This work was supported by a grant from the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT, and Future Planning (2013R1A1A1076125).

伦理学声明:本研究方案被批准由韩国天主教大学（KC12TISI0861）的机构审查委员会。 1.血单核细胞的分离<ol><li>单核细胞的分离（-5天） <ol><li>从在细胞制备管（CPT）抽血获得至少10毫升新鲜血液。 </li><li>血液转移到一个新的50毫升锥形管中，用在一个1的无菌磷酸盐缓冲盐水（PBS）稀释:4的比例。 注:稀释液的比例越高，可用于较高的纯度。 </li><li> 10毫升密度梯度介质添加到一个新的50毫升锥形管，仔细层稀释血液的密度梯度介质的顶部。离心在750 xg离心在室温（RT）下30分钟不离心制动。 </li><li>小心地将血沉棕黄层转移到新的50毫升锥形管中，加入30毫升的PBS至管中，并洗涤细胞。 </li><li>离心将细胞在515×g离心5分钟，在RT &lt;/ LI&gt; <li>丢弃PBS中重悬细胞在0.5ml血细胞介质。 </li><li>计数细胞，和板，每孔1×10 6个细胞在24孔板的。添加PBS到周围井以防止蒸发。 </li><li>转导之前，稳定5天将细胞于37℃在5％CO 2。天3-4添加而增加的0.5毫升的新鲜血液细胞培养基而不干扰细胞。 </li></ol></li></ol> 2.转导仙台病毒<ol><li>转导（第0天） <ol><li>收集和血细胞转移到15毫升锥形管中，并使用血球计数。 </li><li>准备每转3×10 5细胞并离心将细胞在515×g离心在RT 5分钟。 </li><li>丢弃通过抽吸上清，重悬细胞在0.5ml血细胞介质。 </li><li>转移细胞到一个非涂布24孔板的孔中。 </li><li>解冻仙台病毒混合物在冰中，并把它添加到苏spended细胞。仙台病毒添加到根据制造商的建议的细胞。 </li><li>用密封薄膜密封板，并在30℃下在1150 xg离心离心它为30分钟。 </li><li>离心后，孵育细胞在37℃下在5％CO 2过夜（O / N）。 </li></ol></li><li>细胞转移到饲养基质（第1天） <ol><li>第二天，外套一个24孔板与玻。稀释为最终的5微克/毫升的浓度在PBS中的玻连蛋白溶液。 1毫升玻连蛋白添加至24孔平板的孔中，并在室温下孵育，然后至少1小时。使用前取下涂料解决方案。涂覆的板可以存储在室温3天。 </li><li>传送含有细胞和病毒的包被的孔中的所有媒体。 </li><li>收集剩余的细胞用增加的0.5毫升的新鲜血液细胞培养基，并把它添加到井的含细胞。 </li><li>离心板在1150 xg离心35℃10分钟。 </li><li> ％之后rifugation，去除上清，加入1ml的iPSC媒体，并在5％CO 2 O / N保持在37℃。 </li></ol></li><li>第二小区传送（第2天） <ol><li>涂层用5微克/毫升玻连新的24孔板的孔中，如在步骤2.2.1说明。使用该板的一个孔中的每个转导。 </li><li>从第一板细胞悬液转移到新涂玻连板。 注意:如果不需要，悬浮细胞可以被丢弃。在步骤2.3中提到的过程可以与悬浮细胞被重复2-3次。如果该步骤将重复，收获细胞。 </li><li>同时，在5％的CO 2 O / N在37℃下加入1 ml的iPSC介质到第一板的孔，进行维护，孵育它。 </li><li>保持附着的细胞在37℃和5％的CO 2，并执行用新鲜的iPSC媒体每日更换培养基。菌落转导后14-21出现在天。 </li><li> CentrifugÈ含有在1150 xg离心和35℃下10分钟的悬浮细胞的新包被的平板。 </li><li>离心后，孵育细胞在37℃下在5％CO 2 O / N。 </li><li>次日，去除上清，并用新鲜的iPSC媒体更换。 注:在步骤2.3中提到的步骤可以与悬浮细胞被重复2-3次。如果该步骤将重复，收获上清液中的细胞，并重复步骤2.3。 </li><li>保持附着细胞日报传媒的变化，直到达到80％汇合。 </li></ol></li></ol> 3.重编程的细胞维护<ol><li> 24孔板培养后早期维护<ol><li>转导后7-10天，一旦细胞汇合，制备玻连蛋白涂覆的60毫米培养皿中。稀释PBS玻解决方案作最后的5微克/毫升的浓度。 1毫升玻连蛋白添加到培养皿，并在室温下孵育，然后至少1小时。 </li><li>洗细胞用PBS并加入1ml的PBS / 1mM EDTA中的分离的细胞。 </li><li>孵育它们在37℃和5％CO 2的2分钟。 </li><li>收获细胞并在250 xg离心和RT 2分钟离心它们。去除上清，悬浮细胞在3毫升新鲜的iPSC媒体。 </li><li>板上的所有悬浮细胞到新涂60毫米的菜。 </li><li>在37℃下添加10毫Rho激酶抑制剂的细胞，并维持它们在5％CO 2，直到所述细胞是80％汇合。 </li></ol></li><li>拆分菌落外观（亚克隆准备） <ol><li>制备玻连蛋白包被的，100毫米的菜，如在步骤3.1.2说明。 </li><li>清洗细胞，用PBS，并添加1毫升的PBS / 1mM EDTA中的分离的细胞。 </li><li>孵育它们在37℃和5％CO 2的2分钟。 </li><li>收获细胞并在250 xg离心和RT 2分钟离心它们。 </li><li>算使用血球细胞和每培养皿制备1×10 4个细胞。</li><li>离心细胞，在250 xg离心和RT 2分钟。 </li><li>悬浮1×10 4个细胞在6ml的iPSC介质，盘它们涂布后100毫米培养皿，并添加10mM的Rho激酶抑制剂的介质。 </li><li>孵育细胞在37℃，5％CO 2的一个星期，直到大菌落出现。 注意:保持和扩大殖民地的亚克隆。亚克隆通常是在5代做英寸</li></ol></li><li>殖民地采摘使用的iPSC菌落分离解决方案<ol><li>菌落采摘，种子1×10 4个细胞在玻连蛋白包被的，100毫米的菜，如在步骤3.2.7中提到前一个星期。 </li><li>加入2毫升玻连蛋白溶液，并在室温温育至少1小时制备玻璃体结合蛋白涂布的，60毫米培养皿中。 </li><li>通过显微镜（40X或100X放大倍数）观察，标志用记号笔界限清楚的殖民地。从步骤3.3.2取得新盘取出玻的解决方案，并添加6毫升IPSC的介质补充有10毫Rho激酶。 </li><li>从细胞中除去培养基，并用3毫升的PBS洗涤。 </li><li>加入1毫升的iPSC菌落分离解决方案，并培育他们在室温为30秒。 </li><li>除去从板的溶液中并在室温下孵育它为另外的30秒。 </li><li>使用P200移液器，从板块吸取200微升媒体和吹打分离针对性的殖民地。转移殖民地散落到新的100毫米的菜。 </li><li>孵化和在5％CO 2维持在37℃。 注:获得纯的iPSC集落后，将细胞维持，直到它们到达通道10表征为至少10次传代之后进行。抗体的稀释液和引物信息示于表1和2。 </li></ol></li></ol>

