我们要感谢李实验室的成员对稿件有价值的讨论。在李的实验室工作是由纽约干细胞基金会罗伯逊研究奖和美国马里兰干细胞研究基金（TEDCO）资助。

细胞1。制备及媒体（1天） <ol><li>培养和扩增人类成纤维细胞用含有10％FBS的DMEM培养基。 </li><li>板人成纤维细胞（ 图1）到24孔板中，每孔的适当密度的转导的前一天。 注:以下系列稀释建议（200K，100K，50K，25K，12.5K和6.25K），因为不同的细胞类型有不同的附件的能力。 </li><li>孵育在37℃，5％CO2 培养箱细胞多一天，保证了细胞已完全遵守和扩展。 </li></ol> 2执行转导（2天） <ol><li>在转导的一天，检查细胞密度，并选择最高效的密度井（ 图2）。最好是挑三个不同的密度:高（80〜90％），中（50〜70％）和低（20〜40％）。 </li><li>至少1小时前转导，吸出fibrobl从细胞AST介质，并更改新的300微升成纤维细胞培养基。 </li><li>删除一组4个不同的仙台病毒管子从-80°C储存。解冻每管在几秒钟同时在37℃水浴中，然后把试管从水浴。在这之后，它们解冻至室温;离心管中，在6000×g离心10秒，将它们放在冰上，直到使用。不要再冻结和解冻的病毒，因为滴度将无法维持。 </li><li>每四个仙台病毒管（Oct-4的，KLF-4，c-Myc的和Sox-2）所指示的卷添加到微型离心管中。确保将该溶液轻轻吹打混匀。 例如，如果24孔板lookwell转导50K细胞/一井: （效价的基础上分析自Life Technologies的证书，可以从不同的批次） 管hOct-4 = 6.0×10 7 CIU，3 MOI = 5微升 管hSox-2 = 6.5×10 7 CIU，3 MOI = 4.6μ;升 管HKLF-4 = 6.3×10 7 CIU，3 MOI = 4.8微升 管HC-Myc蛋白= 7.8×10 7 CIU，3 MOI = 3.8微升 合计= 18.2微升四个病毒因子的24孔板混合/一井（50K细胞） </li><li>取决于细胞密度;加2倍，1倍，并进入井0.5X体积的病毒的混合物。轻轻摇动板前向后，左，右（2X:36.4微升病毒合剂，1X的:18.2微升和0.5X:9.1微升）。 </li><li>将细胞在37℃，5％CO2 培养箱孵育过夜。 </li></ol> 3，更换培养基（第3天及5天） <ol><li>转导后24小时，用500μl新鲜的成纤维细胞培养基更换培养基。 </li><li>第5天，更换培养基用新鲜的成纤维细胞介质。 </li></ol> 4，准备MEF菜为共培养（8日） <ol><li>制备MEF细胞在60毫米培养皿中有10％FBS的DMEM培养基介质上的一个大传代的成纤维细胞转导到MEF饲养细胞之前年。 注意:我们在各60毫米的菜使用MEF的5×10 5。 </li></ol> 5，开始共培养转导细胞与MEF饲养细胞（9日） <ol><li>电镀前转导的细胞，吸出10％FBS的DMEM培养基介质从MEF饲养的菜肴，并加入新鲜的10％FBS的DMEM培养基介质。 </li><li>从转导的成纤维细胞是在24孔板中并用D-PBS洗细胞一次取出介质。添加200μl的0.25％胰蛋白酶/ EDTA入成纤维细胞在矿井24孔板中。后少于5分钟温育与胰蛋白酶/ EDTA中，当细胞开始从平板分离，收集在15ml锥形管中的细胞生长培养基。然后他们离心6000×g离心4分钟。 注:池2X，1X，0.5X和病毒转导的成纤维细胞，建议。 </li><li>除去上清液介质和洗涤，重新悬浮细胞沉淀4-5毫升新鲜成纤维细胞培养基从胰蛋白酶中和。然后离心它们在135×g离心4分钟。 </li><li>板的细胞作为连续稀释浓度和开始共培养的成纤维细胞介质上的MEF:如1/2，1/4，1/8，1/16，1/32，1/64和1/128每60毫米的菜。取决于转导过程中的第一个星期的细胞死亡速率，1可能不需要稀释至1/2或1/128的密度。放下剩余的细胞中提取总RNA为阳性对照转基因的RT-PCR。 </li><li>将60mm的培养皿在37℃，5％CO2 培养箱过夜。 </li></ol> 6，进给人类胚胎干细胞培养基和监控单元（10日） <ol><li>第二天，用10μM的Y-27632改变成纤维媒体人ES介质。在这之后，与人类ES培养基每天更换介质:800毫升DMEM/F12 +200毫升敲除血清替代+10毫升L-谷氨酰胺+10毫升10毫MEM最小非必需氨基酸+1毫升碱性成纤维细胞生长因子2 - 巯基乙醇+ 10纳克/毫升。 </li><li>做日常的媒体变化与新鲜人类胚胎干媒体。一个星期后，检查菜在显微镜下每一次2〜3天的胚胎干细胞集落样细胞团块的形成。取决于细胞类型，培养物将采取更多或更少的时间菌落被发现之前。 </li></ol> 7，采摘的诱导多能干细胞集落，并展开单元格（20日〜） <ol><li>转三周后，菌落应该会出现适当的生长采摘。 </li><li>采摘殖民地的前一天，准备MEF饲养在24孔板。 注:我们是在一个24孔板使用12.5×10 MEF 5。 </li><li>第二天，更改的MEF菜肴从成纤维细胞介质中到人ES介质用10μM的Y-27632。手动挑选一个菌落在各时间点的采摘罩显微镜下，吹打使较小的团块，转让到新制备的MEF dishes.Try挑几个克隆。 </li><li>通道和一个24孔板扩大每口井的6孔板最初。然后从6孔板至前进一步传播60 mm培养皿中。 </li></ol>人类iPS细胞的8。表征（后10代） <ol><li> 免疫荧光 <ol><li>在4-5天，每天介质变化的24孔板培养板中人类iPSCs的。 </li><li>用于启动免疫荧光测定中，用D-PBS洗板一次。然后在0.4％多聚甲醛立即固定细胞30分钟。后，在D-PBS洗3次，每次5分钟。 </li><li>对待固定的细胞用PBS中的0.1％的Triton X-100溶液中15分钟，在室温下进行。 </li><li>吸出permeablization溶液（0.1％的Triton X-100的溶液），再加入1小时在室温下0.5％的BSA封闭液。 </li><li>执行检测Nanog的Oct-4的，SSEA（阶段特异性胚胎胜光的烯）和TRA-1-81（肿瘤排斥抗原）使用抗人抗体在0.5％牛血清白蛋白溶液（检查抗体稀释率的材料表）。孵育一抗为2小时，在室温下进行。然后在D-PBS洗3次，每次5分钟。 </li><li> 1小时温育的Alexa Fluor 488山羊抗小鼠IgG抗体作为第二抗体，其在室温下的0.5％BSA溶液稀释1:2,000后 ​​检查用荧光显微镜。弄脏每个MEF饲养细胞和人iPS细胞用DAPI的所有核。 </li></ol></li><li> 转基因确认的RT-PCR检测 <ol><li>通过使用RNA提取试剂提取总mRNA从细胞沉淀。 </li><li>做总RNA并将所得的cDNA用于PCR扩增用反转录酶的等分试样上进行逆转录（1微克）。 </li><li>准备PCR混合物含有2微升cDNA模板，10μL2X PCR扩增预混试剂，2微升2皮摩尔引物（向前:1微升和反向:1微升）和6微升的​​DW。来检测基因表达，做RT-PCR方法与表1中所列的引物。 </li><li>用GAPDH基因进行RT-PCR反应作为扩增反应的效率的控制。可视化并通过1％琼脂糖凝胶电泳分析PCR产物。 </li></ol></li></ol>

