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?...

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。

<table border="0" cellpadding="0" cellspacing="0" width="1109">
	<colgroup>
		<col />
		<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?...

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

Research

JoVE Journal

Biology

22.2K 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

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 ...

科研

生物学

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 ...

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, ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

人类多能干细胞的神经元在一个Multititre的板格式的快速和高效地生成

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

人类心肌细胞的生成：从馈线人类诱导多能干细胞的分化协议

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

Episomal Reprogramming of Dermal Fibroblasts to Generate Human-Induced Pluripotent Stem Cells (hiPSCs)

Episomal Reprogramming of Dermal Fibroblasts to Generate Human-Induced Pluripotent Stem Cells (HiPSCs)  

To begin, take a flask of cultured fibroblast cells, coat a 10-centimeter cell culture plate suitable for hiPSC culture with four milliliters of cold coating solution per plate, and incubate the plate for one hour at 37 degrees Celsius. Afterward, remove the media from the fibroblasts and wash them with four milliliters of pre-warmed PBS per flask. Aspirate the PBS and associate the fibroblasts with four milliliters of Trypsin-EDTA per flask by incubating for five to seven minutes at 37 degrees Celsius.Monitor cell detachment using a phase contrast microscope, and if needed, tap against the side of the flask to detach the cells from the plastic surface. Transfer the detached cells to a 50-milliliter conical tube and wash the empty flasks with six milliliters of pre-warmed fibroblast medium. Collect and pool the remaining cells in the conical tube.Then centrifuge the cells at 200 g for five minutes at 20 degrees Celsius. Aspirate the supernatant and re-suspend the cell pellet in three milliliters of fresh, pre-warmed PBS. Count the cells, transfer 1.5 times 10 to the sixth fibroblasts into a new conical tube and fill up with PBS to the five millimeter mark.Centrifuge the tube at 200 g for five minutes at 20 degrees Celsius. Discard the supernatant, re-suspend the cell pellet in five milliliters of electroporation medium, and centrifuge again. In the meantime, aspirate the coating solution from the coated 10-centimeter cell culture plate and add seven milliliters of pre-warmed fibroblast medium.At the end of the centrifugation, discard the supernatant and re-suspend the cell pellet in electroporation medium with a concentration of 1.5 times 10 to the sixth cells in 250 microliters. Transfer the 250 microliter cell suspension to an electroporation cuvette with a four millimeter gap distance. Next, prepare a plasma transfection mix by adding four micrograms of each plasmid to a total volume of 50 microliters of electroporation medium.Transfer the transfection mix to the cuvette, mix gently by flicking, and electroporate with one pulse at 280 volts. Finally, transfer 150 microliters of the electroporated fibroblasts to the prepared 10-centimeter cell culture plate. Agitate the plate thrice in all directions to distribute the cells, and incubate overnight.Fibroblasts present in a long, spindle-like phenotype, the size of 150 to 300 nanometers. After reprogramming, colonies with 50 micrometer small hiPSCs occur displaying distinct borders. The hiPSCs are distinguished by a high nuclei to body ratio and increased proliferation rate.