注意：要保持无菌培养条件下，所有在本议定书的程序进行使用无菌实验室做法，并在层流罩下进行。 开始之前，确保任何媒体平衡至37 ° C和适当的毒气。 准备Geltrex涂层的培养皿注意：使用CELLstart涂层的培养皿见附录。 <ol><li>解冻之一Geltrex（1毫升）慢慢在2-8℃管和1:100，稀释在99毫升淘汰赛D-MEM/F-12。轻轻混合解决方案。 注：有些iPS细胞系可能需要不同Geltrex的最佳生长的稀释。替代稀释见附录。 </li><li>每个培养皿的整个表面覆盖Geltrex溶液（1毫升为35毫米的菜，为60毫米的菜1.5毫升）。 </li><li>密封每盘用封口膜，以防止干燥和1小时的菜在37 ° C。 注意：此时你可能会储存在4 ° C的Geltrex涂层的培养皿中，长达1个月。每个菜用封口膜密封，以防止干燥Geltrex。 </li><li>之前使用，转让Geltrex涂层菜的层流罩，并允许他们平衡至室温（约1小时）。 </li></ol>准备完整的基因敲除SR馈线中<ol><li>为了准备1毫升10微克/毫升的基本的FGF解决方案，在无菌条件下混合下面列出的组件。分装的解决方案，并在-20 ° C存储长达6个月。 <table border="0"><tbody><tr><td>基本的FGF </td><td> 10微克</td></tr><tr><td> D - PBS </td><td> 990μL </td></tr><tr><td> 10％的BSA </td><td> 10微升</td></tr></tbody></table> 注：可与牛血清白蛋白HSA或在相同浓度的基因敲除的SR取代。 </li><li>为了准备50毫升2毫克/毫升Dispase解决方案，在无菌条件下混合下面列出的组件。过滤消毒的解决方案，并储存在4 ° C至2周。 <table border="0"><tbody><tr><td> Dispase </td><td> 100毫克</td></tr><tr><td> D - PBS </td><td> 50毫升</td></tr></tbody></table></li><li>要准备100毫升的完整的​​基因敲除的SR馈线免费（KSR - FF），在无菌条件下表中列出的组件结合起来。 <table border="1"><tbody><tr><td> 组件 </td><td> 股权集中度 </td><td> 终浓度 </td><td> 卷 </td></tr><tr><td>淘汰赛的DMEM/F12（部件没有。12660-012） </td><td> - </td><td> 1X </td><td> 76.8毫升</td></tr><tr><td> GlutaMAX - I（货号35050-061） </td><td> 200毫米</td><td> 2毫米</td><td> 1毫升</td></tr><tr><td>基因敲除SR（货号。10828-028） </td><td> - </td><td> 20％ </td><td> 20毫升</td></tr><tr><td>淘汰赛SR - GFC（部件没有。A10580 - 01） </td><td> 50X </td><td> 1X </td><td> 2毫升</td></tr><tr><td>碱性成纤维细胞生长因子（部件。PHG0024）。 </td><td> 10微克/毫升</td><td> 20毫微克/毫升</td><td> 200微升</td></tr></tbody></table> 您可能会储存在2-8℃KSR - FF介质长达一个星期。 </li><li>就在预平衡温度和气体的完全培养基，在无菌条件下加入终浓度为0.1毫米2巯基乙醇所需的量（55毫米股票浓度） 。例如，要准备100毫升KSR - F的F培养基中添加182μL，2 - 巯基乙醇（1:550稀释），另外55毫米，2 - 巯基乙醇可添加到1X完成介质和存储长达一个星期在2-8 ° C。 </li></ol>准备冻结/冷冻保存介质冻存介质包括两个媒体;冷冻介质，并冻结中等B.他们是在不同时期的细胞，并且必须保持分离。他们每个冻存中等总量的50％需要。需要中等的超低温冷冻保存的总体积为1毫升为每个被冻结的小瓶。 90％融合60毫米菜的胚胎干细胞可用于银行2瓶的人类胚胎干细胞冻存媒体。 一个额外的2 - 5ML准备足够的量，以确保每个冻存液盘盈。 <table border="1"><tbody><tr><td>冷冻介质中，终体积（50％）： </td><td> 50％的DMEM/F12 </td><td> 50％KSR </td></tr><tr><td>冷冻培养基B（终体积的50％）： </td><td> 80％的DMEM/F12 </td><td> 20％DMSO </td></tr></tbody></table>例如： 如果你冻结了20瓶细胞，你会需要20ML超低温冷冻保存的介质。共24mL冷冻保存液中添加盘盈4ML。这意味着你将需要冻存液12mL（24mL的50％）和冷冻培养基B（50％），24mL 12mL。 <table border="1"><tbody><tr><td>冷冻介质中，（12毫升）： </td><td> 6毫升的DMEM/F12 </td><td> 6毫升KSR </td></tr><tr><td>冷冻培养基B（12毫升）： </td><td> 9.6毫升的DMEM/F12 </td><td> 2.4毫升二甲基亚砜</td></tr></tbody></table>最终组成的超低温冷冻保存中等，65％的DMEM/F12，25％KSR，和10％DMSO。 Cryopreserving iPS细胞<ol><li>预温的Dispase所需的音量在37℃水浴。所需的卷的详细信息，请参阅下面的表1。 </li><li>在37℃水浴for15分钟中预平衡KSR - FF所需的量。所需的卷的详细信息，请参阅表1below。 </li><li>从文化的船只使用吸管吸用过的介质，并与D - PBS冲洗细胞两次。 </li><li>轻轻预热Dispase解决方案添加到培养容器（例如，1毫升每60毫米培养皿Dispase解决方案）。摇动培养容器的整个细胞表面涂层。 </li><li> 3分钟，在37℃孵育培养容器。 </li><li>从孵化器中取出容器，吸Dispase解决方案，并轻轻的D - PBS洗涤细胞。 </li><li>轻轻刮去使用细胞刮刀的文化菜表面的细胞，细胞转移到无菌的15ml离心管。 </li><li> KSR - FF冲洗培养皿两次，轻轻的“喷涂”不沾边的任何细胞。池冲洗15ML管细胞的媒介。 </li><li>在200 XG离心管在室温下5分钟沉淀细胞。 </li><li>小心吸出，而不会干扰细胞沉淀上清，将它丢弃。 </li><li>准备足够数量的离心管。一旦细胞在用DMSO接触，他们应该被分装，冷冻2-3分钟内。 </li><li>轻轻一抖管，完全逐出试管底部的细胞沉淀，轻轻吹打向上和向下，使用5毫升血清吸管]重新暂停在冻存液A.中的细胞。团块均匀的混悬液，同等体积的冷冻培养基B中添加它，在下拉明智的方式，同时轻轻摇动混合细胞悬液。 注：在这一点上，细胞在用DMSO接触，和必须进行有效的工作。一旦细胞接触与二甲基亚砜，他们应该被分装，冷冻2-3分钟内。 </li><li>分装到每个离心管的细胞悬液1毫升。 </li><li>快速放置小瓶先生冷若冰霜，迅速冻结]，并将其转移至-80 ° C过夜。 </li><li>经过隔夜存放于-80 ° C，细胞转移到液氮罐长期贮存。 </li></ol> IPSC的小瓶的解冻和细胞恢复注：KSR - FF可取代含KSR - MEF的条件培养基，或KSR与馈线的使用语言。为媒体准备的说明见附录。 KSR - FF培养基，在50毫升管预温10ML。 <ol><li>室温平衡Geltrex涂层菜肴适当数量和吸电镀前你从菜品的任何液体R细胞。 </li><li>小心地取出胚胎干细胞（或IPSC）从液氮储存罐瓶。 </li><li>在37℃水浴快速解冻冷冻细胞小瓶，直到小瓶仍然只是一个小冷冻块。 </li><li>喷雾小瓶，用70％异丙醇净化它，并将其转移到无菌组织培养罩。 </li><li>无菌操作转移到15 mL锥形管使用5毫升吸管的小瓶中的全部内容。 </li><li>冲洗用1毫升KSR - FF的小瓶和添加15 mL离心管。 </li><li>慢慢地，添加4毫升KSR - FF下拉明智的做法是在15毫升的锥形管的细胞。虽然加入培养基，轻轻地来回移动管混合的细胞。 （这减少了细胞的渗透压冲击）。 </li><li>离心15毫升细胞悬液，在1000RPM（200 × G）为2分钟，在室温下管。 </li><li>小心吸上清液丢弃，而不会干扰细胞沉淀。 </li><li>重悬细胞沉淀在预热KSR - FF（如4mL/60毫米菜）用5毫升吸管轻轻吸液管细胞的向上和向下，直至细胞沉淀完全散去。预计大于70％的可行性，但是如果生存能力是低于70％，它可能采取的细胞更长的时间才能达到汇合。 </li><li>使用相同的吸管，转移下拉明智的预平衡到室温Geltrex涂层的培养皿中的细胞悬液。 </li><li>放置在37℃孵化器的培养皿中，用4至6％，空气中的CO 2饱和湿度。小心漩涡船只在北到南，东，西图案均匀地分布在细胞。 </li><li>轻轻流体改变培养皿中解冻后24小时，此后每天以去除细胞碎片，并提供新鲜的养分，直到大约70-80％的菜是融合。对于您的iPS细胞的持续培养和传代，请参阅我们的协议，名为“免费接驳文化和传代使用完整的基因敲除血清替代的人类iPS细胞直属免费中等” </li></ol>预期结果细胞应该是大约70-80％汇合前冷冻保存。 Dispase消化的结果，在卷曲和折叠沿菌落边缘的殖民地。收获后的细胞，它们是冷冻冷冻保存媒体的小团块。小瓶解冻后，细胞恢复，并附加小团块预涂菜。他们迅速成长，导致在5-6天的融合菜。 表1 - 推荐量<table border="1"><tbody><tr><td> 组件 </td><td> 35毫米菜 </td><td> 60毫米菜 </td><td> 100毫米菜 </td></tr><tr><td>完整的基因敲除简中​​等</td><td> 2毫升</td><td> 4毫升</td><td> 10毫升</td></tr><tr><td> Geltrex解决方案</td><td> 1毫升</td><td> 1.5毫升</td><td> 4-5毫升</td></tr><tr><td> Dispase </td><td> 0.5毫升</td><td> 1毫升</td><td> 3-4毫升</td></tr><tr><td> D - PBS漂洗</td><td> 2毫升</td><td> 4毫升</td><td> 10毫升</td></tr></tbody></table>请<a href="https://www.jove.com/files/ftp_upload/2237/2237_appendix.pdf">点击这里查看附录</a> 。