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The authors have nothing to disclose.

由于胚胎干细胞（ESCs）显示若干缺点，需要一种替代的工具的需要。因此，诱导多能干细胞由山开发（iPS细胞）在国际聚光灯下的来。它已经将近十年，因为山中发现多能性可以通过添加只有四个基因导入成年体细胞被诱导。由于iPS细胞是由成熟的体细胞"诱导"，他们可以逃避那个曾经是与胚胎干细胞的关注伦理问题。不像胚胎干细胞，可以从每个生成的iPSC。因此，它们可以在个性化医学研究和...

干细胞一直是最有吸引力的材料在临床治疗在过去几十年1中的一个。干细胞的吸引人的特性是多能性和能力的自我更新。在1981年，第一个胚胎干细胞（ESCs）从小鼠胚胎2隔离。然而，当该技术被应用到人的胚胎，它面临几个伦理问题。 在2006年，当山博士和他的团队重新编程小鼠体细胞第一多能细胞，干细胞领域恢复了可能性和兴趣再次被点燃3。通过提供几个限定的因素，多能干细胞成功地从成年体细胞"诱导"，并由此命名为"诱导多能干细胞（iPS细胞）。"在2007年，该技术被应用到人类细胞4，得到具有胚胎干细胞的确切特征的细胞，但没有伦理争论。从理论上讲，IPS铯可以从任何个体或患者获得的任何细胞类型中产生。患者特异性iPSCs的上升作为能够模拟疾病表型和每个个体患者的后生条件的潜在工具。使用基因的编辑或可逆转的致病条件的其它方法，患者专用的iPSC也可在个性化用药5中使用。此外，iPS细胞较少与免疫排斥反应相关的，因为他们有相同的免疫标识作为供体，使得自动移植更为可行6。因此，iPSCs的已成为疾病建模，药物筛选，并再生疗法的最有希望的平台。考虑到这些好处，改善了协议，可以从最小的细胞来源给予纯净和较高的收益率在最短的时间内量都在不断发展之中。发现为将来应用的最有效的协议中的一个重要的考虑是主要的细胞类型。大多数早期的iPSC产生的原COLS是为贴壁细胞，因为原来的iPSC系从皮肤成纤维细胞诱导4优化。然而，这些细胞的分离和制备是劳动密集型的。此外，皮肤成纤维细胞的隔离包括微创手术过程，可以成为更广泛应用的一大缺憾。 因此，对于进一步使用的iPSC的，则需要用采集方便的细胞来源。由于它是通过一个相当微创手术7-9获得的血液被视为理想的细胞来源。在这项研究中，我们开发了一个简单的修改协议产生外周血单个核细胞（PBMC）iPS细胞。没有特定的细胞类型，例如CD34 +细胞的困难扩张过程中，全血细胞或外周血单个核细胞中连续通过离心用含有山因子的仙台病毒转导之后镀上涂覆基质的平板。这种方法减少了所需的时间转导的漂浮细胞的附着和重编程的细胞是不能够自己附加的损失减少。

