Name: efficient generation human induced pluripotent stem cells from human somatic cells with sendai-virus
Uploaded: 2014-04-23T22:00:00
Description: Watch this Scientific Journal Video about Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus Video at JoVE.com

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><...

<ol>
	<li>Yu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cell+lines+derived+from+human+somatic+cells.">Induced pluripotent stem cell lines derived from human somatic cells.</a> Science. 318, 1917-1920 (2007).</li><li>Okita, K., Nakagawa, M., Hyenjong, H., Ichisaka, T., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+mouse+induced+pluripotent+stem+cells+without+viral+vectors.">Generation of mouse induced pluripotent stem cells without viral vectors.</a> Science. 322, 949-953 (2008).</li><li>Zhou, W., Freed, C. 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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。

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			<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>
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		<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>
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			<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>
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			<td height="21" style="height:21px;">DMEM/F-12 medium</td>
			<td style="width:195px;">&#160;invitrogen</td>
			<td>11330-032</td>
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			<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>
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		<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>
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		<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>
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		<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>
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			<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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</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 Video at JoVE.com

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus Video (Video) | JoVE

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 ...

Research

JoVE Journal

Biology

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

Generation of Human CD40-activated B cells

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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 ...

科研

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

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set Video (Video) | JoVE

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

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人类诱导多能干细胞的转染和Nucleofecting

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

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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

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

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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

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

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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

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells Video (Video) | JoVE

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

Generating Mice from Induced Pluripotent Stem Cells | Protocol (Video) | JoVE

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

Generating Human Induced Pluripotent Stem Cells from Peripheral Blood Uing the STEMCCA Lentiviral Vector | Protocol (Video) | JoVE

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

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format Video (Video) | JoVE

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 ...