这些研究的部分经费由美国国立卫生研究院UO1HL107443-01奖，郭敬明和GM。

1。外周血单个核细胞（PBMC）的分离和扩展 0天<ol><li>画出4毫升外周血的BD真空采血管CPT细胞下游管与柠檬酸钠。倒置管8至10倍，并在1,800 xg离心30分钟，在室温下离心。在理想的情况下，这一步应在采集后2小时内完成。 </li><li>收集的单核细胞（MCs），通过移液到一个无菌的15毫升锥形离心管中的血沉棕黄层（小区凝胶屏障和血浆层之间）。到10毫升，用无菌磷酸盐缓冲的盐水（PBS），将总体积，倒置几次并在300×g离心15分钟离心。 </li><li>重悬在10毫升无菌PBS中的细胞，并进行细胞计数。 1至2×10 6个细胞转移到一个无菌的15毫升锥形离心管中，并在300×g离心10分钟离心。 </li><li>重悬细胞的膨胀介质在2毫升（EM）（QBSF-60干细胞培养基含有50微克/毫升阿斯科40毫微克/毫升50毫微克/毫升SCF，山梨酸，10 ng / ml的IL-3，2 U / ml的EPO，IGF-1，1μM地塞米松和100微克/毫升primocin的1％青/链霉素），并转移到一个孔的12孔板中。孵育的细胞中，在37℃下，5％CO 2培养箱中。 </li><li>离心剩余的细胞在300× 克并冷冻10分钟〜2×10 6个细胞/ FBS的含10％DMSO的样品瓶。 </li><li>要启动协议冻结的外周血单个核细胞，细胞解冻1瓶10毫升QBSF介质和离心机，在300×g离心10分钟。在2ml的EM和重悬细胞，转移到12孔板的一个阱。孵育的细胞中，在37℃下，5％CO 2培养箱中。 </li></ol> 3天，第6天<ol start="17"><li>将细胞转移到无菌的15毫升锥形管和洗一次的QBSF-60干细胞中等至收集贴壁细胞用1ml。 </li><li>离心细胞在300×g离心10分钟。 </li><li>在2ml的EM和重悬细胞，以及一个12转移到一个孔板中。孵育的细胞中，在37℃下，5％CO 2培养箱中。 </li></ol> 2。与STEMCCA慢病毒转导对外周血单核细胞（PBMC） 第九天<ol><li>将细胞转移到无菌的15毫升锥形管和洗一次的QBSF-60干细胞中等至收集贴壁细胞用1ml。 </li><li>离心细胞在300×g离心10分钟。 </li><li>细胞重悬在1毫升含5微克/毫升的聚凝胺和STEMCCA慢病毒新鲜EM（MOI = 1到10）和转移到12孔板的一个孔。 （慢病毒颗粒的协议描述生产可以发现在<a href="http://www.bumc.bu.edu/stemcells/protocols/" target="_blank">http://www.bumc.bu.edu/stemcells/protocols/的</a> ） </li><li>旋转板上，在2250转速在25℃下90分钟。 </li><li>分拆后，添加一个额外的1毫升的新鲜EM 2毫升培养基共孵育的板在37℃的含5微克/毫升的聚凝胺（polybrene），5％CO 2培养箱中。 </li></ol> 10天<ol start="16"><li>将细胞转移到无菌的15毫升锥形管和洗一次的QBSF-60干细胞中等至收集贴壁细胞用1ml。 </li><li>离心细胞在300×g离心10分钟。 </li><li>在2ml的EM和重悬细胞，转移到1井的12孔板。孵育的细胞中，在37℃下，5％CO 2培养箱中。 </li></ol> 3。电镀转染细胞上的MEF 第11天<ol><li>涂在6孔板的孔中有0.1％明胶和板灭活小鼠胚胎成纤维细胞（MEF中）为2×10 5个细胞/孔在MEF培养基（IMDM中含有10％FBS，1％非必需氨基酸，100μM的β-巯基乙醇，2mM的L-谷氨酰胺，100微克/毫升primocin或1％青/链霉素）。准备感染每3个井。 </li></ol> 12天<ol start="2"><li>将细胞转移到无菌的15毫升锥形管<html>洗井一次的QBSF-60干细胞中等至收集贴壁细胞，用1ml。 </li><li>重悬细胞的MEF培养基中含有10纳克/毫升碱性成纤维细胞生长因子，和抗坏血酸和在相同浓度的生长因子，如使用在EM在3ml培养基（50μg/ ml的抗坏血酸，50 ng / ml的SCF，10 ng / ml的IL -3，2 U / ml的促红细胞生成素，40 ng / ml的IGF-1，1μM地塞米松）。 </li><li>板1毫升细胞每孔的6孔板中，含有的MEF。共2.5毫升/孔的介质，添加1.5毫升的MEF媒体，抗坏血酸，碱性成纤维细胞生长因子和生长因子。 </li><li>旋转板在500rpm下在25℃下30分钟。板在37℃，5％CO 2培养箱中孵育。 </li></ol> 14天<ol start="6"><li>饲料细胞每隔一天用2.5毫升含有10 ng / ml的碱性成纤维细胞生长因子和50μg/ ml的抗坏血酸（无生长因子）的MEF媒体。每个饲料漂浮的细胞吸出并丢弃。 </li><li>添加额外的MEF中，根据需要（通常每周一次）。 </li></ol>P“〜20天<ol start="8"><li>一旦小群出现，饲料的细胞每天用2毫升的人类胚胎干细胞（hESC）培养基（DMEM/F12含20％淘汰赛血清替代物，1％非必需氨基酸，100μM的β-巯基乙醇，2毫米L-谷氨酰胺，100微克/毫升primocin或1％青/链霉素和10 ng / ml的碱性成纤维细胞生长因子）。 </li></ol> 4。采摘的IPSC的克隆和扩展 〜30天 - 40 <ol><li>拾取每个菌落预先接种含有1毫升/孔的胚胎干细胞介质中加10μMY27632（ROCK抑制剂）Stemolecule与灭活的MEF的12孔板在个别井。 </li><li>此后每天饲料细胞的胚胎干细胞培养基（无ROCK抑制剂）与1毫升。 </li></ol> 5。多能性标记免疫荧光染色法<ol><li>使用工具包“ES细胞标记物样品套件”自Millipore，并按照制造商的协议进行染色。 </li></ol> 6。切除INTEG的额定STEMCCA向量<ol><li>切除磁带重新编程实现，利用一个酶Cre-IRES-PURO表达载体，转染后的和短暂的嘌呤霉素筛选的说明6。 </li></ol>