<table><tbody><tr><td colspan="4">&lt;strong&gt;Plasticware&lt;/strong&gt;</td></tr><tr><td>100 mm Dish</td><td>TPP&amp;nbsp;</td><td>93100</td><td></td></tr><tr><td>6-well Plate</td><td>TPP</td><td>92006</td><td></td></tr><tr><td>50 ml Cornical Tube</td><td>SPL</td><td>50050</td><td></td></tr><tr><td>15 ml Cornical Tube</td><td>SPL</td><td>50015</td><td></td></tr><tr><td>10 ml Disposable Pipette</td><td>Falcon</td><td>7551</td><td></td></tr><tr><td>5 ml Disposable Pipette</td><td>Falcon</td><td>7543</td><td></td></tr><tr><td>12-well Plate</td><td>TPP</td><td>92012</td><td></td></tr><tr><td>24-well Plate</td><td>TPP</td><td>92024</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;PBMC Isolation Materials&lt;/strong&gt;</td></tr><tr><td>DPBS</td><td>Life Technologies</td><td>14190-144</td><td></td></tr><tr><td>Ficoll</td><td>GE Healthcare</td><td>17-1440-03</td><td></td></tr><tr><td>StemSpan</td><td>STEMCELL Technologies</td><td>9805</td><td>Blood cell media</td></tr><tr><td>CC110</td><td>STEMCELL Technologies</td><td>8697</td><td>Blood cell media supplement (100x)</td></tr><tr><td colspan="4">&lt;strong&gt;iPSC Generation and Culture Materials&lt;/strong&gt;</td></tr><tr><td>CytoTune-iPSC Sendai Reprogramming Kit</td><td>Life Technologies</td><td>A16518</td><td></td></tr><tr><td>TeSR-E8 Media</td><td>STEMCELL Technologies</td><td>5940</td><td>iPSC media</td></tr><tr><td>Vitronectin</td><td>Life Technologies</td><td>A14700</td><td></td></tr><tr><td>ROCK Inhibitor</td><td>Sigma Aldrich</td><td>Y0503</td><td></td></tr><tr><td>TrypLE express (TrypLE)</td><td>Life Technologies</td><td>12604-039</td><td></td></tr><tr><td>ReleSR</td><td>STEMCELL Technologies</td><td>12604-039</td><td>Colony detaching solution</td></tr><tr><td colspan="4">&lt;strong&gt;Quality Control Materials&lt;/strong&gt;</td></tr><tr><td>18 mm Cover Glass</td><td>Superior</td><td>HSU-0111580</td><td></td></tr><tr><td>4% Paraformaldyhyde</td><td>Tech &amp;amp; Innovation</td><td>BPP-9004</td><td></td></tr><tr><td>Triton X-100</td><td>BIOSESANG</td><td>9002-93-1</td><td></td></tr><tr><td>Bovine Serum Albumin&amp;nbsp;</td><td>Vector Lab</td><td>SP-5050&amp;nbsp;</td><td></td></tr><tr><td>Anti-SSEA4 Antibody</td><td>Millipore</td><td>MAB4304</td><td></td></tr><tr><td>Anti-Oct4 Antibody</td><td>Santa Cruz</td><td>SC9081</td><td></td></tr><tr><td>Anti-TRA-1-60 Antibody</td><td>Millipore</td><td>MAB4360</td><td></td></tr><tr><td>Anti-Sox2 Antibody</td><td>Biolegend</td><td>630801</td><td></td></tr><tr><td>Anti-TRA-1-81 