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作者什么都没有透露。

重新编程人类体细胞，以人iPS细胞拥有在基础生物学，个人药品，移植12空前的承诺。先前，人类iPSC的世代所需DNA病毒，具有整合风险入宿主基因组，它可以产生不期望的基因突变，限制进一步的临床应用，如药物开发和移植疗法13。与这个原因，许多研究已经报道几种可供选择的方法来生成向量和转基因的无系统人类iPSC，但在同一时间，隔离&#39;脚打印自由&#39;的效率人类iPSC应予以考虑。例如，RNA病毒应该具有的遗传畸变比较其他方法14最低水平，虽然目前还没有公布的全基因组测序，以证明仙台病毒介导的iPSC生成的"基因安全"呢。在这里，我们提出了人iPS细胞生成的方法仙台病毒具有成本效益的方式高重编程效率。由此产生的人类iPS细胞可以自由转基因与维护细胞和分子相似性增生和发育潜能的胚胎干细胞。 我们可以在同一时间重新编程成纤维细胞（&gt; 4）不同的起源，只有一组仙台病毒。而在我们的方法，我们应用各种细胞汇流和病毒剂量的差异。这种&#39;混搭&#39;组合技术可以最大限度地重编程的疗效。成纤维细胞（从单个病人）在每口井有不同的病毒感染水平，但汇集在一起​​，使得人iPS细胞集落的可能性可以根据我们的经验增加。其中hiPSC线克隆变异的其他问题是增殖率，细胞附着/生存的X染色体失活15状态，分化电势16等的确，我们观察到，每个克隆有一个不同耳鼻喉科神经的倾向，这可以通过测量的mir-371簇的表达水平来预测。此外，我们已经成功地从人成肌细胞产生人类iPSC使用这种方法，这表明仙台病毒可以用于在重编程过程中的许多不同的细胞类型。此外，使用我们的方法，我们已经在超过10个不同的疾病相关成纤维细胞产生人iPS细胞。的平均值，从5至10 hiPSC克隆可以从一个成纤维细胞而得到。 尽管转基因 - 自由人iPS细胞的仙台病毒的推导是这可能是最实际的路径的最有效的重新编程的方法，也有在该协议被认为是一些限制; - 根据每个仙台病毒批次，病毒滴度需要计算为一致的MOI（如2.4所述）。 - 我们观察到感染性的变化不同的体细胞中，它也需要额外的滴定为ROBUST病毒感染。 - 仙台病毒介导的重编程可以被视为用于研究目的的最好的选择之一。但对于临床试验，它可能不会是第一选择，因为有一个发牌的问题与最初开发的仙台病毒系统的公司。 - 它需要大约两个月，直到重新编程体细胞不受转基因，这就是为什么我们需要至少10次传代后，检查转基因表达。 - 这似乎是使原代培养的成纤维细胞的传代次数可能会影响重新编程与仙台病毒的效率，虽然我们没有直接的比较。增殖率也需要确定。还需要进一步研究，以确定成纤维细胞传代次数以及它们对重编程效率增殖的效果。 - 在本研究中，我们使用了一个鼠饲养层hiPSC产生和维持，但无饲养系统可以是一种了。Alternativê方式在未来的研究中。 我们目前的协议已经走向研究特定病人的人iPS细胞用于疾病模型，再生医学和其他应用的重要一步。

人类胚胎干细胞（胚胎干细胞）有能力的自我更新体外和具有多能性，这可能是对疾病的建模可能有用的，用于药物筛选和开发基于细胞的疗法来治疗疾病和组织损伤。然而，人类胚胎干细胞有细胞替代治疗的局限性，因为免疫学，肿瘤学和道德的障碍，并研究疾病相关基因，疾病特异性胚胎干细胞可以通过植入前遗传学诊断（PGD）的方法进行分离，但它仍然是技术上的挑战和胚胎捐赠是相当罕见的。这些问题都涉及到干细胞生物学，这导致了诱导性多能干细胞（人iPS细胞）的发展进程。 人类iPSC在遗传自人成年体细胞的重新编程，和怀有多能性干细胞样特征类似于人类胚胎干细胞，这使得它们对再生医学的如d的有用来源地毯的发现，疾病模型和细胞治疗的病人的具体方式1,2。 到目前为止，有几种方法来产生人类iPSC，包括病毒介导的（逆转录病毒和腺病毒）3，非病毒介导的（BAC系统和载体转染）4的基因转导和蛋白质递送系统5-7。 虽然递送的病毒介导的基因可以确保效率的某一水平时，病毒载体可以离开遗传足迹，因为它们整合到宿主染色体中表达重编程基因以不受控制的方式。甚至当病毒融合转录因子可以激活或失活的宿主基因8，它可以导致意想不到的遗传畸变和肿瘤发生5,9的风险。另一方面，直接导入蛋白质或RNA导入体细胞的报道，但也有一些缺点，如劳动密集，反复被传挠度，并重新编程7,10水平低。即使附加型和非整合型腺病毒，腺相关病毒，和质粒载体仍然相对效率较低11。由于这些原因，它是合理选择非集成方法重编程与IPSC的新一代高疗效和较少的遗传异常。在这项研究中，我们使用了仙台病毒基础的重新编程。这种方法是已知的非整合到宿主基因组中，并持续产生人类iPSC无转基因的整合。