<ol><li>Takahashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131, 861-872 (2007).</li>
<li>Yu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induced+pluripotent+stem+cell+lines+derived+from+human+somatic+cells%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Induced pluripotent stem cell lines derived from human somatic cells.</a> Science. 318, 1917-1920 (2007).</li>
<li>Maherali, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Guidelines+and+techniques+for+the+generation+of+induced+pluripotent+stem+cells%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Guidelines and techniques for the generation of induced pluripotent stem cells.</a> Cell Stem Cell. 3, 595-605 (2008).</li>
<li>Li, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+rat+and+human+induced+pluripotent+stem+cells+by+combining+genetic+reprogramming+and+chemical+inhibitors%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Generation of rat and human induced pluripotent stem cells by combining genetic reprogramming and chemical inhibitors.</a> Cell Stem Cell. 4, 16-19 (2009).</li>
<li>Liao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+induced+pluripotent+stem+cell+lines+from+adult+rat+cells%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Generation of induced pluripotent stem cell lines from adult rat cells.</a> Cell Stem Cell. 4, 11-15 (2009).</li>
<li>Dimos, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induced+pluripotent+stem+cells+generated+from+patients+with+ALS+can+be+differentiated+into+motor+neurons%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons.</a> Science. 321, 1218-1221 (2008).</li>
<li>Aasen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+and+rapid+generation+of+induced+pluripotent+stem+cells+from+human+keratinocytes%5BTitle%5D)+AND+%22Nat+Biotechnli%22%5BJournal%5D)">Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes.</a> Nat Biotechnli. 26, 1276-1284 (2008).</li>
<li>Park, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+human-induced+pluripotent+stem+cells%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Generation of human-induced pluripotent stem cells.</a> Nat Protoc. 3, 1180-1186 (2008).</li>
</ol>

