
		</tr>
		<tr>
			<td>200 ul tips</td>
			<td>Sarstedt&#160;</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&#160;</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)&#160;</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

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

多能干细胞的海藻酸钠封装使用同轴喷嘴

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

科研

发育生物学

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

从人多能干细胞的黑素细胞的无饲养推导

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

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 ...

Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent state. Now, reprogramming is done with various types of adult somatic cells: keratinocytes, urine cells, fibroblasts, etc. Early experiments were usually done with dermal fibroblasts. However, this required an invasive surgical procedure to obtain fibroblasts from the patients. Therefore, suspension cells, such as blood and urine cells, were considered ideal for reprogramming because of the convenience of obtaining the primary cells. Here, we report an efficient protocol for iPSC generation from peripheral blood mononuclear cells (PBMCs). By plating the transduced PBMCs serially to a new, matrix-coated plate using centrifugation, this protocol can easily provide iPSC colonies. This method is also applicable to umbilical cord blood mononuclear cells (CBMCs). This study presents a simple and efficient protocol for the reprogramming of PBMCs and CBMCs.

We propose a protocol for reprogramming peripheral blood mononuclear cells (PBMCs) into induced pluripotent stem cells (iPSCs). By plating the transduced blood cells onto matrix-coated plates with centrifugation, iPSCs are successfully induced from floating cells. This technique suggests a simple and effective reprogramming protocol for cells such as PBMCs and CBMCs.

从血细胞使用仙台病毒和离心诱导多能干细胞代

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent ...

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 ...

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 ...

Semi-automated Production of Hepatocyte Like Cells from Pluripotent Stem Cells

Human pluripotent stem cells represent a renewable source of human tissue. Our research is focused on generating human liver tissue from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). Current differentiation procedures generate human hepatocyte-like cells (HLCs) displaying a mixture of fetal and adult traits. To improve cell phenotype, we have fully defined our differentiation procedure and the cell niche, resulting in the generation of cell populations which display improved gene expression and&#160;function. While these studies mark progress, the ability to generate large quantities of multi well plates for screening has been limited by labour intensive procedures and batch to batch variation. To tackle this issue, we have developed a semi-automated platform to differentiate pluripotent stem cells into HLCs. Stem cell seeding and differentiation were performed using liquid handling and automatic pipetting systems in&#160;96-well plate format. Following the differentiation, cell phenotype was analyzed using automated microscopy and a multi well luminometer.

This protocol describes a semi-automated approach to produce hepatocyte-like cells from human pluripotent stem cells in a 96 well plate format. This process is rapid and cost-effective, allowing the production of quality assured batches of hepatocyte-like cells for basic and applied human research.

多能干细胞半自动化生产肝样细胞的研究

Human pluripotent stem cells represent a renewable source of human tissue. Our research is focused on generating human liver tissue from human embryonic ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

从脐带血源性多能干细胞中提取三维皮肤有机细胞

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C） 损伤的反应

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...