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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">CytoTune-iPS Reprogramming Kit</td>
			<td style="width:195px;">invitrogen</td>
			<td style="width:119px;">A1378002</td>
			<td style="width:253px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">CF-6,MEFs, neomycin-resistant, mitomycin C treated&#160;</td>
			<td>Global stem</td>
			<td>GSC-6105M&#160;</td>
			<td>5X105/6cm or 12.5X105/24 well plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin EDTA 0.25% Trypsin with EDTA 4Na 1X</td>
			<td style="width:195px;">invitrogen</td>
			<td>25200114</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F-12 medium</td>
			<td style="width:195px;">&#160;invitrogen</td>
			<td>11330-032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-well Cell Culture Plate, flat-bottom with lid</td>
			<td style="width:195px;">BD</td>
			<td>353935</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Y-27632</td>
			<td style="width:195px;">TOCRIS</td>
			<td style="width:119px;">1254</td>
			<td>10 uM (Stock: 10 mM)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">basic fibroblast growth factor</td>
			<td>LIFE TECHNOLOGIES&#160;</td>
			<td>PHG0263</td>
			<td>10 ng (Stock : 100 ug)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Knock-out serum replacement</td>
			<td>Gibco</td>
			<td>10828028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s Modified Eagle Medium (D-MEM, DMEM) (1X), liquid (high glucose)</td>
			<td style="width:195px;">&#160;invitrogen</td>
			<td>11965118</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Fetal bovine serum</td>
			<td style="width:195px;">Thermo Scientific Fermentas</td>
			<td style="width:119px;">SH30071.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Glutamine-200 mM (100X), liquid</td>
			<td style="width:195px;">GIBCO</td>
			<td>25030-081</td>
			<td>1/100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM Non-Essential Amino Acids Solution, 100X</td>
			<td>LIFE TECHNOLOGIES&#160;</td>
			<td>11140050</td>
			<td>1/100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-Mercaptoethanol (1,000X), liquid</td>
			<td style="width:195px;">GIBCO</td>
			<td>21985023</td>
			<td>1/1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hausser Phase Contrast Hemacytometers</td>
			<td>Hausser Scientific</td>
			<td>02-671-54</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EmbryoMax 0.1% Gelatin Solution</td>
			<td style="width:195px;">Millipore</td>
			<td style="width:119px;">ES-006-B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">SSEA-4</td>
			<td style="width:195px;">DSHB</td>
			<td>MC-813-70</td>
			<td>1/200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-Tra-1-81</td>
			<td style="width:195px;">cell signaling</td>
			<td>4745S</td>
			<td>1/200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse monoclonal Oct4 antibody</td>
			<td>Santa Cruz</td>
			<td>SC-5279</td>
			<td>1/1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Nanog</td>
			<td>R&#38;D</td>
			<td>AF1997</td>
			<td>1/1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">Alexa Flouor 488 goat anti-mouse</td>
			<td>invitrogen</td>
			<td>948492</td>
			<td>1/2000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DPBS (Dulbecco&#39;s Phosphate-Buffered Saline), 1X without calcium &#38; magnesium</td>
			<td style="width:195px;">cellgro</td>
			<td style="width:119px;">21-031-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">QuantiTect Reverse Transcription Kit</td>
			<td style="width:195px;">QIAGEN</td>
			<td>205313</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">PCR Master Mix [2X]</td>
			<td style="width:195px;">Thermo Scientific Fermentas</td>
			<td style="width:119px;">K0171</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trizol</td>
			<td style="width:195px;">invitrogen</td>
			<td>15596018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">picking hood</td>
			<td>NuAire</td>
			<td style="width:119px;">&#160;NU-301&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:543px;">dissecting scope&#160;</td>
			<td style="width:195px;">Nikon</td>
			<td>SMZ745</td>
			<td></td>
		</tr>
	</tbody>
</table>