<table><tbody><tr><td>Knockout DMEM/F12</td><td></td><td>12660-012</td><td>Note: see appendix for the use of alternative DMEM products GlutaMAX-I (Cat. No. 35050-061)</td></tr><tr><td>KnockOut Serum Replacement</td><td></td><td>10828-028</td><td></td></tr><tr><td>KnockOut Serum Replacement Growth Factor Cocktail</td><td></td><td>A10580-01</td><td></td></tr><tr><td>FGF-basic, human recombinant protein, 10 &amp;mu;g</td><td></td><td>PHG0024</td><td>Note: see appendix for alternative bFGF pack sizes.</td></tr><tr><td>2-Mercapt&amp;#8211;thanol</td><td></td><td>21985-023</td><td></td></tr><tr><td>Dispase</td><td></td><td>17105-041</td><td>Note: see appendix for alternative passaging methods</td></tr><tr><td>Geltrex hESC-Qualified Reduced Growth Factor Basement Matrix, 1 mL</td><td></td><td>A10480-01</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline (D-PBS) without calcium and magnesium</td><td></td><td>14190-144</td><td></td></tr><tr><td>DMSO, 500mL</td><td>JT Baker</td><td>JT9224-1</td><td></td></tr><tr><td>Sterile Tissue Culture Hood</td><td></td><td></td><td></td></tr><tr><td>Incubator set at 37&amp;#x00B0;C</td><td></td><td></td><td></td></tr><tr><td>Pipette-Aid</td><td></td><td></td><td></td></tr><tr><td>Water Bath set at 37&amp;#x00B0;C</td><td></td><td></td><td></td></tr><tr><td>Sterile serological pipettes (5 mL, 10 mL)</td><td></td><td></td><td></td></tr><tr><td>Centrifuge</td><td></td><td></td><td></td></tr><tr><td>15 mL centrifuge tubes</td><td></td><td></td><td></td></tr><tr><td>60 mm Tissue Culture treated dishes</td><td></td><td></td><td></td></tr><tr><td>Liquid nitrogen storage tank</td><td></td><td></td><td></td></tr><tr><td>Isopropanol freezing containers</td><td></td><td></td><td></td></tr><tr><td>1.5 mL Cryovials</td><td></td><td></td><td></td></tr></tbody></table>

