在这个手稿中描述的工作成为可能，资金从美国加州再生医学研究所（CIRM）UCR干细胞核心。

我们的协议，开始与人类iPSCs的方法，以适应无饲养层培养，GeneJuice（EMD）和人类iPSCs使用Amaxa公司nuclefector设备的核转染转染人类iPSCs的协议。 注：下列程序进行，在无菌层流罩。所有的媒体和解决方案是平衡至37℃或室温，然后再启动，除非另有规定。 1。馈线系统上建立人类iPSCs 人类iPSCs可以保持原来的饲养层细胞分裂，转移到Geltrex涂层的菜和维护馈线转染前两个通道。 <ol><li>在4°C过夜解冻Geltrex要准备Geltrex涂料，冷DMEM的解冻Geltrex 1:50稀释。混合轻轻的解决方案。 </li></ol>注：Geltrex，类似基底膜是可溶形式的基底膜MATR九纯化Engelbreth - 霍尔姆群（EHS）从小鼠肿瘤细胞。或者，基底膜可以用作一种细胞外基质，建立人类无馈线IPSC的文化。 <ol start="2"><li>覆盖培养孔与Geltrex溶液（1毫升，35毫米）的整个表面上。外套井Geltrex在37℃培养箱中培养1小时。 </li><li>要通过人类iPSCs，1毫升的accutase中，每孔置于37°C 1分钟，直到大部分细胞开始分离。 </li></ol>要通过人类iPSCs，1毫升的accutase中，每孔置于37°C 1分钟，直到大部分细胞开始分离。 <ol start="4"><li>将添加10-15玻璃珠的细胞，轻轻地旋转板。加入2毫升的淘汰赛DMEM / F12，轻轻捣碎。细胞悬浮液转移到10毫升离心管中。 </li><li>附带细胞以800rpm的转速在室温下3分钟。 </li><li>吸液管从上清液，留下人类IPSC的颗粒完好无损。轻轻一抖管，分散细胞沉淀。 </li><li>从涂好删除Geltrex。 </li><li>轻轻重悬人类IPSC的颗粒在适当体积的STEMPRO。分发井间的进料器，取决于增殖率）。人类iPS细胞可以传代的分路比为1:2到1:6。 </li><li>小心地放入5％CO 2培养箱中，涡流板仔细横跨孔中的细胞，以确保均匀分布。 </li><li>饲料，直到细胞的细胞每天都准备好再次被分裂（当细胞达到80％融合）。 </li><li>流路到新的Geltrex涂覆以及人类iPSCs（步骤1.3到1.9）中的分流比为1:2。小菌落形成上Geltrex转染前涂好且分布均匀。 </li></ol> 2。 GeneJuice转染的人类iPSCs 细胞（6孔板上生长）应该是约40 -50％汇合的转染当天，以达到最佳的转染效率。这是没有必要改变的细胞培养基中，直到第二天。 <ol><li>准备的100微升KO-DMEM/F12在无菌的1.5毫升微量离心管。加入27微升GeneJuice转染试剂。拌匀。在室温下孵育5分钟。 </li><li>加入4μg质粒DNA。管，在室温下孵育15分钟。质粒的选择是至关重要的最佳的转染效率。我们使用的质粒与CAG启动子5（pCAG-eGFP的）驱动的增强型绿色荧光蛋白（eGFP）。 CAG启动子是一个强启动子的转录活性在人类iPSCs，并因此可以被用来驱动在这些细胞中的转基因表达。在我们的手中，线性化的质粒转染效率似乎并没有影响。 </li><li>加入GeneJuice-DNA混合物的细胞和旋流板。旋转板，在1200 rpm离心5分钟（&#39;spinoculation&#39;方法），以增加接触就做好了人类iPSCs转染混合物。 </li><li>培养细胞在37及度C过夜。 </li><li>监测转染效率的第二天。 </li><li>对于稳定转染，改变介质的第二天与新鲜STEMPRO。 24 - 48小时后，加入适当的抗生素选择。 </li></ol> 3。核转染的人类iPSCs 馈线或Geltrex上生长的人类iPSCs可以使用核转染。