<ol>
	<li>Lowry, W. E., Richter, L., Yachechko, R., 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=Generation+of+human+induced+pluripotent+stem+cells+from+dermal+fibroblasts.">Generation of human induced pluripotent stem cells from dermal fibroblasts.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 2883-2888 (2008).</li><li>Park, I. H., Zhao, R., West, J. A., 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=Reprogramming+of+human+somatic+cells+to+pluripotency+with+defined+factors.">Reprogramming of human somatic cells to pluripotency with defined factors.</a> Nature. 451, 141-146 (2008).</li><li>Takahashi, K., Tanabe, K., Ohnuki, M., 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=Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors.">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131, 861-872 (2007).</li><li>Yu, J., Vodyanik, M. A., Smuga-Otto, K., 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>Stadtfeld, M., Hochedlinger, K., 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+pluripotency:+history,+mechanisms,+and+applications.">Induced pluripotency: history, mechanisms, and applications.</a> Genes Dev. 24, 2239-2263 (2010).</li><li>Somers, A., Jean, J. C., Sommer, C. A., 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=Generation+of+transgene-free+lung+disease-specific+human+induced+pluripotent+stem+cells+using+a+single+excisable+lentiviral+stem+cell+cassette.">Generation of transgene-free lung disease-specific human induced pluripotent stem cells using a single excisable lentiviral stem cell cassette.</a> Stem Cells. 28, 1728-1740 (2010).</li><li>Loh, Y. H., Hartung, O., Li, H., 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=Reprogramming+of+T+cells+from+human+peripheral+blood.">Reprogramming of T cells from human peripheral blood.</a> Cell Stem Cell. 7, 15-19 (2010).</li><li>Seki, T., Yuasa, S., Oda, M., 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=Generation+of+induced+pluripotent+stem+cells+from+human+terminally+differentiated+circulating+T+cells.">Generation of induced pluripotent stem cells from human terminally differentiated circulating T cells.</a> Cell Stem Cell. 7, 11-14 (2010).</li><li>Staerk, J., Dawlaty, M. M., Gao, Q., 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=Reprogramming+of+human+peripheral+blood+cells+to+induced+pluripotent+stem+cells.">Reprogramming of human peripheral blood cells to induced pluripotent stem cells.</a> Cell Stem Cell. 7, 20-24 (2010).</li><li>Chou, B. K., Mali, P., Huang, X., 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=Efficient+human+iPS+cell+derivation+by+a+non-integrating+plasmid+from+blood+cells+with+unique+epigenetic+and+gene+expression+signatures.">Efficient human iPS cell derivation by a non-integrating plasmid from blood cells with unique epigenetic and gene expression signatures.</a> Cell Res. 21, 518-529 (2011).</li><li>vanden Akker, E., Satchwell, T. J., Pellegrin, S., Daniels, G., Toye, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+majority+of+the+in+vitro+erythroid+expansion+potential+resides+in+CD34(-)+cells,+outweighing+the+contribution+of+CD34(+)+cells+and+significantly+increasing+the+erythroblast+yield+from+peripheral+blood+samples.">The majority of the in vitro erythroid expansion potential resides in CD34(-) cells, outweighing the contribution of CD34(+) cells and significantly increasing the erythroblast yield from peripheral blood samples.</a> Haematologica. 95, 1594-1598 (2010).</li></ol>