efficient generation human induced pluripotent stem cells from human somatic cells with sendai-virus

几年前，建立人类诱导多能干细胞（iPS细胞）的迎来了在生物医学的新时代。人类iPSC的潜在用途包括人类遗传病的发病机制的建模，基因校正后的自体细胞治疗，以及通过提供特定病人的症状和相关细胞来源的个性化药物筛选。但是，也有一些困难需要克服，如消除了人类iPSC产生后剩下的重新编程因子的转基因表达。更重要的是，在未分化的人类iPS细胞残余的转基因表达可能妨碍适当的分化和误导性疾病相关的体外表型的解释。与此原因，集成无和/或转基因的无人类iPSC已经开发了使用几种方法，如腺病毒，在piggyBac转系统，小环载体，附加型载体，直接蛋白质送货和合成的mRNA。然而，reprogra效率使用集成自由方法mming是相当低的，在大多数情况下。 在这里，我们提出了一个方法，用仙台病毒（RNA病毒）的重编程系统来隔离人类iPSCs的。这种重编程方法显示一致的结果和高效率的成本效益的方式。

通常感染的成纤维细胞没有显示出仙台病毒转导后的任何形态的变化在数天，但五天后，便开始有不同的形状（ 图1）。如在图1的右面板中所述，细胞不具有典型的成纤维细胞形态的任何更多。它们具有一个圆形的形状和更大的细胞核比细胞质。即使在80％的细胞汇合时进行转导，看起来他们是在井少汇合，他们开始重新编程后。如果这些种形态的变化开始在最井待观察，成纤维细胞转导准备在MEF馈线培养皿被镀。 在我们的协议中，我们最小化细胞培养规模和也给各个重编程的可能性，例如使用不同的成纤维细胞密度和病毒滴度，和随后的几个重电镀比MEF，这会增加人iPS细胞产生效率（ <sTRONG&gt;图2）。传递之前，可以选择进一步仙台病毒转导井用不同的融合（低，中，高）;表示为红，绿，及黄色的颜色的板。 在重新编程的MEF饲养的开始，所以难以区分转导的细胞类型，但在一周内共培养后，部分地重新编程成纤维细胞的出现和看起来像松动ES样形态（ 图3）。因为这个原因，当人类iPSC集落并准备用作传代到新的MEF饲养盘，手工采摘是更有效的方式进行传递，而不是与酶的解离。 在图3和图4，在显微镜下唯一的人类iPS细胞具有明显的优势，这使人类iPS细胞（被视为"完全重新编程的细胞&#39;）和&#39;不完全重编程细胞的形态学之间有明显的区别。完全重新编程的细胞卤味确定像紧凑，排列紧密。特别是殖民地有明确的边界，而部分重编程的细胞看起来像聚集在一起，使殖民地，但非常失去的，脆弱的打破。即使我们做一个移液轻轻很容易脱落。即使不包括部分重编程人类iPS细胞集落（扩大他们和传代过程中），完全重新编程人类iPS细胞需要由人工采摘来维持。 采摘殖民地和扩大hiPSC克隆在几个星期后，需要确定的干性标记物的表达。扩张hiPSC克隆后，我们用不同组的测试，包括抗体染色（SSEA4 - ，OCT-4，TRA-81和Nanog）和多能性标记物的RT-PCR验证它们（数据未示出）作为一种质量控制。 相比H9，两个不同的人iPS细胞是阳性SSEA4 - ，OCT-4，TRA-81和Nanog。该结果表明，分离的人iPS细胞的多能获得的质量（ <strong&gt;的图5）。 最后， 图6显示，我们不能用RT-PCR检测到任何转基因表达在hiPSC克隆。至少在10代人iPS细胞，特异性引物（ 表1）对于外源Oct-4的，SOX-2，KLF-4和c-Myc可以使用阳性对照样品具有较强的转基因表达水平。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51406/51406fig1highres.jpg" src="/files/ftp_upload/51406/51406fig1.jpg" /> 图1的形态变化后的仙台病毒转导。转导之前，成纤维细胞显示典型的纺锤状（左图 ）。转导后，大约八天，好像更圆的形状（ 右 ）转导的细胞变化的形态。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51406/51406fig1highres.jpg" target="_blank">请点击ħ</a>埃雷查看此图的放大版本。 <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/51406/51406fig2highres.jpg" src="/files/ftp_upload/51406/51406fig2.jpg" /> 图感应计划2。图，以确保最高的效率 。在四个不同的细胞类型可以在24孔板中进行电镀（从1到4）。因为它们的附着能力和细胞存活可能是不同的依赖于细胞类型，因此建议在板不同的细胞密度（从200K到6.25K细胞每孔）。此外，病毒浓度可以调整（0.5X，1X和2X）。 <img alt="图3" fo:content-width="5in" fo:src="/files/ftp_upload/51406/51406fig3highres.jpg" src="/files/ftp_upload/51406/51406fig3.jpg" /> 图3:从转F的形成人类诱导多能干细胞集落ibroblast。