然而，我们强烈建议replating，nucleofected人类iPSCs到小鼠胚胎成纤维细胞（MEF），人包皮成纤维细胞（HFF）馈线，以确保高细胞活力和恢复。应使用至少2百万个细胞，以实现更高的后核转染的细胞的存活。 <ol><li>准备饲养层细胞的条件培养基（CM）培育人类iPSC的Knockout血清的更换（KOSR）的媒体与饲养层细胞过夜。收集CM，每24小时。 </li></ol>注：任何用于制备饲养层（如BLK6，CF1和MF1）的小鼠品系是适合用于R在CM。 <ol start="2"><li>核转染的当天，预治疗人类iPSCs用10μMROCK抑制剂至少为1小时。 </li><li>准备人类干细胞nucleofector溶液的82μl的在无菌的1.5毫升微量离心管中。加入18微升补充1。拌匀。溶液在37℃下孵育5分钟。 </li><li>预温暖的饲养细胞的条件培养基（CM）和0.25％胰蛋白酶/ EDTA至37℃。引擎盖下，prelabel 1无菌15毫升锥形管。 </li><li>小心地取出媒体从人类iPSC的文化被nucleofected。轻轻洗2毫升1X磷酸盐缓冲溶液（PBS）中，每孔细胞。吸取PBS。 0.25％胰蛋白酶，每孔加入1 ml。细胞在37℃下孵育3分钟。 </li><li>细胞用1000毫升移液管尖轻轻捣碎。洗井的底部，以确保所有人类iPSCs完全脱离。细胞悬浮液转移到标记的15毫升锥形管。 </li></ol>注：胰酶消化成单个C英语学习者应严格避免。细胞应该只被抛下小由约三胞胎细胞组成的细胞团块，。在随后的处理的细胞小的细胞团块（小区三元组），将解离成单细胞。 <ol start="7"><li>加9毫升的MEF媒体灭活胰蛋白酶。附带细胞以800rpm，3分钟。小心吸出上清液，并留下细胞沉淀完好。 </li><li>重悬细胞预热100μl的人干细胞nucleofector的解决方案，从步骤3.3。 </li><li>将细胞转移到一个nucleofector的比色皿，用1毫升移液管尖端部。 </li><li>到试管中的细胞悬浮液中加入4μg质粒DNA。细胞和DNA混合，轻轻回荡。轻按两次比色皿的引擎盖上表面。 </li><li>将试管中到nucleofector持有人。方案B-016。 Nucleofect细胞按按钮X. </li></ol> &lt; <ok> &gt;时，将显示的核转染过程是COMPLET的ED（通常只需要1-5秒）。使用其他核转染方案的（A-023，A-033和U-023），得到小于10％的从​​人类iPSCs的eGFP阳性细胞。 </ok><ol start="12"><li>从试管中使用所提供的巴斯德塑料吸管检索nucleofected细胞。恢复细胞，再悬浮到无菌的1.5毫升微量离心管中预热的CM和ROCK抑制剂。 10分钟，在37°C培养细胞，让细胞恢复。 </li><li>逐滴传递细胞的饲养层上使用1毫升的移液枪枪头。孵育细胞于37℃培养过夜。 </li><li>监测转染效率的第二天。为了获得稳定的转基因人类iPSCs克隆稳定表达的转基因，改变介质的第二天，CM和ROCK抑制剂。 24 - 48小时后，加入适当的抗生素选择。 </li></ol> 4。代表性的成果： <img alt="图1" src="/files/ftp_upload/3110/3110fig1.jpg" /> <st荣图1。的RIV1人类iPSCs与pCAG-EGFP转染的显微照片。 （A）-EGFP表达RIV1细胞转染使用GeneJuice在Geltrex转染后12小时。电镀和馈线后，核转染（B）B-016和A-023（C）程序上形成的RIV1人类iPSCs的殖民地。 （D）稳定EGFP表达人类iPSC的殖民地无处不在的绿色荧光蛋白表达来自GeneJuice。 （E）稳定转染RIV1人的iPS细胞保留构的绿色荧光蛋白表达在胚体分化。

