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.remotexs.ntu.edu.sg/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/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. <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. <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>Maherali, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+and+techniques+for+the+generation+of+induced+pluripotent+stem+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+rat+and+human+induced+pluripotent+stem+cells+by+combining+genetic+reprogramming+and+chemical+inhibitors.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+induced+pluripotent+stem+cell+lines+from+adult+rat+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cells+generated+from+patients+with+ALS+can+be+differentiated+into+motor+neurons.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+and+rapid+generation+of+induced+pluripotent+stem+cells+from+human+keratinocytes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+human-induced+pluripotent+stem+cells.">Generation of human-induced pluripotent stem cells.</a> Nat Protoc. 3, 1180-1186 (2008).</li></ol>

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>Knockout DMEM/F12</td>
<td>&nbsp;</td>
<td>&nbsp;</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>&nbsp;</td>
<td>&nbsp;</td>
<td>10828-028</td>
<td>&nbsp;</td>
<tr>
<td>KnockOut Serum Replacement Growth Factor Cocktail</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>A10580-01</td>
<td>&nbsp;</td>
<tr>
<td>FGF-basic, human recombinant protein, 10 &mu;g</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>PHG0024</td>
<td>Note: see appendix for alternative bFGF pack sizes.</td>
</tr>
<tr>
<td>2-Mercaptoethanol</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>21985-023</td>
<td>&nbsp;</td>
<tr>
<td>Dispase</td>
<td>&nbsp;</td>
<td>&nbsp;</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>&nbsp;</td>
<td>&nbsp;</td>
<td>A10480-01</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco&#x2019;s Phosphate Buffered Saline (D-PBS) without calcium and magnesium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>14190-144</td>
<td>&nbsp;</td>
<tr>
<td>DMSO, 500mL</td>
<td>&nbsp;</td>
<td>JT Baker</td>
<td>JT9224-1</td>
<td>&nbsp;</td>
<tr>
<td>Sterile Tissue Culture Hood</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Incubator set at 37&deg;C</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pipette-Aid</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Water Bath set at 37&deg;C</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sterile serological pipettes (5 mL, 10 mL)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>15 mL centrifuge tubes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>60 mm Tissue Culture treated dishes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Liquid nitrogen storage tank</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Isopropanol freezing containers</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1.5 mL Cryovials</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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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

Nanyang Technological University

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

Predicting the Effectiveness of Population Replacement Strategy Using Mathematical Modeling

Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy.  Insight is given into how computational models can provide information on the population dynamics of mosquitoes and the spread of transposable elements through A. gambiae subspecies.  The ethical considerations of releasing genetically modified mosquitoes into the wild are discussed.

Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy. Insight is given into how computational models can provide information on the population dynamics of mosquitoes and the spread of transposable elements through A. gambiae subspecies. The ethical considerations of releasing genetically modified mosquitoes into the wild are discussed.

利用数学建模预测的人口更替策略的有效性

Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy.  ...

科研

生物学

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., ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

隔离和动物血清自由膨胀的人脐血源性间质基质细胞（MSCs），内皮细胞集落形成祖细胞（ECFCs）

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

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

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 ...

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 &#181;l of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/&#181;l from serum but can also be adapted for other biological tissues.

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

小非编码RNA从人血清中分离

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

人类iPS细胞的微小RNA表达谱，视网膜色素上皮衍生自IPS和胎儿视网膜色素上皮

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

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 ...

Bone Conditioned Medium: Preparation and Bioassay

Autologous bone grafts are widely used in oral and maxillofacial surgery, orthopedics, and traumatology. Autologous bone grafts not only replace missing bone, they also support the complex process of bone regeneration. This favorable behavior of autografts is attributed to the three characteristics: osteoconductivity, osteogenicity, and osteoinductivity. However, there is another aspect: Bone grafts release a myriad of molecules, including growth factors, which can target mesenchymal cells involved in bone regeneration. The paracrine properties of bone grafts can be studied in vitro by the use of bone-conditioned medium (BCM). Here we present a protocol on how to prepare bone-conditioned medium from native pig cortical bone, and bone that underwent thermal processing or demineralization. Cells can be directly exposed to BCM or seeded onto biomaterials, such as collagen membranes, previously soaked with BCM. We give examples for in vitro bioassays with mesenchymal cells on the expression of TGF-&#946; regulated genes. The presented protocols should encourage to further reveal the paracrine effects of bone grafts during bone regeneration and open a path for translational research in the broad field of reconstructive surgery.

We describe here how to prepare bone-conditioned medium (BCM) and test its activity in vitro.

骨条件培养基的制备和生物测定

Autologous bone grafts are widely used in oral and maxillofacial surgery, orthopedics, and traumatology. Autologous bone grafts not only replace missing ...

Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 and minimum levels of 0.03 ng ml&#8722;1) in circulating blood are essential as variable levels may indicate altered physiology. For example, growth disorders occurring in childhood can be diagnosed by measuring levels of hGH in blood. Also, the misuse of recombinant hGH in sports not only poses an ethical issue it also presents serious health threats to the abuser. One popular strategy for measuring hGH misuse, relies on the detection of the ratio of 22 kDa hGH to total hGH, as non-22 kDa endogenous levels drop after exogenous recombinant hGH (rhGH) administration. Surface plasmon resonance imaging (SPRi) is an analytical tool that allows direct (label-free) monitoring and visualization of biomolecular interactions by recording changes of the refractive index adjacent to the sensor surface in real time. In contrast, the most frequently used colorimetric method, enzyme-linked immunosorbent assay (ELISA) uses enzyme labeled detection antibodies to indirectly measure analyte concentration after the addition of a substrate that induces a color change. To increase detection sensitivity, amplified SPRi uses a sandwich assay format and near infrared quantum dots (QDs) to increase signal strength. After direct SPRi detection of recombinant rhGH in spiked human serum, the SPRi signal is amplified by the sequential injection of detection antibody coated with near-infrared QDs (Nano-SPRi). In this study, the diagnostic potential of direct and amplified SPRi was assessed for measuring rhGH spiked in human serum and compared directly with the capabilities of a commercially available ELISA kit.

The proposed work assesses the diagnostic potential of direct and amplified surface plasmon resonance imaging (SPRi) assays, particularly for the detection of recombinant human growth hormone in spiked human serum, by comparing SPRi results directly with commercially available enzyme-linked immunosorbent assay (ELISA) kit.

人生长激素测定血清SPRI，纳米单层屋面和酶联免疫吸附测定比较分析

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 ...