这项工作得到了来自美国国立卫生研究院的资助（CA127574 CA107040和CA09071）与wm

1。收获从小鼠移植瘤<ol><li>准备细胞解离缓冲液：贝科的改良Eagle培养基（DMEM），辅以10％小牛血清（FCS），青霉素，链霉素，200 U / mL胶原酶IV型，和0.6 U / mL的dispase。 </li><li>一个裸鼠（NU + / NU +）窝藏皮下人胰腺癌移植瘤，是最大的直径小于1厘米的鼠标是安乐死二氧化碳窒息和颈椎脱位。 </li><li>皮下肿瘤是收获从鼠标的侧面使用镊子和剪刀钝清扫，放置在细胞分解缓冲区立即。 </li><li>肿瘤称重，每1克肿瘤组织放入10 mL细胞解离缓冲。 </li></ol> 2。生成的移植瘤单细胞悬液<ol><li>在无菌的生物安全柜的工作，肿瘤转移与细胞分离缓冲液1毫升100 × 20毫米培养皿。肿瘤是机械肉末用无菌刀片和镊子。通过5毫升血清吸管中的肿瘤细胞悬液磨碎10倍。肿瘤件应足够小，不堵塞5毫升血清吸管。 </li><li>肿瘤细胞悬液转移到50 mL锥形管中的细胞分解缓冲区的剩余量（填补低于50％），并在1分钟的最大速度振荡。 </li><li>肿瘤细胞悬液，然后在37℃2小时1分钟，每20分钟的间歇振荡。 </li></ol> 3。消耗细胞悬浮组织碎片和死细胞<ol><li> 2小时孵化后的细胞悬液，振荡1分钟的最高速度。 </li><li>细胞悬液，然后通过一个70微米的过滤器和收集在一个新的50 - mL锥形管和调整最终体积至15毫升每管。 </li><li>为了进一步分离可行的细胞死亡的细胞和组织碎片，肿瘤细胞悬液密度使用聚蔗糖 - 帕确加离心分离。聚蔗糖 - 帕确加衬垫pipeting加上缓慢的单元格下方小心，不要混合两​​层悬挂15毫升的聚蔗糖 - 帕确。 </li><li>细胞悬液和聚蔗糖层是在500X克离心30分钟，与刹车关闭。 </li><li>聚蔗糖 - 帕确Plus和细胞解离缓冲接口的细胞被收集并转移到一个新的50 mL锥形管。 </li><li>新鲜的DMEM含10％FCS的补充，加入细胞悬液稀释1:3（如20毫升新鲜媒体加入到10 ml的细胞悬液）。 </li><li>细胞收集在450X克离心10分钟，倒出的媒体，和细胞沉淀重悬于1毫升ALDEFLUOR缓冲区。 </li><li> 10微升的细胞悬液稀释10μL，台盼蓝活细胞计数使用一个血球。如果在这一步中指出大量的死细胞或组织碎片，然后可重复细胞密度离心分离（步骤3.3 - 3.7）。 </li></ol> 4。染色荧光激活细胞分选的单元格<ol><li>标签七个12 × 75毫米的聚苯乙烯试管作为如下： <ol><li>不染</li><li> CD44 - APC </li><li> CD24 - PE </li><li>小鼠CD31的抗生物素蛋白+鼠标沿袭-生物素+鼠标的H - 2Kd -生物素</li><li> ALDEFLUOR </li><li> ALDEFLUOR + DEAB + IgG抗体2bκ- APC + IgG抗体2aκ聚乙烯</li><li> ALDEFLUOR + CD44 - APC + CD24 - PE +鼠标CD31的抗生物素蛋白+鼠标沿袭抗生物素蛋白+鼠标的H - 2Kd -生物素。 </li></ol></li><li> ALDEFLUOR缓冲区在2毫升细胞悬液稀释到终浓度为5-10万细胞/ mL，置于冰上。添加100μL的细胞悬液，以管＃1，2，3和4，并保持他们在冰上。添加1μL，DEAB＃6管，并保持在冰上。 ALDEFLUOR试剂加入到剩余的细胞悬液，稀释1000 - 倍（如添加1.6μLALDEFLUOR试剂1.6 mL细胞悬液），拌匀，并置于冰上。 100μL这种混合物管＃5和6，其余的混合物（1.4毫升）传输管＃7。在37℃水浴40分钟中孵育管。混合细胞悬液，每10分钟一班，以防止沉淀到管底部的细胞。 </li><li>离心机450X克细胞和4 ° C下10分钟，然后倒出的缓冲区。重悬于100μLALDEFLUOR缓冲区管＃1和5的颗粒。要管＃2，3，4和6，添加100μLALDEFLUOR缓冲区包含如下稀释相应抗体，IgG 抗体 2bΚ- APC，IgG 抗体 2aΚ- PE，CD44 - APC和CD24 - PE： 1:20抗体; 1:100的小鼠CD31的抗生物素蛋白，鼠标沿袭抗生物素蛋白和鼠标的H - 2Kd生物素抗体。 7＃管加入1.4毫升的ALDEFLUOR含有CD44 - APC的1:20稀释的缓冲，CD24 - PE抗体和1:100稀释的小鼠CD31 -生物素，鼠标沿袭抗生物素蛋白，小鼠的H - 2Kd -生物素抗体。在冰上孵育10分钟的细胞悬液。 </li><li>离心机450X克细胞和4 ° C下10分钟，然后倒出的缓冲区。重悬浮颗粒在管1＃，2，3，5，6和100μLALDEFLUOR缓冲区。准备1.5毫升含1:100稀释的链霉亲和PerCP蛋白ALDEFLUOR缓冲区。加入100μL＃4管，并加入1.4毫升管＃7。在冰上孵育10分钟的细胞悬液。 </li><li>离心机450X克细胞和4 ° C下10分钟，然后倒出的缓冲区。重悬浮颗粒在管1＃，2，3，4，5，和6 200μLALDEFLUOR缓冲区。重悬在2.8毫升含有2μg/ mL的碘化丙啶ALDEFLUOR缓冲区＃7管沉淀。在任何时候都保持冰管。 </li><li>通过使用12 × 75毫米的聚苯乙烯试管过滤器，过滤器盖35微米的细胞。 </li></ol> 5。荧光激活细胞分选分离肿瘤干细胞<ol><li>一个FACSAria流式细胞仪细胞分选的准备。 </li><li>补偿控制是从管＃1，2，3，4和5使用的细胞。 </li><li>盖茨创建正向和侧面散射参数的基础上，收集细胞汗衫。 </li><li>非滑鼠（PerCP负）和可行的（非碘化丙啶染色）细胞使用FL - 3通道的门控。 </li><li> ALDH +细胞收集的基础上，盖茨创建使用DEAB处理的细胞（＃6管）和FL - 1通道。 </li><li> CD44 + CD24 +细胞是基于对盖茨收集，使用与ALDEFLUOR，IgG 抗体 2bΚ- APC和IgG2aκ- PE（＃6管），染色细胞，使用FL - 4和FL - 2通道创建。 </li><li>细胞收集在12 × 75毫米聚苯乙烯测试含有1毫升辅以10％FCS的DMEM管。 </li><li>细胞已被排序后，离心10分钟，倒出媒体450X克细胞悬液，500μL新鲜的DMEM重悬细胞与10％FCS的补充。 </li><li>计数排序使用在步骤3.8中所描述的血球细胞的数量。 </li></ol> 6。代表性的成果该协议将导致ALDH +和CD44 + CD24 +胰腺癌社区体育的隔离。鼠源性细胞的比例是可变的，但我们发现，大多数人胰腺癌移植瘤包含20-60％的鼠源性细胞CD31的使用鼠标鼠标的血统鸡尾酒，和鼠标的H - 2Kd生物素标记的抗体（图1A ）。同样，每次从不同的异种移植的CSC的人口的频率是可变的，但一般我们发现，细胞总人口的1-4％是乙醛脱氢酶（图1B）和0.2-5％的CD44 + CD24 +（图1C）。 我们经常手动FACSAria机计数排序细胞使用血球计数，因为往往是由于不准确的FACSAria检测非细胞颗粒的。 <img src="/files/ftp_upload/2169/2169fig1.jpg" alt="图1" /> 图1。流式细胞仪的情节描绘从胰腺癌移植瘤的特定人群（一）积极在FL - 3通道的细胞染色（ 碘化丙啶+鼠标CD31 +鼠标沿袭+或小鼠的H - 2Kd）被排除在外，以便隔离只可行的和非鼠源性细胞。 （二）在人胰腺癌移植瘤的乙醛脱氢酶活动ALDEFLUOR试剂ALDH1抑制剂二乙氨基苯甲醛（DEAB）的存在和缺乏流动使用流式细胞仪测定。帧代表盖茨描绘ALDH +已创建的基础上与DEAB处理的细胞，然后用于未经处理的细胞的细胞。 ALDH +细胞的百分比显示在门上。 （三）CD44 + CD24 +门是创建的基础上与ALDEFLUOR染色的细胞，IgG 抗体 2bκ- APC（CD44 - APC的isotypic控制）和IgG2aκ- PE（CD24 - PE isotypic控制）抗体。 CD44 + CD24 +细胞的百分比显示在门上。

<ol>
	<li>Clarke, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells--perspectives+on+current+status+and+future+directions:+AACR+Workshop+on+cancer+stem+cells.">Cancer stem cells--perspectives on current status and future directions: AACR Workshop on cancer stem cells.</a> Cancer Res. 66, 9339-9344 (2006).</li><li>Al-Hajj, M., Wicha, M. S., Benito-Hernandez, A., Morrison, S. J., Clarke, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+identification+of+tumorigenic+breast+cancer+cells.">Prospective identification of tumorigenic breast cancer cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 100, 3983-3988 (2003).</li><li>Ginestier, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ALDH1+Is+a+Marker+of+Normal+and+Malignant+Human+Mammary+Stem+Cells+and+a+Predictor+of+Poor+Clinical+Outcome.">ALDH1 Is a Marker of Normal and Malignant Human Mammary Stem Cells and a Predictor of Poor Clinical Outcome.</a> Cell stem cell. 1, 555-567 (2007).</li><li>Jones, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+clonotypic+B+cells+in+classic+Hodgkin+lymphoma.">Circulating clonotypic B cells in classic Hodgkin lymphoma.</a> Blood. 113, 5920-5926 (2009).