cryopreserving and recovering of human ips cells using complete knockout serum replacement feeder-free medium

在2006年的发现，人类和小鼠成纤维细胞可以重新编程，以产生iPS细胞与胚胎干细胞非常相似的特质1-3创造了一个有价值的新来源，为药物开发，细胞治疗和基础研究的多能干细胞。 GIBCO公司媒体和试剂已在多年的多能干细胞研究的前沿。淘汰赛的DMEM淘汰赛血清替代补充是胚胎干细胞的生长和现在的iPS细胞培养3-9的首选媒体。这个黄金标准的媒体系统现在可以用于馈线自由的文化，除了与基因敲除的SR生长因子鸡尾酒。 传统的人类ES和iPS细胞培养的方法需要使用鼠标或人类成纤维细胞饲养层，或馈线条件培养基。这些文化的方法是劳动密集型，规模是难以维持臀部细胞的未分化由于未定义的条件。 Invitrogen公司开发了淘汰赛的SR生长因子鸡尾酒，让你轻松转换你的臀部和胚胎干细胞培养，馈线，同时仍保持了您的使用基因敲除的SR。

Watch this Scientific Journal Video about Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium at JoVE.com

Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

这一协议描述为cryopreserving淘汰赛SR冻存介质的人类iPS细胞，这些细胞恢复完整的基因敲除SR直属免费（KSR - FF）介质或进纸器为基础的基因敲除的SR介质的详细过程。

Cryopreserving和恢复人类iPS细胞血清替代馈线中使用完整的基因敲除

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem ...