<ol>
	<li>Liew, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cells:+possibilities+for+human+cell+transplantation.">Human embryonic stem cells: possibilities for human cell transplantation.</a> Ann Med. 37, 521-532 (2005).</li><li>Eiges, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+human+embryonic+stem+cell-transfected+clones+carrying+a+marker+for+undifferentiated+cells.">Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells.</a> Curr Biol. 11, 514-518 (2001).</li><li>Zwaka, T. P., Thomson, 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=Homogous+recombination+in+human+embryonic+stem+cells.">Homogous recombination in human embryonic stem cells.</a> Nat Biotechnol. 21, 319-321 (2003).</li><li>Siemen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleofection+of+human+embryonic+stem+cells.">Nucleofection of human embryonic stem cells.</a> Stem Cells Dev. 14, 378-383 (2005).</li><li>Liew, C. G., Draper, J. S., Walsh, J., Moore, H., Andrews, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+and+stable+transgene+expression+in+human+embryonic+stem+cells.">Transient and stable transgene expression in human embryonic stem cells.</a> Stem Cells. 25, 1521-1528 (2007).</li><li>Xia, X., Ayala, M., Thiede, B. R., Zhang, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro-+and+in+vivo-induced+transgene+expression+in+human+embryonic+stem+cells+and+derivatives.">In vitro- and in vivo-induced transgene expression in human embryonic stem cells and derivatives.</a> Stem Cells. 26, 525-533 (2008).</li><li>Braam, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+genetic+manipulation+of+human+embryonic+stem+cells.">Improved genetic manipulation of human embryonic stem cells.</a> Nat Methods. 5, 389-392 (2008).</li><li>Braam, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeder-free+culture+of+human+embryonic+stem+cells+in+conditioned+medium+for+efficient+genetic+modification.">Feeder-free culture of human embryonic stem cells in conditioned medium for efficient genetic modification.</a> Nat Protoc. 3, 1435-1443 (2008).</li><li>Hohenstein, K. A., Pyle, A. D., Chern, J. Y., Lock, L. F., Donovan, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleofection+mediates+high-efficiency+stable+gene+knockdown+and+transgene+expression+in+human+embryonic+stem+cells.">Nucleofection mediates high-efficiency stable gene knockdown and transgene expression in human embryonic stem cells.</a> Stem Cells. 26, 1436-1443 (2008).</li><li>Placantonakis, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BAC+transgenesis+in+human+embryonic+stem+cells+as+a+novel+tool+to+define+the+human+neural+lineage.">BAC transgenesis in human embryonic stem cells as a novel tool to define the human neural lineage.</a> Stem Cells. 27, 521-532 (2009).</li></ol>

