1。重新编程<ol><li>种子北京1 × 10 5 6孔板每孔细胞密度细胞。文化在2毫升培养液（450毫升与50毫升ES合格的胎牛血清，5毫升10毫米非必需氨基酸，5 ml青霉素/链霉素，5毫升200毫米L -谷氨酰胺和0.9毫升55毫米βEMEM补充细胞β-巯基乙醇）一夜之间在37 ° C和5％的CO 2。 </li><li>就在同一天，开始准备加入0.1％明胶加水稀释到6孔板和孵化一夜之间在37 ° C和5％的CO 2 MEF的接驳板。 </li><li>孵化后，取出从BJ细胞的培养基，并添加2毫升6微克/毫升的聚凝胺为辅的生长介质中，500μLhOct4慢病毒，50μLhSox2的慢病毒，50μLhNanog慢病毒和50μLhLin28慢病毒，每孔。轻轻摇动，以确保中期甚至涂层板。孵育过夜，在37 ° C和5％的CO 2。 </li><li>就在同一天，取出1小瓶CF - 1的MEF细胞从液氮和解冻。取出液体从明胶涂井（见步骤1.2），并添加在MEF饲养层细胞密度0.2 × 10 5细胞/孔。每孔总体积应2毫升细胞MEF生长培养基（DMEM培养液450毫升与50毫升FBS和5毫升的非必需氨基酸补充）。 </li><li>次日，0.05％胰蛋白酶/ EDTA和200 XG为5分钟的离心分离与北京的细胞。吸在生长介质的介质和悬浮。删除从MEF馈线板（见第1.4步）中，加2毫升/ BJ细胞悬液。细胞浓度应约5 × 10 4细胞/孔。孵育过夜，在37 ° C和5％的CO 2。 </li><li>更换与人类ES / iPS细胞培养液（100毫升淘汰赛血清替代，5毫升10毫米非必需氨基酸，5毫升200毫米L -谷氨酰胺，0.9毫升补充400毫升的DMEM/F12培养24小时后重新播种β-巯基乙醇55毫米和20 ng / ml的重组人碱性成纤维细胞生长因子）。改变介质为7天，每24小时。 </li><li>七天后，改变中MEF条件培养基（见第2节）。 </li></ol> 2。准备MEF的条件培养基<ol><li>种子的CF - 1 MEF饲养层细胞，2 × 10 5细胞/孔在2毫升MEF生长培养基在6孔板。孵育过夜，在37 ° C和5％的CO 2。 </li><li>改变人类ES / iPS细胞培养液中的介质。孵育过夜，在37 ° C和5％的CO 2。 </li><li>收集上清为4天，每24小时。通过0.22微米过滤器的过滤上清。 </li><li>与碱性成纤维细胞生长因子50毫微克/毫升上清补充。 </li></ol> 3。 IPS殖民地的选择和传代<ol><li>选择一个ES -殖民地在人类胚胎干/ I​​PS文化与10μMStemolecule Y27632 CF - 1 MEF饲养层细胞种子细胞培养菜培养基和重新播种。孵育过夜，在37 ° C和5的CO 2。 </li><li>中期的第7天（不含Stemolecule Y27632补充），每24小时变化的文化。七天后，改变中MEF条件培养基（见准备第2条）。我们继续传代细胞，直到他们表现出典型的人类胚胎干形态。 </li></ol> 4。多潜能性标志物的免疫细胞化学考试<ol><li>清洗细胞，用PBS轻轻三次。 </li><li>修复细胞在室温下20分钟的固定液500μL。 </li><li>清洗细胞，用PBS轻轻三次。 </li><li>阻止非特异性结合，用500μl堵在室温下一个小时的缓冲。 </li><li>用250μL的特定抗体，4℃过夜孵育细胞° C（我们使用1:100 TRA - 1 - 81，TRA - 1 - 60，SSEA - 4，SSEA - 3，OCT4，SOX2，Nanog和Lin28稀释）。 </li><li>清洗细胞，用PBS轻轻三次。 </li><li>用250μL二次抗体孵育的细胞在室温下1小时，保持避光（我们用羊抗小鼠IgM Cy3标记共轭，羊抗鼠IgG Cy3标记山羊抗大鼠IgM抗体Cy3标记共轭轭，和山羊抗兔Cy3标记物）在1:300稀释。 </li><li>清洗细胞，用PBS轻轻三次。 </li><li>添加的DAPI（终浓度为1微克/毫升）的最后一次洗涤，孵育5分钟，以可视化核。 </li><li>分析下放大的细胞。 </li></ol>第5部分：代表结果 1。形态学结果： 人包皮成纤维细胞（北京）分别与Oct4的合作转，SOX2，NANOG和Lin28。早转导后第4天观察形态学变化，以及细胞集群成为更紧密的第17天（图1）包装。菌落手工采摘25天，CF - 1 MEF饲养层细胞培养。为了方便后重新编程的iPS细胞集落形成，我们使用Stemolecule Y27632岩石抑制剂，在每个通道的初始隔夜播种。三个殖民地的顺序轮采摘和传代后的iPS细胞克隆具有良好的形态观察。 2。多能性标志物的表达： 为了进一步孤立的iPS细胞集落，我们期待在ES细胞中表达的共同的全能性的标志物存在。殖民地表现出较强的碱性磷酸酶（AP）的活性（图2）。此外，免疫细胞化学（ICC）的iPS细胞克隆具有多能性的分子标记的特异性抗体的面板，包括表面标志物TRA - 1 - 81，TRA - 1 - 60，SSEA - 4和SSEA - 3以及核标记Oct4，Sox2和Nanog的。隔离IPS殖民地的所有标记（图3）呈阳性反应。国际刑事法院的结果显示，iPS细胞表现出适当的多能性标志物表达模式，表明这些iPS细胞非常类似于未分化的人类胚胎干细胞。 <img src="/files/ftp_upload/1553/1553fig1.jpg" alt="图1" /> 图1：形态学变化转BJ细胞。一个典型的iPS细胞集落形成（一）（二）4天，17天后转明场图像。 <img src="/files/ftp_upload/1553/1553fig2.jpg" alt="图2" /> 图2：AP的北京重新编程细胞的活动。三种不同的菌落染色Stemgent碱性磷酸酶染色试剂盒。 <img src="/files/ftp_upload/1553/1553fig3.jpg" alt="图3" /> 图3：人类iPS细胞表达高水平以下的ES细胞特异性标志物：表面标志物TRA - 1 - 81，TRA - 1 - 60，SSEA - 4和SSEA - 3，10月4日，NANOG和Sox2核标记。