在大约两个星期再镀成的MEF后，圆形菌落应该会出现，看起来像人类胚胎干细胞集落。通常情况下，完全重新编程的人iPSC集落有非常明确的界限。 （箭:完全重新编程的人iPSC集落，慈姑:部分重编程人类iPS细胞）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51406/51406fig3highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图4" fo:content-width="5in" fo:src="/files/ftp_upload/51406/51406fig4highres.jpg" src="/files/ftp_upload/51406/51406fig4.jpg" /> 图4。拿起人类诱导多能干细胞集落 。人类iPSC集落经常被包围部分地重编程的细胞（ 左 ）。在显微镜下，采摘类ES细胞集落只，分解成小块，然后转移（ 右 ）。经过2〜3代，<html>有未分化hiPSC殖民地（ 底部和左侧 ）。 （箭:完全重新编程的人iPSC集落，慈姑:部分重编程人类iPS细胞） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51406/51406fig4highres.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图5" fo:content-width="5in" fo:src="/files/ftp_upload/51406/51406fig5highres.jpg" src="/files/ftp_upload/51406/51406fig5.jpg" /> 图5。免疫荧光染色与干细胞多能性标志物。免疫荧光检测表明人类iPS细胞具有干性features.SSEA-4，TRA-81，Nanog的10月，香港4As广告多能性标记和DAPI对细胞核染色对照。（A）表示H9染色作为对照样品（B）和（C）示出了人类iPSC的克隆。等="_blank"&gt;点击这里查看大图。 <img alt="图6" fo:content-width="5in" fo:src="/files/ftp_upload/51406/51406fig6highres.jpg" src="/files/ftp_upload/51406/51406fig6.jpg" /> 图6。确认的转基因表达水平，至少以后人类iPS细胞，外源性Sox2的沉默，KLF-4，c-Myc的，和Oct-4基因表达水平的10通道的文化是由逆转录-聚合酶链反应证实。 hGAPDH是内部控制。对于阳性对照，RNA是从剩余的转导成纤维细胞（仙台病毒感染后第7天）萃取。 （泳道1:标记物，泳道2 - 6:GAPDH，KLF4，c-Myc的，OCT-4和Sox-2阳性对照，泳道7 - 11:GAPDH，KLF4，c-Myc的，OCT-4和Sox -2在人iPS细胞）。 <table border="0" cellpadding="0" cellspacing="0" width="305"><colgroup><col /><col /></colgroup><tbody><tr><td>引物名称</td> &lt;TD&gt;序</td></tr><tr><td> hOCT4（F） </td><td> CCC GAA AGA GAA AGC GAA CCAĞ </td></tr><tr><td> hOCT4（R） </td><td> GTA AAT AAG TCG GTG CTC AA </td></tr><tr><td> hMYC（F） </td><td> TAA CTG ACT AGC CTT AGG GTCĞ </td></tr><tr><td> hMYC（R） </td><td> TCC ACA TAC AGT CCT GGA TGA热重分析TG </td></tr><tr><td> hSOX2（F） </td><td> ACA AGA GAA AAA ACA TGT ATGĞ </td></tr><tr><td> hSOX2（R） </td><td> ATG CGC TGG TTC ACG GCG CCC认证CCC AGG </td></tr><tr><td>物hKLF4（F） </td><td> ACA AGA GAA AAA ACA TGT ATGĞ </td></tr><tr><td>物hKLF4（R） </td><td> CGC GCT GGC AGG GCC GCT GCT CGAç </td></tr><tr><td> hGAPDH（F） </td><td> AGG TCG GAG TCA ACG GAT TTG </td></tr><tr><td> hGAPDH（R） </td><td> GTG ATG GCA TGG ACT GTG GT </td></tr></tbody></table> 表1 RT-PCR引物对转基因的确认 ， 这些都是用于TRA的引物序列nsgene确认。 Tm值是50.6℃，在所有的引物。