Antibody</td><td>Millipore</td><td>MAB4381</td><td></td></tr><tr><td>Anti-Klf4 Antibody</td><td>Abcam</td><td>ab151733</td><td></td></tr><tr><td>Alexa Fluor 488 goat anti-mouse IgG (H+L) antibody</td><td>Molecular Probe</td><td>A11029</td><td></td></tr><tr><td>Alexa Fluor 594 goat anti-rabbit IgG (H+L) antibody</td><td>Molecular Probe</td><td>A11037</td><td></td></tr><tr><td>DAPI</td><td>Molecular Probe</td><td>D1306</td><td></td></tr><tr><td>Prolong gold antifade reagent</td><td>Invitrogen</td><td>P36934</td><td></td></tr><tr><td>Slide Glass, Coated&amp;nbsp;</td><td>Hyun Il Lab-Mate</td><td>HMA-S9914</td><td></td></tr><tr><td>Trizol</td><td>Invitrogen</td><td>15596-018</td><td></td></tr><tr><td>Chloroform</td><td>Sigma Aldrich</td><td>366919</td><td></td></tr><tr><td>Isoprypylalcohol</td><td>Millipore</td><td>109634</td><td></td></tr><tr><td>Ethanol</td><td>Duksan</td><td>64-17-5</td><td></td></tr><tr><td>RevertAid First Strand cDNA Synthesis kit</td><td>Thermo Scientfic</td><td>K1622</td><td></td></tr><tr><td>i-Taq DNA Polymerase</td><td>iNtRON BIOTECH</td><td>25021</td><td></td></tr><tr><td>UltraPure 10X TBE Buffer&amp;nbsp;</td><td>Life Technologies</td><td>15581-044</td><td></td></tr><tr><td>loading star</td><td>Dyne Bio</td><td>A750</td><td></td></tr><tr><td>Agarose</td><td>Sigma-Aldrich</td><td>9012-36-6</td><td></td></tr><tr><td>1kb (+) DNA ladder marker</td><td>Enzynomics</td><td>DM003</td><td></td></tr><tr><td>Alkaline Phosphatase</td><td>Millipore</td><td>SCR004</td><td></td></tr><tr><td>Tris base</td><td>Fisher Scientific</td><td>BP152-1</td><td>Rinse Buffer</td></tr><tr><td>Sodium Chloride</td><td>Duchefa Biochemie</td><td>S0520.1000</td><td>Rinse Buffer</td></tr><tr><td>Tween-20</td><td>BIOSESANG</td><td>T1027</td><td>Rinse Buffer</td></tr><tr><td>Hydrochloric Acid</td><td>Duksan</td><td>1129</td><td>Rinse Buffer</td></tr></tbody></table>

induced pluripotent stem cell generation from blood cells using sendai virus and centrifugation

最近的人类诱导多能干细胞（iPS细胞）的发展证明，成熟的体细胞可以返回到未分化，多能状态。角质形成细胞，尿细胞，成纤维等早期实验一般用真皮成纤维细胞进行:现在，重新编程各种类型成年体细胞进行。然而，这需要侵入性手术，以获得从病人的成纤维细胞。因此，悬浮细胞，例如血液和尿液细胞，分别因为得到原代细胞的方便的认为是理想的重新编程。这里，我们报告从外周血单核细胞（PBMC）对的iPSC产生一个有效的协议。通过串联电镀转导的PBMC到一个新的，包衣基质使用板离心，该协议可以很容易地提供的iPSC集落。这种方法也适用于脐带血单核细胞（CBMCs）。这项研究提出了一个简单而有效的PROTocol的外周血单个核细胞和CBMCs的重新编程。

该协议提供了一个简单的方法来重新编程从血液中分离外周血单个核细胞。利用串行电镀和离心的组合，成功地产生的iPSC。用这种方法，可以与全血细胞的少量生成的iPSC无需分离或扩大特定的细胞类型。我们在一个小细胞培养板成功生成iPS细胞仅1×10 4个细胞。 重新编程之前，血细胞用密度梯度介质隔离。血细胞分离...