cryopreserving-recovering-human-ips-cells-using-complete-knockout

Research

JoVE Journal

Biology

18.2K 次观看.  Life Technologies.  中文 日本語 

视频: Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006; Wernig, et al., 2007; Okita, et al., 2007; Maherali, et al., 2007). We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors (Brambrink et al., 2008). Using these inducible constructs, we can derive induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs). In this video, we demonstrate the procedure for the generation of inducible lentiviruses that express the four transcription factors and show how to infect MEFs with these viruses in order to produce iPS cells. By using inducible lentiviruses, the expression of the four factors in controlled by the addition of doxycyline to the culture medium. The advantage of this system over the traditional retroviral infection is the ability to turn the genes on and off so that the kinetics of reprogramming and gene expression requirements can be analyzed in detail.

This video shows the procedure for generating induced pluripotent stem cells using inducible lentivirus that express Oct4, Sox2, c-Myc and Klf4.

从MEFS iPS细胞生成通过强制SOX - 2的表达，10月4，c - Myc基因和Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006;  Wernig, et al., ...

科研

生物学

Small-scale Propagation of Human iPSCs in Serum-free Conditions for Routine Immunocytochemical Characterization

There is great interest in utilizing human induced pluripotent stem cells (hiPSCs) for disease modeling and cell therapeutics due to their patient specificity and characteristic stemness. However, the pluripotency of iPSCs, which is essential to their functionality, must be confirmed before these cells can be used in such applications. While a rigorous characterization of iPSCs, through different cellular and functional assays is necessary to establish their pluripotency, routine assessment of pluripotency maintenance can be achieved more simply and effectively through immunocytochemical techniques. Here, we present a systematic protocol for culturing hiPSCs, in a scaled-down manner, to particularly facilitate the verification of their pluripotent state using immunocytochemistry. More specifically, this methodology encompasses an efficient and cost-effective means of growing iPSCs in serum-free conditions and plating them on small chamber slides or glass coverslips ideal for immunocytochemistry.

Regular characterization of induced pluripotent stem cells (iPSCs), to ascertain maintenance of their pluripotent state, is an important step before these cells are used for other applications. Here we describe a method for the small-scale propagation of human iPSCs specifically designed to enable their easy and routine characterization via immunocytochemistry.

在常规免疫细胞化学鉴定无血清条件下人类iPS细胞的小规模的传播

There is great interest in utilizing human induced pluripotent stem cells (hiPSCs) for disease modeling and cell therapeutics due to their patient ...

发育生物学

Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of human-induced pluripotent stem cells (iPSCs) is particularly attractive for neurodegenerative disease studies, including retinal dystrophies, where iPSC-derived retinal cell models mark a major step forward to understand and fight blindness. In this paper, we describe a simple and scalable protocol to generate, mature, and cryopreserve retinal organoids. Based on medium changing, the main advantage of this method is to avoid multiple and time-consuming steps commonly required in a guided differentiation of iPSCs. Mimicking the early phases of retinal development by successive changes of defined media on adherent human iPSC cultures, this protocol allows the simultaneous generation of self-forming neuroretinal structures and retinal pigmented epithelial (RPE) cells in a reproducible and efficient manner in 4 weeks. These structures containing retinal progenitor cells (RPCs) can be easily isolated for further maturation in a floating culture condition enabling the differentiation of RPCs into the seven retinal cell types present in the adult human retina. Additionally, we describe quick methods for the cryopreservation of retinal organoids and RPE cells for long-term storage. Combined together, the methods described here will be useful to produce and bank human iPSC-derived retinal cells or tissues for both basic and clinical research.

The production of specialized retinal cells from pluripotent stem cells is a turning point in the development of stem cell-based therapy for retinal diseases. The present paper describes a simple method for an efficient generation of retinal organoids and retinal pigmented epithelium for basic, translational, and clinical research.

定义的无异和无饲养培养条件的人 iPSC 视网膜细胞模型的生成

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of ...

Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS cell cultures to feeder-free while still maintaining your use of Knockout SR.

The following protocol provides instruction for adapting human induced Pluripotent Stem (iPS) Cells to feeder-free culture using complete KnockOut Serum Replacement Feeder-Free medium (KSR-FF). Once adapted, instructions for continual maintenance are also provided.