</li><li>Matsui, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonogenic+multiple+myeloma+progenitors,+stem+cell+properties,+and+drug+resistance.">Clonogenic multiple myeloma progenitors, stem cell properties, and drug resistance.</a> Cancer Res. 68, 190-197 (2008).</li><li>Li, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+pancreatic+cancer+stem+cells.">Identification of pancreatic cancer stem cells.</a> Cancer Res. 67, 1030-1037 (2007).</li><li>Hermann, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+populations+of+cancer+stem+cells+determine+tumor+growth+and+metastatic+activity+in+human+pancreatic+cancer.">Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer.</a> Cell stem cell. 1, 313-323 (2007).</li><li>Rasheed, Z. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+significance+of+tumorigenic+cells+with+mesenchymal+features+in+pancreatic+adenocarcinoma.">Prognostic significance of tumorigenic cells with mesenchymal features in pancreatic adenocarcinoma.</a> J Natl Cancer Inst. 102, 340-351 (2010).</li><li>Rubio-Viqueira, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vivo+platform+for+translational+drug+development+in+pancreatic+cancer.">An in vivo platform for translational drug development in pancreatic cancer.</a> Clin Cancer Res. 12, 4652-4661 (2006).</li><li>Jones, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+aldehyde+dehydrogenase+in+viable+cells.">Assessment of aldehyde dehydrogenase in viable cells.</a> Blood. 85, 2742-2746 (1995).</li></ol>

本协议描述了人类异种移植胰腺社区体育隔离。在此过程中的几个关键步骤，将可行的社区体育会提高产量。的胰腺癌社区体育纯人口的复苏是依赖于FACSAria的排序前的细胞悬液中的活细胞的纯度。同时注意使用小于最大的直径1厘米的异种移植，有助于减少肿瘤中心坏死组织量。此外，消耗组织碎片和死细胞的细胞悬液，在聚蔗糖的帕确梯度离心法是至关重要的，如果大量的组织碎片或非活菌检测细胞的血球计数时可重复。 染色协议的描述，这里利用ALDEFLUOR试剂系统检测到乙醛脱氢酶的活动以及荧光标记的抗体来检测特定的细胞表面抗原。 ALDEFLUOR染色是在37 ° C和，因此，需要完成前抗体染色（冰），以防止抗体封顶的潜力，它可以发生在37 ° C由于细胞外排ALDEFLUOR试剂，重要的是保持ALDEFLUOR缓冲区的细胞在4 ° C已经完成ALDEFLUOR染色后。 ALDEFLUOR缓冲区包含了药物外排抑制剂，有助于维持细胞内的ALDEFLUOR水平。 由于这种方法分离可行的胰腺癌社区体育，他们可以应用于实验，测量这些细胞的生理功能。我们这些细胞用于肿瘤开始利用免疫功能低下小鼠的实验和体外细胞迁移/入侵检测。此外，RNA，DNA和蛋白质可以收集这些细胞的分析研究，比较CSC和非CSC人口。

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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>DMEM </td>
<td>&nbsp;</td>
<td>Invitrogen (Carlsbad, CA) </td>
<td>11965-118 </td>
<td>&nbsp;</td>
<tr>
<td>Fetal calf serum </td>
<td>&nbsp;</td>
<td>Harlan (Indianapolis, IN)</td>
<td>BT-9501</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-streptomycin </td>
<td>&nbsp;</td>
<td>Invitrogen </td>
<td>15140-122 </td>
<td>&nbsp;</td>
<tr>
<td>Collagenase type IV </td>