<ol>
	<li>Takahashi, K., 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=Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126, 663-676 (2006).</li><li>Takahashi, K., 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=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., Antosiewicz-Bourget, J., Frane, J. L., Tian, S., Nie, J., Jonsdottir, G. A., Ruotti, V., Stewart, R., Slukvin, I. I., Thompson, J. A. <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 (1917).</li><li>Watanabe, K., Ueno, M., Kamiya, D., Nishiyama, A., Matsumura, M., Wataya, T., Takahashi, J. B., Nishikawa, S., Muguruma, K., Sasai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+ROCK+inhibitor+permits+survival+of+dissociated+human+embryonic+stem+cells.">A ROCK inhibitor permits survival of dissociated human embryonic stem cells.</a> Nat. Biotechnol. 25, 681-686 (2007).</li><li>Koyanagi, M., Takahashi, J., Arakawa, Y., Doi, D., Fukuda, H., Hayashi, H., Narumiya, S., Hashimoto, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+the+Rho/ROCK+pathway+reduces+apoptosis+during+transplantation+of+embryonic+stem+cell-derived+neural+precursors.">Inhibition of the Rho/ROCK pathway reduces apoptosis during transplantation of embryonic stem cell-derived neural precursors.</a> J. Neurosci. Res. 86, 270-280 (2008).</li></ol>

The authors of this article are employed by Stemgent that produces reagents and instruments used in this article.

这些结果表明，Stemgent人类TF慢病毒设置，可以用来有效地产生异位表达诱导的iPS殖民地转染人成纤维细胞中的转录因子。设计重新编程实验时，应考虑几个变量，重新编程，以优化效率。首先，积极的病毒靶比（多重感染的教学语言），可能需要在修改的主要传导步骤，以达到最佳的转染效率。其次，靶细胞的生长条件可能会影响重新编程。健康和增殖细胞更适合重新编程。第三，修改为不同的手机号码的协议时，建议按比例调整到培养皿的表面区域，目标手机号码。最后，如Y27632应用岩石抑制剂应被视为最近的研究已经证明，在提高胚胎干细胞集落生存的效用，以帮助确保成功地重编程。4,5

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<td>Human TF Lentivirus Set</td>
<td>&nbsp;</td>
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<td>Stemolecule Y27632</td>
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<td>TRA-1-81 antibody</td>
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generation of induced pluripotent stem cells by reprogramming human fibroblasts with the stemgent human tf lentivirus set