没有利益冲突的声明。

我们的协议简单，可靠，重现性好技术引进转基因人类iPSCs没有显着的毒性作用和细胞死亡。人类iPSCs应成更小的细胞团块，（5-10个细胞）传代，并镀上Geltrex在高密度（1:2），以确保最佳的转染效率在众多的小菌落。对于人类IPSC的线更容易发生分化和细胞死亡，更高数量的人类iPSCs（最多至4×10 6个细胞），应使用一个单一的核转染实验。瞬时转染1天之内产生大量的转基因表达人类iPSCs。稳定转染的iPSC的克隆通常出现在7天，应该是在三个星期内随时可以拿起这些转基因的殖民地。这里所描述的CAG启动子的使用确保无处不在表达绿色荧光蛋白记者。在这样的改善，我们的协议可以被输入到其他应用程序，包括过度表达，Conditional归纳，推导系特异性记者，短发夹RNA，siRNA敲除，基因打靶技术和同源重组。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> 0.25％胰蛋白酶与EDTA </td><td> Invitrogen公司</td><td> 25200056 </td><td></td></tr><tr><td> 2 - 巯基乙醇</td><td> Invitrogen公司</td><td> 21985-023 </td><td></td></tr><tr><td> 3毫米的玻璃珠</td><td> Fisher Scientific则</td><td> 11-312A </td><td>玻璃珠应用盐酸（HCl）过夜，用氢氧化钠（NaOH）和蒸馏水冲洗掉，并在使用前用高压灭菌器灭菌。 </td></tr><tr><td> Accutase细胞分离试剂</td><td> Invitrogen公司</td><td> A11105-01 </td><td></td></tr><tr><td>碱性成纤维细胞生长因子（bFGF） </td><td> Invitrogen公司</td><td> PHG0263 </td><td></td></tr><tr><td> DMEM（高糖） </td&gt; <td>龙沙</td><td> 12-741 F </td><td></td></tr><tr><td>胎牛血清的</td><td> Invitrogen公司</td><td> 16000044 </td><td></td></tr><tr><td> Geltrex </td><td> Invitrogen公司</td><td> 12760013 </td><td>生长因子减少</td></tr><tr><td> GeneJuice转染试剂</td><td> EMD Biosciences公司</td><td> 70967 </td><td></td></tr><tr><td> Glutamax-I（100X） </td><td> Invitrogen公司</td><td> 35050061 </td><td> L-谷氨酰胺（Invitrogen公司，25030081）也可以被代替使用。 </td></tr><tr><td>人类iPSC的KOSR媒体</td><td></td><td></td><td> 500毫升人ES媒体由390毫升基因敲除的DMEM / F12，100毫升Knockout血清替代品，将5ml Glutamax-I（100X），5毫升的NEAA（100X），500微升，2 - 巯基乙醇（55毫米），用10 ng / ml的碱性成纤维细胞生长因子和补充。 </td></tr><tr><td>淘汰赛DMEM/F12 </td><td> Invitrogen公司</td><td> 12660-012 </td><td></td></tr><tr><td>Knockout血清替代品（KOSR） </td><td> Invitrogen公司</td><td> 10828-028 </td><td></td></tr><tr><td>小鼠胚胎成纤维细胞介质</td><td></td><td></td><td> MEF媒体包括90％FBS，1×NEAA Glutamax-I，1X和1X丙酮酸钠的DMEM（高糖） </td></tr><tr><td>非必需氨基酸，NEAA（100X） </td><td> Invitrogen公司</td><td> 11140050 </td><td></td></tr><tr><td>人类干的细胞nucleofector解决方案与补充</td><td>龙沙</td><td> VAPH-5012 </td><td></td></tr><tr><td>磷酸盐缓冲液，不含Ca 2 +和Mg 2 + </td><td> Invitrogen公司</td><td> 10010023 </td><td></td></tr><tr><td>丙酮酸钠（100X） </td><td> Invitrogen公司</td><td> 11360070 </td><td></td></tr><tr><td> STEMPRO介质工具包</td><td> Invitrogen公司</td><td> A1000701 </td><td> 500毫升STEMPRO媒体组成的454毫升敲满分的DMEM / F12，10毫升STEMPRO无血清生长添加物（50X），5毫升Glutamax-I（100X），将5ml NEAA（100X），36毫升BSA（牛血清白蛋白，25％），909微升2 - 巯基乙醇（55MM用10 ng / ml的碱性成纤维细胞生长因子）和补充。 </td></tr></tbody></table>

transfecting and nucleofecting human induced pluripotent stem cells

转基因（GM）将继续是一个重要的工具，在研究干细胞生物学和提出潜在的临床应用人类胚胎干细胞（胚胎干细胞）1。尽管仍有改进的在几个基因传递方法已被描述2-9，转染仍然是一个反复无常的过程对人类胚胎干细胞，尚未据报道，在人类诱导多能干细胞（iPSCs）。在这段视频中，我们展示了我们的实验室常规转染和利用质粒增强型绿色荧光蛋白（EGFP）记者nucleofects人类iPSCs。人类iPS细胞无饲养层培养适应和维护，以消除可能的饲养细胞转染，并允许稳定的转基因IPSC的克隆，转染后的有效选择。对于核转染，人类iPSCs预先处理与ROCK抑制剂11，胰蛋白酶消化成小的细胞团块，上馈线nucleofected和再接种在饲养细胞空调中，增强细胞恢复。 6小时后，可以得到转基因表达的人类iPSCs。抗生素的选择被施加后24小时和稳定的转基因品系1星期内出现。本协议的鲁棒性和重现性好，可用于人类iPSC的线，在不改变这些细胞的多能性。

