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><tbody><tr><td>CytoTune-iPS Reprogramming Kit</td><td>Invitrogen</td><td>A1378002</td><td></td></tr><tr><td>CF-6,MEFs, neomycin-resistant, mitomycin C treated&amp;nbsp;</td><td>Global stem</td><td>GSC-6105M</td><td>5 x 10&lt;sup&gt;5&lt;/sup&gt;/6 cm or 12.5 x 10&lt;sup&gt;5&lt;/sup&gt;/24-well plate</td></tr><tr><td>Trypsin EDTA 0.25% Trypsin with EDTA 4Na 1X</td><td>Invitrogen</td><td>25200114</td><td></td></tr><tr><td>DMEM/F-12 medium</td><td>Invitrogen</td><td>11330-032</td><td></td></tr><tr><td>24-well Cell Culture Plate, flat-bottom with lid</td><td>BD</td><td>353935</td><td></td></tr><tr><td>Y-27632</td><td>TOCRIS</td><td>1254</td><td>10 &amp;#956;M (Stock: 10 mM)</td></tr><tr><td>basic fibroblast growth factor</td><td>LIFE TECHNOLOGIES</td><td>PHG0263</td><td>10 ng (Stock : 100 ug)</td></tr><tr><td>Knock-out serum replacement</td><td>Gibco</td><td>10828028</td><td></td></tr><tr><td>Dulbecco&amp;#039;s Modified Eagle Medium (D-MEM, DMEM) (1X), liquid (high glucose)</td><td>Invitrogen</td><td>11965118</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Thermo Scientific Fermentas</td><td>SH30071.03</td><td></td></tr><tr><td>L-Glutamine-200 mM (100X), liquid</td><td>GIBCO</td><td>25030-081</td><td>1/100</td></tr><tr><td>MEM Non-Essential Amino Acids Solution, 100X</td><td>LIFE TECHNOLOGIES</td><td>11140050</td><td>1/100</td></tr><tr><td>2-Mercaptoethanol (1,000X), liquid</td><td>GIBCO</td><td>21985023</td><td>1/1,000</td></tr><tr><td>Hausser Phase Contrast Hemacytometers</td><td>Hausser Scientific</td><td>02-671-54</td><td></td></tr><tr><td>EmbryoMax 0.1% Gelatin Solution</td><td>Millipore</td><td>ES-006-B</td><td></td></tr><tr><td>SSEA-4</td><td>DSHB</td><td>MC-813-70</td><td>1/200</td></tr><tr><td>anti-Tra-1-81</td><td>Cell Signaling</td><td>4745S</td><td>1/200</td></tr><tr><td>mouse monoclonal Oct4 antibody</td><td>Santa Cruz</td><td>SC-5279</td><td>1/1,000</td></tr><tr><td>Nanog</td><td>R&amp;amp;D</td><td>AF1997</td><td>1/1,000</td></tr><tr><td>Alexa Flouor 488 goat anti-mouse</td><td>Invitrogen</td><td>948492</td><td>1/2,000</td></tr><tr><td>DPBS (Dulbecco&amp;#039;s Phosphate-Buffered Saline), 1X without calcium &amp;amp; magnesium</td><td>Cellgro</td><td>21-031-CV</td><td></td></tr><tr><td>QuantiTect Reverse Transcription Kit</td><td>QIAGEN</td><td>205313</td><td></td></tr><tr><td>PCR Master Mix [2X]</td><td>Thermo Scientific Fermentas</td><td>K0171</td><td></td></tr><tr><td>Trizol</td><td>Invitrogen</td><td>15596018</td><td></td></tr><tr><td>picking hood</td><td>NuAire</td><td>NU-301</td><td></td></tr><tr><td>dissecting scope&amp;nbsp;</td><td>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

22.5K 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 from Frozen Buffy Coats using Non-integrating Episomal Plasmids

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

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

使用非整合附加型质粒从冷冻血沉棕黄层诱导多能干细胞的产生

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

科研

发育生物学

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on dermal fibroblasts obtained by invasive biopsy and retroviral genomic insertion of transgenes, there have been many efforts to avoid these disadvantages. Human peripheral T cells are a unique cell source for generating iPSCs. iPSCs derived from T cells contain rearrangements of the T cell receptor (TCR) genes and are a source of antigen-specific T cells. Additionally, T cell receptor rearrangement in the genome has the potential to label individual cell lines and distinguish between transplanted and donor cells. For safe clinical application of iPSCs, it is important to minimize the risk of exposing newly generated iPSCs to harmful agents. Although fetal bovine serum and feeder cells have been essential for pluripotent stem cell culture, it is preferable to remove them from the culture system to reduce the risk of unpredictable pathogenicity. To address this, we have established a protocol for generating iPSCs from human peripheral T cells using Sendai virus to reduce the risk of exposing iPSCs to undefined pathogens. Although handling Sendai virus requires equipment with the appropriate biosafety level, Sendai virus infects activated T cells without genome insertion, yet with high efficiency. In this protocol, we demonstrate the generation of iPSCs from human peripheral T cells in feeder-free conditions using a combination of activated T cell culture and Sendai virus.