2006年，Yamanaka和他的同事首先证明了逆转录病毒介导的传递和表达Oct4的，SOX2，c - Myc和KLF4能够诱导小鼠成纤维细胞的多能状态。1同组还报告了人体细胞成功地重编程到诱导多能干细胞（iPS）细胞的逆转录病毒载体提供相同的转录因素中使用的人类版本。此外， 詹姆斯汤姆森等报道的慢病毒介导的另一因素的共同表达（OCT4，SOX2， NANOG和Lin28 ）是人体细胞重编程为iPS细胞的能力。 iPS细胞是类似ES细胞的形态，增殖和分化成人体的所有组织类型的能力。人类iPS细胞对ES细胞的一个明显的优势，他们表现出不破坏胚胎的伦理困境的ES细胞的关键特性。病人特异性的iPS细胞的生成个性化的再生医学疗法规避的一个重要障碍，消除非自体移植的细胞的免疫排斥反应的可能性。 在这里，我们展示的协议进行重新编程人类成纤维细胞，使用Stemgent人类TF慢病毒设置。我们也表明，这个设置重新编程的细胞开始显示IPS形态四天后传导。使用Stemolecule Y27632，我们选择了三个殖民地的顺序轮采摘和传代后的iPS细胞，并观察到正确的形态。我们还证明，细胞重新编程后显示的全能性的标志AP，表面标志物TRA - 1 - 81，TRA - 1 - 60，SSEA - 4和SSEA - 3，和核标记OCT4，SOX2和Nanog。

Watch this Scientific Journal Video about Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set at JoVE.com

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

我们展示了为生成诱导多能干的干细胞，人类体细胞用慢病毒介导的人为因素OCT4，SOX2，NANOG和Lin28交付协议。多能性证实了形态学和胚胎干（ES）细胞特异性标志物的存在。

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 ...

generation-induced-pluripotent-stem-cells-reprogramming-human

Nanyang Technological University

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Biology

Unit 1

Embryonic and Induced Pluripotent Stem Cells

SCIENTISTS IN ACTION

29.8K 次观看.  Stemgent. 我们展示了为生成诱导多能干的干细胞，人类体细胞用慢病毒介导的人为因素OCT4，SOX2，NANOG和Lin28交付协议。多能性证实了形态学和胚胎干（ES）细胞特异性标志物的存在。

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

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

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

生物学

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

文化和维护人类胚胎干细胞

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

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 ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

人类胚胎干细胞冷冻和解冻

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Transduction of Human Cells with Polymer-complexed Ecotropic Lentivirus for Enhanced Biosafety

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for laboratory personnel. Pantropic lentiviruses, such as the commonly used VSV-G pseudotype, are a valuable tool for studying gene function because they can transduce many cell types, including non-dividing cells. However, researchers may wish to avoid production and centrifugation of pantropic viruses encoding oncogenes due to higher biosafety level handling requirements and safety issues. Several potent oncogenes, including c-Myc and SV40 large T antigen, are known to enhance production of induced pluripotent stem cells (iPSC). All other known iPSC-inducing genetic changes (OCT4, SOX2, KLF4, NANOG, LIN28, and p53 loss of function) also have links to cancer, making them of relatively high safety concern as well. 
While these cancer-related viruses are useful in studying cellular reprogramming and pluripotency, they must be used safely. To address these biosafety issues, we demonstrate a method for transduction of human cells with ecotropic lentivirus, with additional emphasis on reduced cost and convenient handling. We have produced ecotropic lentivirus with sufficiently high titer to transduce greater than 90% of receptor-expressing human cells exposed to the virus, validating the efficacy of this approach. 
Lentivirus is often concentrated by ultracentrifugation; however, this process takes several hours and can produce aerosols infectious to human biomedical researchers. As an alternative, viral particles can be more safely sedimented onto cells by complexation with chondroitin sulfate and polybrene (CS/PB). This technique increases the functional viral titer up to 3-fold in cells stably expressing murine retrovirus receptor, with negligible added time and cost. Transduction of human dermal fibroblasts (HDFs) is maximally enhanced using CS/PB concentrations approximately 4-fold lower than the optimal value previously reported for cancer cell lines, suggesting that polymer concentration should be titrated for the target cell type of interest. We therefore describe the use of methylthiazolyldiphenyl-tetrazolium bromide (MTT) to assay for polymer toxicity in a new cell type. We observe equivalent viability of HDFs after viral transduction using either polymer complexation or the standard dose of polybrene (PB, 6 &mu;g/ml), indicating minimal acute toxicity.
In this protocol, we describe the use of ecotropic lentivirus for overexpression of oncogenes in human cells, reducing biosafety risks and increasing the transduction rate. We also demonstrate the use of polymer complexation to enhance transduction while avoiding aerosol-forming centrifugation of viral particles.

Lentiviruses are a valuable research tool for exploring gene function; however, researchers may wish to avoid production of pantropic lentivirus encoding known or suspected oncogenes. As an alternative, we present a safer protocol for use of ecotropic lentivirus on human cells modified to express the ecotropic receptor mSlc7a1.

增强生物安全与聚合物复合物Ecotropic慢病毒的人体细胞的转导

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for ...

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 ...

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, ...