没有利益冲突的声明。

我们在此描述的STEMCCA慢病毒载体的使用，以产生从几毫升的新鲜收集的外周血单核细胞分离的人类iPSCs。协议也可以用于利用从一个遥远的位置获得的供体细胞重编程冷冻的外周血单个核细胞（直接得自血沉棕黄层）时，显着的实际影响的细节的。分离的外周血单个核细胞重编程诱导前，必须经过一个关键的扩展步骤，使一个健康的增殖人口更适合核重编程的细胞。由&#39;膨胀时在造血的培养条?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> BD真空采血管CPT细胞制备管柠檬酸钠</td><td> BD Biosciences公司</td><td> 362760 </td><td></td></tr><tr><td> QBSF-60干细胞培养基</td><td>质量生物</td><td> 160-204-101 </td><td></td></tr><tr><td> IMDM </td><td> Invitrogen公司</td><td> 12440 </td><td></td></tr><tr><td> DMEM/F12 </td><td> Invitrogen公司</td><td> 11330 </td><td></td></tr><tr><td> FBS </td><td>亚特兰大生物</td><td> S10250 </td><td></td></tr><tr><td> Knockout血清替代品</td><td> Invitrogen公司</td><td> 10828 </td><td></td></tr><tr><td> Primocin </td><td> Invivogen </td><td> ANT-PM-2 </td><td></td></tr><tr><td>青/链霉素</td><td> Invitrogen公司</td><td> 15140 </td><td></td></tr><tr><td> L-谷氨酰胺</td><td> Invitrogen公司</td><td> 25030 </td><td></td></tr><tr><td>非必需氨基酸</td><td> Invitrogen公司</td><td> 11140 </td><td></td></tr><tr><td> β-巯基乙醇</td><td> MP Biomedicals </td><td> 190242 </td><td></td></tr><tr><td>抗坏血酸</td><td>西格玛</td><td> A4544 </td><td></td></tr><tr><td> IGF-1的</td><td> R＆D Systems公司</td><td> 291-G1 </td><td></td></tr><tr><td> IL-3的</td><td> R＆D Systems公司</td><td> 203-IL </td><td></td></tr><tr><td> SCF </td><td> R＆D Systems公司</td><td> 255-SC </td><td></td></tr><tr><td> EPO </td><td> R＆D Systems公司</td><td> 286-EP </td><td></td></tr><tr><td>地塞米松</td><td>西格玛</td><td> D4902 </td><td></td></tr><tr><td>聚凝胺</td><td>西格玛</td><td> H-9268 </td><td></td></tr><tr><td>碱性成纤维细胞生长因子</td><td> R＆D Systems公司</td><td> 233-FB </td><td></td></tr><tr><td> Stemolecule Y27632 </td><td> Stemgent </td><td> 04-0012 </td><td></td></tr><tr><td> ES细胞标记物样品套件</td><td>密理博</td><td> SCR002 </td><td></td></tr></tbody></table>

generation of human induced pluripotent stem cells from peripheral blood using the stemcca lentiviral vector