Watch this Scientific Journal Video about Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells at JoVE.com

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

尽管基因改造的最新进展，转染的人类胚胎干细胞（胚胎干细胞）保持一个反复无常的过程。据我们所知，系统和有效的方法，转染人诱导多能干细胞（iPSCs）尚未见报道。在这里，我们描述了可靠的协议，有效地转染和nucleofect人类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 ...

transfecting-and-nucleofecting-human-induced-pluripotent-stem-cells

Research

JoVE Journal

Biology

21.4K 次观看.  University of California Riverside. 尽管基因改造的最新进展，转染的人类胚胎干细胞（胚胎干细胞）保持一个反复无常的过程。据我们所知，系统和有效的方法，转染人诱导多能干细胞（iPSCs）尚未见报道。在这里，我们描述了可靠的协议，有效地转染和nucleofect人类iPSCs。

视频: Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Transfecting Human Neural Stem Cells with the Amaxa Nucleofector

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection reagents has often resulted in poor cell viability and low transfection efficiency.  Other mechanical methods of introducing a gene of interest, such as a &#8220;gene gun&#8221; or microinjection, are also limited by poor cell viability and low numbers of transfected cells.  The strategy of using viral constructs to introduce an exogenous gene into primary cells has been constrained by both the amount of time and labor required to create viral vectors and potential safety concerns.  We describe here a step-by-step protocol for transfecting hNSPCs using Amaxa's Nucleofector device and technology with electrical current parameters and buffer solutions specifically optimized for transfecting neural stem cells.  Using this protocol, we have achieved initial transfection efficiencies of ~35% and ~70% after stable transfection.  The protocol entails combining a high number of hNSPCs with the DNA to be transfected in the appropriate buffer followed by electroporation with the Nucleofector device.

Introducing a gene of interest into a cell is a powerful method for elucidating its function in vivo. This protocol describes an efficient method of transfecting a culture of human neural stem/precursor cells (hNSPCs) using the Nucleofector electroporation apparatus made by Amaxa.

人类神经干细胞转染与Amaxa Nucleofector

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection ...

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 ...

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 ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

适用于培养人类胚胎和诱导多能干细胞的小鼠胚胎成纤维细胞的制备

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

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 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 ...

Episomal Reprogramming of Dermal Fibroblasts to Generate Human-Induced Pluripotent Stem Cells (hiPSCs)

Episomal Reprogramming of Dermal Fibroblasts to Generate Human-Induced Pluripotent Stem Cells (HiPSCs)  

真皮成纤维细胞的游离体重编程以产生人诱导的多能干细胞 （hiPSC）

Generation and Maintenance of Primate Induced Pluripotent Stem Cells Derived from Urine

Cross-species approaches studying primate pluripotent stem cells and their derivatives are crucial to better understand the molecular and cellular mechanisms of disease, development, and evolution. To make primate induced pluripotent stem cells (iPSCs) more accessible, this paper presents a non-invasive method to generate human and non-human primate iPSCs from urine-derived cells, and their maintenance using a feeder-free culturing method.
The urine can be sampled from a non-sterile environment (e.g., the cage of the animal) and treated with a broad-spectrum antibiotic cocktail during primary cell culture to reduce contamination efficiently. After propagation of the urine-derived cells, iPSCs are generated by a modified transduction method of a commercially available Sendai virus vector system. First&#160;iPSC colonies may already be visible after 5 days, and can be picked after 10 days at the earliest. Routine clump passaging with enzyme-free dissociation buffer supports pluripotency of the generated iPSCs for more than 50 passages.

The present protocol describes a method to isolate, expand, and reprogram human and non-human primate urine-derived cells to induced pluripotent stem cells (iPSCs), as well as instructions for feeder-free maintenance of the newly generated iPSCs.