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> 990微升</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毫升完整的基因敲除SR馈线免费（KSR - FF）培养基无菌结合下表中列出的组件。 <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 - FF培养基中添加55毫米，182μL，2 - 巯基乙醇（1:550稀释），另外，2 - 巯基乙醇可添加到1X完成中型，储存在2-8℃一个星期。 </li></ol>使iPSCs的KSR - FF中等<ol><li>开始前，预先平衡你的Geltrex涂菜室温在引擎盖。 </li><li>文化在MEF饲养层细胞的iPS细胞，直到他们70％-80％融合。请参阅Gibco公司小鼠胚胎成纤维细胞MEF的文化协议手册。 </李&gt; <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，请参阅下面的表1。 </li><li>吸从培养皿中，并添加适量Dispase解决方案。在37 ° C孵育3-5分钟的菜肴。 </li><li>从每一种文化船吸Dispase解决方案，并洗掉用D - PBS（2至3倍）的MEF饲养层细胞轻轻。 </li><li>添加适量的完整的基因敲除SR载体，以每个培养容器。使用细胞刮刀或5毫升吸管轻轻刮去细胞培养容器的表面。 注：参见附录很难适应的iPS细胞系的替代性适应协议， </li><li>从每个培养皿中收集到单独的15 mL锥形管的细胞悬液。冲洗每个培养容器，一个完整的基因敲除SR介质适量添加的D - PBS漂洗中期到15 mL锥形管含有细胞悬液。要谨慎，不要分解成单个细胞的细胞团块</li><li>离心5分钟沉淀iPSCs的200 XG 15 ml锥形管。 </li><li>吸上清IPSC的颗粒。 KSR - FF媒介分流比（见表1）适量的重悬沉淀。不要打破细胞团块尺寸更小，因为较小的团块，表面不重视。 请注意：我们建议的第3通道的一个分流比为1:2，iPSCs的IPSC的MEF培养液传代，从直接向KSR - FF培养基。通常情况下，分流比为1:3和1:5之间是适当的，但在1:2传代，确保成馈线的文化适应时需要较高的细胞密度。 </li><li>电镀Geltrex涂菜的iPS细胞在此之前，吸出剩余的Geltrex解决方案，从预涂的菜，和每个培养皿中慢慢加入适量的细胞悬液。注意：不要冲洗菜之前电镀。 </li><li>将培养皿中来回方方多次驱散整个表面的菜细胞。轻轻地放置在37℃孵化器的培养皿中，用4至6％，空气中的CO 2饱和湿度。更换花KSR - FF中每天。 </li></ol>传代人类iPS细胞使用KSR - FF <ol start="13"><li>观察确认70-80％汇合，准备传代细胞在显微镜下完成KSR - FF增长的人类iPS细胞。请参考图1。 注：如果殖民地成为太密或过大，出现分化加剧。 </li><li>切出来，删除任何区别的IPSC殖民地前传代的文化。 </li><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，请参阅下面的表1。 </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>轻轻一抖管完全逐出试管底部的细胞沉淀。 </li><li>使用5毫升的血清吸管轻轻重悬在预平衡KSR - FF的细胞。不要磨碎。注意：它是在关键的一步，轻轻重悬细胞，而不使用武力，以避免损坏。 </li><li>细胞转移到一个新的60毫米Geltrex涂层所需的分割比率菜和移动培养皿中来回方方多次分散在其表面的细胞。 </li><li>与加湿的气氛中，培养皿放置在37℃孵化器4％至6％，空气中一氧化碳 2 。 </li><li>第二天，轻轻取代KSR - FF用过的介质，去除细胞碎片。此后更换介质的花费每天。每日观察iPS细胞，通过他们的需要（约每4-5天）。当细胞达到70％至80％汇合的传代是建议。对于iPS细胞的冷冻保存和解冻，参考我们的协议名为“Cryopreserving和人类iPS细胞恢复使用完整的基因敲除血清替代馈线免费中等”。 </li></ol>预期结果<img src="/files/ftp_upload/2236/2236fig1.jpg" alt="图1" /> 图1下面的图片显示相衬Geltrex涂层的文化，使用完整的基因敲除血清替代馈线免费中等菜种植iPSCs的。 iPSCs的展出形态类似人类胚胎干细胞，细胞核大和缺乏细胞质。 （40X放大倍率） <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 valign="top"><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 valign="top"><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 valign="top"><td> Dispase </td><td> 0.5毫升</td><td> 1毫升</td><td> 3-4毫升</td></tr><tr valign="top"><td> D - PBS漂洗</td><td> 2毫升</td><td> 4毫升</td><td> 10毫升</td></tr></tbody></table> 表1。推荐卷 <a href="http://www.jove.com/files/ftp_upload/2236/2236appendix.pdf">请点击此处，见附录</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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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Knockout DMEM/F12</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Cat. no. 12660-012</td>
<td>Note: see appendix for the use of alternative DMEM products</td>
</tr>
<tr>
<td>GlutaMAX-I</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Cat. No. 35050-061</td>
<td>&nbsp;</td>
<tr>
<td>KnockOut Serum Replacement</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>KnockOut SR, Cat. no. 10828-028</td>
<td>&nbsp;</td>
<tr>
<td>KnockOut Serum Replacement Growth Factor Cocktail</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>KnockOut SR-GFC, Cat. no. A10580-01</td>
<td>&nbsp;</td>
<tr>
<td>FGF-basic, human recombinant protein, 10 &mu;g</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>bFGF, Cat. no. 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>Cat. no. 21985-023</td>
<td>&nbsp;</td>
<tr>
<td>Dispase</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Cat. no. 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>Cat. no. 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>Cat. no. 14190-144</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>60mm Tissue Culture treated dishes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