通过四个转录因子，即Oct4，Klf4导入正常的异位表达，Sox2和cMyc，人的体细胞可以被转换到多能干状态，产生所谓的诱导多能干细胞（iPS细胞）1-4。特定病人的iPS细胞缺乏的伦理问题，围绕胚胎干细胞（ES细胞），并会绕过可能的免疫排斥反应。因此，iPS细胞疾病模型研究，药效成分的筛选和再生疗法5，已经引起相当大的关注。 我们已经证实了代转基因人类iPSCs从不同的肺部疾病的患者使用一个单一的应纳税多顺反子慢干细胞盒式录音带（STEMCCA）的编码山因素6。这些的IPSC的线产生的皮肤成纤维细胞中，最常见的用于重新编程的细胞类型。通常情况下，获得成纤维细胞，需要皮肤活检冲头，然后通过细胞的膨胀秒，文化几段。更重要的是，若干组报道人外周血细胞的重新编程到iPSCs的7-9。在一项研究中，采用的TET诱导版本的STEMCCA载体9，这需要同时感染的血细胞组成性激活的慢病毒编码的逆四环素反式激活。与成纤维细胞，外周血细胞可以收集通过微创手术，大大减少了病人的不适和痛苦的。一个简单而有效的协议可能会加速血细胞重新编程的一个组成单应纳税载体IPSC的技术应用到更广泛的研究。此外，外周血细胞重新编程，允许从个人产生的iPS细胞在皮肤活检应该是可以避免的（ 即 。异常疤痕），或由于预先存在的疾病状况preventi纳克冲的活组织切片检查。 在这里，我们展示了一个协议，用于人类iPSCs从外周血单核细胞（PBMCs）使用一个单一的两侧装接loxP的消费税慢病毒载体的组成型表达的4个因子的产生。被用的STEMCCA慢病毒转导之前，新鲜收集的或解冻的外周血单个核细胞被扩展为9，10,11天的描述在介质中含有抗坏血酸，SCF，IGF-1，IL-3和EPO。然后将细胞培养皿上MEFs和感染两周后，可以可视化ESC样集落。最后，所选的克隆被扩展和测试的多能性干细胞标记SSEA-4，TRA-1-60和Tra-1-81的表达。该协议是简单的，鲁棒的和高度一致，提供了一个可靠的方法用于产生人类iPSCs从易于接触的4毫升血液。

我们展示了一个简单而有效的协议，用于人类iPSCs从使用一个单一的慢病毒载体的外周血单个核细胞的产生。 图1A示出了示意性表示的协议。血液被收集到的BD真空采血管CPT细胞下游管与柠檬酸钠，和单核细胞，离心后，可以收集到的聚酯凝胶和血浆（血沉棕黄层）（ 图1B）之间的界面。然后将分离的外周血单个核细胞扩大培养9天。 图2比较了在第0天，第9天?...

Watch this Scientific Journal Video about Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector at JoVE.com

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

这里，我们表明一个简单而有效的协议，用于从外周血3-4毫升，使用一个单一的慢病毒重新编程矢量代人类iPSCs。现成的血细胞重新编程的承诺，加快利用IPSC的技术，使其接触到更广泛的研究团体。

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, ...

generation-human-induced-pluripotent-stem-cells-from-peripheral-blood

Research

JoVE Journal

Biology

26.4K 次观看.  Boston University School of Medicine. 这里，我们表明一个简单而有效的协议，用于从外周血3-4毫升，使用一个单一的慢病毒重新编程矢量代人类iPSCs。现成的血细胞重新编程的承诺，加快利用IPSC的技术，使其接触到更广泛的研究团体。

视频: Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc, and Klf4 is capable of inducing pluripotency in mouse fibroblasts.1 Subsequent reports have demonstrated the utility of the doxycycline (DOX) inducible lentiviral delivery system for the generation of both primary and secondary iPS cells from a variety of other adult mouse somatic cell types.2,3
Induced pluripotent stem (iPS) cells are similar to embryonic stem (ES) cells in morphology, proliferation and ability to induce teratoma formation. Both types of cell can be used as the pluripotent starting material for the generation of differentiated cells or tissues in regenerative medicine.4-6 iPS cells also have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction.
Here we demonstrate the protocol for reprogramming mouse embryonic fibroblast (MEF) cells with the Stemgent DOX Inducible Mouse TF Lentivirus Set. We also demonstrate that the Stemgent DOX Inducible Mouse TF Lentivirus Set is capable of expressing each of the four transcription factors upon transduction into MEFs thereby inducing a pluripotent stem cell state that displays the pluripotency markers characteristic of ES cells.

The Stemgent Dox Inducible Mouse TF Lentivirus Set can reprogram mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. Here we demonstrate the protocol for DOX-inducible expression of mouse reprogramming transcription factors Oct4, Sox2, Klf4 and c-Myc to generate iPS colonies that express common mES pluripotency markers.

重新用四个转录因子的小鼠胚胎成纤维细胞诱导多能干细胞的产生，强力霉素诱导慢病毒转导系统

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and ...

科研

生物学

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

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

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

由Stemgent人类TF慢病毒重新编程人类成纤维细胞生成诱导多能干细胞

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

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

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

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

人类诱导多能干细胞的转染和Nucleofecting

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

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 ...

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 ...

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 and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.

This protocol allows for the reliable generation and characterization of blood outgrowth endothelial cells (BOECs) from a small volume of adult peripheral blood. BOECs can be used as a surrogate for endothelial cells from patients with vascular disorders and as a substrate for the generation of induced pluripotent stem cells.