This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.

从人外周T细胞无饲养条件下，使用仙台病毒诱导多能干细胞的生成

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on ...

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets of patients with metastatic melanoma. Major obstacles of this approach are the reduced viability of transferred T cells, caused by telomere shortening, and the limited number of TILs obtained from patients. Less-differentiated T cells with long telomeres would be an ideal T cell subset for adoptive T cell therapy;however, generating large numbers of these less-differentiated T cells is problematic. This limitation of adoptive T cell therapy can be theoretically overcome by using induced pluripotent stem cells (iPSCs) that self-renew, maintain pluripotency, have elongated telomeres, and provide an unlimited source of autologous T cells for immunotherapy. Here, we present a protocol to generate iPSCs using Sendai virus vectors for the transduction of reprogramming factors into TILs. This protocol generates fully reprogrammed, vector-free clones. These TIL-derived iPSCs might be able to generate less-differentiated patient- and tumor-specific T cells for adoptive T cell therapy.

The goal of this protocol is to show the protocol for reprogramming melanoma tumor-infiltrating lymphocytes into induced pluripotent stem cells.

从人黑色素瘤肿瘤浸润淋巴细胞诱导多能干细胞的产生

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets ...

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 ...

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

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

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

从人体外周血单个核细胞融合，无诱导多能干细胞的生成方法游离型载体

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

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 ...

生物学

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral mesencephalon (VM). Cell replacement therapy holds great promise for treatment of PD. Recently, induced neural stem cells (iNSCs) have emerged as a potential candidate for cell replacement therapy due to the reduced risk of tumor formation and the plasticity to give rise to region-specific neurons and glia. iNSCs can be reprogrammed from autologous somatic cellular sources, such as fibroblasts, peripheral blood mononuclear cells (PBMNCs) and various other types of cells. Compared with other types of somatic cells, PBMNCs are an appealing starter cell type because of the ease to access and expand in culture. Sendai virus (SeV), an RNA non-integrative virus, encoding reprogramming factors including human OCT3/4, SOX2, KLF4 and c-MYC, has a negative-sense, single-stranded, non-segmented genome that does not integrate into host genome, but only replicates in the cytoplasm of infected cells, offering an efficient and safe vehicle for reprogramming. In this study, we describe a protocol in which iNSCs are obtained by reprogramming PBMNCs, and differentiated into specialized VM DA neurons by a two-stage method. Then DA precursors are transplanted into unilaterally 6-hyroxydopamine (6-OHDA)-lesioned PD mouse models to evaluate the safety and efficacy for treatment of PD. This method provides a platform to investigate the functions and therapeutic effects of patient-specific DA neural cells in vitro and in vivo.

The protocol presents the reprogramming of peripheral blood mononuclear cells to induce neural stem cells by Sendai virus infection, differentiation of iNSCs into dopaminergic neurons, transplantation of DA precursors into the unilaterally-lesioned Parkinson&#39;s disease mouse models, and evaluation of the safety and efficacy of iNSC-derived DA precursors for PD treatment.

从外周单核细胞生成诱导神经干细胞,分化至多巴胺能神经元前体,用于移植研究

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral ...

Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with muscular dystrophy is an invaluable cellular source for in&#160;vitro disease model systems and can be used for drug screening studies. Patient-derived urine cells have been used in successful reprogramming into induced pluripotent stem cells in order to model dystrophic cardiomyopathy1. Addressing the safety concerns of integrating vector systems, we present a protocol using a non-integrating Sendai virus vector for transduction of Yamanaka factors into urine cells collected from patients with muscular dystrophy. This protocol generates fully reprogrammed clones within 2&#8211;3 weeks. The pluripotent cells are vector-free by passage-13. These dystrophic iPSCs can be differentiated into cardiomyocytes and used either to study disease mechanisms or for drug screening.

This protocol entails detailed procedures for isolation of urine derived cells from muscular dystrophy patients; their efficient and rapid reprogramming through Sendai virus transduction.

从生成性肌营养不良患者诱导多能干细胞的:尿源性细胞的有效整合，免费重编程

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with ...

医学

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 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, ...