免费接驳适应，文化和传代使用完整的基因敲除血清替代馈线中的人类iPS细胞

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 ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

使用非整合方法分离成人人体皮肤纤维细胞与诱导多能干细胞的生成

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

生物工程

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 ...

Cell Surface Marker Mediated Purification of iPS Cell Intermediates from a Reprogrammable Mouse Model

Mature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of the body and consequently have vast potential for regenerative medicine applications. Traditionally iPS cells are generated by viral introduction of transcription factors Oct-4, Klf-4, Sox-2, and c-Myc (OKSM) into fibroblasts. However, reprogramming is an inefficient process with only 0.1-1% of cells reverting towards a pluripotent state, making it difficult to study the reprogramming mechanism. A proven methodology that has allowed the study of the reprogramming process is to separate the rare intermediates of the reaction from the refractory bulk population. In the case of mouse embryonic fibroblasts (MEFs), we and others have previously shown that reprogramming cells undergo a distinct series of changes in the expression profile of cell surface markers which can be used for the separation of these cells. During the early stages of OKSM expression successfully reprogramming cells lose fibroblast identity marker Thy-1.2 and up-regulate pluripotency associated marker Ssea-1. The final transition of a subset of Ssea-1 positive cells towards the pluripotent state is marked by the expression of Epcam during the late stages of reprogramming. Here we provide a detailed description of the methodology used to isolate reprogramming intermediates from cultures of reprogramming MEFs. In order to increase experimental reproducibility we use a reprogrammable mouse strain that has been engineered to express a transcriptional transactivator (m2rtTA) under control of the Rosa26 locus and OKSM under control of a doxycycline responsive promoter. Cells isolated from these mice are isogenic and express OKSM homogenously upon addition of doxycycline. We describe in detail the establishment of the reprogrammable mice, the derivation of MEFs, and the subsequent isolation of intermediates during reprogramming into iPS cells via fluorescent activated cells sorting (FACS).

Mouse embryonic fibroblast can be reprogrammed into induced pluripotent stem cells at low efficiency by the forced expression of transcription factors Oct-4, Sox-2, Klf-4, c-Myc. The rare intermediates of the reprogramming reaction are FACS isolated via labeling with antibodies against cell surface makers Thy-1.2, Ssea-1, and Epcam.

的iPS细胞从中间体可重编程小鼠模型的细胞表面标志物介导的净化

Mature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of ...

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

从血液高效的iPS细胞生成使用附加体和HDAC抑制剂

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

Advances in Human Induced Pluripotent Stem Cell-Derived Chimeric Antigen Receptor-Expressing Natural Killer Cells

Natural killer (NK) cells are innate immune cells that play a crucial role in the body's defense against tumors and viral infections. The generation of human induced pluripotent stem cell (iPSC)-derived chimeric antigen receptor (CAR) expressing NK cells has emerged as a promising avenue for "off the shelf" cancer immunotherapy. Here, we utilized an NK cell-optimized CAR construct that includes the transmembrane domain of NKG2D, the 2B4 co-stimulatory domain, and the CD3&#950; signaling domain, which has been demonstrated to stimulate robust antigen-specific NK cell-mediated antitumor activity. The use of iPSCs for CAR NK cell generation offers several advantages, including homogenous CAR expression, scalability, reproducibility, and the potential for clinical application. This detailed step-by-step protocol from cell engineering to differentiation enables the generation of NK cell-optimized iPSC-derived CAR-expressing NK cells, providing a standardized and targeted cancer immunotherapy with improved antitumor activity and highlighting their potential as a promising treatment option for various malignancies.

Here, we present a method to differentiate and expand human iPSC-derived chimeric antigen receptor (CAR)-expressing natural killer cells with improved killing against various malignancies. This protocol demonstrates the differentiation and expansion of natural killer (NK) optimized iPSC-derived CAR-NK cells and the measurement of antitumor activity against various tumor cell lines.

人诱导多能干细胞衍生的嵌合抗原受体表达自然杀伤细胞的研究进展

Natural killer (NK) cells are innate immune cells that play a crucial role in the body's defense against tumors and viral infections. The generation of ...