Watch this Scientific Journal Video about Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus at JoVE.com

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

在这里，我们提出我们建立的方法重新编程人类体细胞转基因成无人类iPS细胞与仙台病毒，这说明一致的结果，并提高效率。

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 ...

efficient-generation-human-induced-pluripotent-stem-cells-from-human

Nanyang Technological University

Research

JoVE Journal

Biology

22.3K 次观看.  Johns Hopkins University School of Medicine. 在这里，我们提出我们建立的方法重新编程人类体细胞转基因成无人类iPS细胞与仙台病毒，这说明一致的结果，并提高效率。 

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

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

从人类胚胎干细胞的染色质免疫沉淀

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

科研

生物学

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

人CD40激活的B细胞的生成

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing the pluripotent state in mouse fibroblasts.1 The same group also reported the successful reprogramming of human somatic cells into induced pluripotent stem (iPS) cells using human versions of the same transcription factors delivered by retroviral vectors.2 Additionally, James Thomson et al. reported that the lentivirus-mediated co-expression of another set of factors (Oct4, Sox2, Nanog and Lin28) was capable of reprogramming human somatic cells into iPS cells.3
iPS cells are similar to ES cells in morphology, proliferation and the ability to differentiate into all tissue types of the body. Human iPS cells have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction. The generation of patient-specific iPS cells circumvents an important roadblock to personalized regenerative medicine therapies by eliminating the potential for immune rejection of non-autologous transplanted cells.
Here we demonstrate the protocol for reprogramming human fibroblast cells using the Stemgent Human TF Lentivirus Set. We also show that cells reprogrammed with this set begin to show iPS morphology four days post-transduction. Using the Stemolecule Y27632, we selected for iPS cells and observed correct morphology after three sequential rounds of colony picking and passaging. We also demonstrate that after reprogramming cells displayed the pluripotency marker AP, surface markers TRA-1-81, TRA-1-60, SSEA-4, and SSEA-3, and nuclear markers Oct4, Sox2 and Nanog.

We demonstrate the protocol for the generation of induced pluripotent stem cells from human somatic cells using lentivirus-mediated delivery of the human factors Oct4, Sox2, Nanog, and Lin28. Pluripotency was confirmed by morphology and the presence of embryonic stem (ES) cell-specific markers.

由Stemgent人类TF慢病毒重新编程人类成纤维细胞生成诱导多能干细胞

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

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

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

人类诱导多能干细胞的转染和Nucleofecting

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

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

高效的推导人类心脏的前体和从多能干的小分子诱导人类胚胎干细胞的心肌细胞

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

选择和隔离殖民地的人类诱导多能干细胞重新编程成年成纤维细胞

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

人类体细胞重编程为诱导多能干细胞（iPS细胞）用绿色荧光蛋白逆转录病毒载体

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

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

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

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

适用于培养人类胚胎和诱导多能干细胞的小鼠胚胎成纤维细胞的制备

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

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, ...