feeder-free adaptation, culture and passaging 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，让您可以轻松地转型你的臀部细胞培养馈线，同时仍保持了您的使用基因敲除的SR。

Watch this Scientific Journal Video about Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium at JoVE.com

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

以下协议提供馈线自由的文化，使用完整的基因敲除血清替代馈线培养基（KSR - FF）为适应人类诱导多能干细胞（iPS）细胞的指令。一旦适应，也提供了不断维护的说明。

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

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 ...

feeder-free-adaptation-culture-passaging-human-ips-cells-using

Research

JoVE Journal

Biology

22.3K 次观看.  Life Technologies.  中文 日本語 

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

Passaging HuES Human Embryonic Stem Cell-lines with Trypsin.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts of cell culture, and tend to acquire karyotypic abnormalities with high population doublings.  Proper passaging is essential for maintaining a healthy, undifferentiated, karyotypically normal HuES human embryonic stem cell culture.  First, an expanding culture is washed in PBS to remove residual media and cell debris, then cells are overlaid with a minimal volume of warm 0.05% Trypsin-EDTA.  Trypsin is left on the cells for up to five minutes, then cells are gently dislodged with a 2mL serological pipette.  The cell suspension is collected and mixed with a large volume of HuES media, then cells are collected by gentle centrifugation.  The inactivated trypsin media mixture is removed, and cells resuspended in pre-warmed HuES media.  An appropriate split ratio is calculated (generally 1:10 to 1:20), and cells re-plated onto a 1-2 day old plate containing a monolayer of irradiated mouse embryonic fibroblast feeder cells.  The newly seeded HuES culture plate is left undisturbed for 48 hrs, then media is changed every day thereafter.  It is important not to trpsinize down to a single cell suspension, as this increases the risk of introducing karyotypic abnormalities.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin. Brought to you by JoVE.

人类胚胎干细胞传代的色调线与胰蛋白酶

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts ...

科研

生物学

Human ES cells: Starting Culture from Frozen Cells

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator.  A frozen vial of cells from long term liquid nitrogen storage or a -80C freezer is sourced and quickly submerged in a 37C water bath for quick thawing.  Cells in freezing media are then removed from the vial and placed in a large volume of HuES media.  The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate.  Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate.  It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate.  The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.

人类胚胎干细胞：启动文化冷冻细胞

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  ...

Passaging Human Neural Stem Cells

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for therapeutic purposes as well as to explore many fundamental processes of human neural development and pathology.  This protocol presents a simple method of culturing and passaging hNSPCs in hopes of standardizing this technique and increasing reproducibility of human stem cell research.  The hNSPCs we use were isolated from cadaveric postnatal brain cortices by the National Human Neural Stem Cell Resource and grown as adherent cultures on flasks coated with fibronectin (Palmer et al., 2001; Schwartz et al., 2003).  We culture our hNSPCs in a DMEM:F12 serum-free media supplemented with EGF, FGF, and PDGF and passage them 1:2 approximately every seven days.  Using these conditions, the majority of the cells in the culture maintain a bipolar morphology and express markers of undifferentiated neural stem cells (such as nestin and sox2).

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro allows to investigate their utility as cell transplants for therapeutic purposes and to explore human neural development. This protocol presents a method of culturing and passaging hNSPCs in hopes of increasing reproducibility of human stem cell research.

传代人类神经干细胞

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for ...

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 ...

Cryopreserving and Recovering 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 and hES cell cultures to feeder-free while still maintaining your use of Knockout SR.

This protocol describes the detailed procedure for cryopreserving human iPS cells in KnockOut SR cryopreservation medium and recovering these cells in complete KnockOut SR Feeder Free (KSR-FF) medium or feeder-based KnockOut SR medium.

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

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 ...

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 ...