Watch this Scientific Journal Video about Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation at JoVE.com

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

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 ...

induced-pluripotent-stem-cell-generation-from-blood-cells-using

Research

JoVE Journal

Developmental Biology

14.2K 次观看.  The Catholic University of Korea. 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.

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

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

人类神经干细胞从外周血造血祖细胞直接诱导

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

科研

发育生物学

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, ...

Direct Induction of Hemogenic Endothelium and Blood by Overexpression of Transcription Factors in Human Pluripotent Stem Cells

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro is essential for mechanistic studies of the endothelial-hematopoietic transition and hematopoietic specification. Here, we describe a method for the efficient induction of HE from human pluripotent stem cells (hPSCs) by way of overexpression of different sets of transcription factors. The combination of ETV2 and GATA1 or GATA2 TFs is used to induce HE with pan-myeloid potential, while a combination of GATA2 and TAL1 transcription factors allows for the production of HE with erythroid and megakaryocytic potential. The addition of LMO2 to GATA2 and TAL1 combination substantially accelerates differentiation and increases erythroid and megakaryocytic cells production. This method provides an efficient and rapid means of HE induction from hPSCs and allows for the observation of the endothelial-hematopoietic transition in a culture dish. The protocol includes hPSCs transduction procedures and post-transduction analysis of HE and blood progenitors.

This protocol describes the efficient induction of hemogenic endothelium and multipotential hematopoietic progenitors from human pluripotent stem cells via the forced expression of transcription factors.

Hemogenic内皮细胞直接诱导和血液中转录因子在人多能干细胞表达

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro ...

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

集成的无人类诱导多能干细胞的生成方法头发衍生角质形成细胞

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

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 ...

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 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, ...

生物学

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

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 include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

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 ...

Generation of Induced Neural Stem Cells (iNSCs) from Peripheral Blood Mononuclear Cells (PBMNCs): A Sendai Virus-based Procedure to Reprogram PBMNCs into iNSCs

Source: Zheng, W. et al. <a href="https://www.jove.com/video/59690" target="_blank">Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies.</a>&#160;J. Vis. Exp.&#160;(2019)
This video demonstrates the reprogramming of peripheral blood mononuclear cells (PBMNCs) into induced neural stem cells (iNSCs) using Sendai virus. Sendai virus is a non-integrating virus that offers an efficient and safe vehicle for reprogramming cells.

从血细胞使用仙台病毒和离心诱导多能干细胞代

摘要

探索更多视频

此视频中的章节

人类神经干细胞从外周血造血祖细胞直接诱导

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

Hemogenic内皮细胞直接诱导和血液中转录因子在人多能干细胞表达

集成的无人类诱导多能干细胞的生成方法头发衍生角质形成细胞

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

从人黑色素瘤肿瘤浸润淋巴细胞诱导多能干细胞的产生

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

代人类诱导多能干细胞从外周血中使用STEMCCA慢病毒载体

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

从外周血单核细胞（PBMNCs）产生诱导神经干细胞（iNSC）:一种基于仙台病毒的程序，将 PBMNCs 重编程为 iNSC

从血细胞使用仙台病毒和离心诱导多能干细胞代

摘要

探索更多视频

此视频中的章节

人类神经干细胞从外周血造血祖细胞直接诱导

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

Hemogenic内皮细胞直接诱导和血液中转录因子在人多能干细胞表达

集成的无人类诱导多能干细胞的生成方法头发衍生角质形成细胞

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

从人黑色素瘤肿瘤浸润淋巴细胞诱导多能干细胞的产生

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

代人类诱导多能干细胞从外周血中使用STEMCCA慢病毒载体

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

从外周血单核细胞 （PBMNCs） 产生诱导神经干细胞 （iNSC）:一种基于仙台病毒的程序，将 PBMNCs 重编程为 iNSC

从外周血单核细胞（PBMNCs）产生诱导神经干细胞（iNSC）:一种基于仙台病毒的程序，将 PBMNCs 重编程为 iNSC