<td>&nbsp;</td>
<td>Invitrogen </td>
<td>17104-019 </td>
<td>&nbsp;</td>
<tr>
<td>Dispase </td>
<td>&nbsp;</td>
<td>Sigma (St. Louis, MO) </td>
<td>D4818 </td>
<td>&nbsp;</td>
<tr>
<td>100 x 20 mm culture dish </td>
<td>&nbsp;</td>
<td>BD Biosciences (San Jose, CA) </td>
<td>353003 </td>
<td>&nbsp;</td>
<tr>
<td>70-&mu;m filter </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>352350 </td>
<td>&nbsp;</td>
<tr>
<td>50-mL conical tube </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>352070 </td>
<td>&nbsp;</td>
<tr>
<td>Ficoll-Paque Plus </td>
<td>&nbsp;</td>
<td>GE Healthcare (Upsala, Sweden) </td>
<td>17-1440 </td>
<td>&nbsp;</td>
<tr>
<td>ALDEFLUOR reagent system </td>
<td>&nbsp;</td>
<td>Stem Cell Technologies (Vancouver, BC, Canada) </td>
<td>01700 </td>
<td>&nbsp;</td>
<tr>
<td>Trypan blue </td>
<td>&nbsp;</td>
<td>Invitrogen </td>
<td>15250-061 </td>
<td>&nbsp;</td>
<tr>
<td>12 x 75 mm polystyrene test tubes </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>352054 </td>
<td>&nbsp;</td>
<tr>
<td>CD44-APC (clone G44-26) </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>559942 </td>
<td>&nbsp;</td>
<tr>
<td>CD24-PE (clone ML5) </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>555428 </td>
<td>&nbsp;</td>
<tr>
<td>Mouse CD31-biotin </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>553371 </td>
<td>&nbsp;</td>
<tr>
<td>Mouse lineage-biotin </td>
<td>&nbsp;</td>
<td>Miltenyi Biotec (Auburn, CA) </td>
<td>130-092-613 </td>
<td>&nbsp;</td>
<tr>
<td>Mouse H-2Kd-biotin </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>553564 </td>
<td>&nbsp;</td>
<tr>
<td>IgG2b&#x03BA;-APC</td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>555745 </td>
<td>&nbsp;</td>
<tr>
<td>IgG2a&#x03BA;-PE</td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>555574 </td>
<td>&nbsp;</td>
<tr>
<td>Streptavidin-PerCP protein </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>554064 </td>
<td>&nbsp;</td>
<tr>
<td>Propidium iodide </td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>P4170 </td>
<td>&nbsp;</td>
<tr>
<td>12 x 75 mm polystyrene test tubes with strainer caps </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>352235 </td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

isolation of stem cells from human pancreatic cancer xenografts

在越来越多的恶性肿瘤和癌症干细胞（CSCS）已确定在功能上进行自我更新的能力，并产生有区别的后代1的定义。这些特性使社区体育重述注射到免疫功能低下小鼠时，原发肿瘤。社区体育会内上皮恶性肿瘤首次描述在乳腺癌和发现，以显示特定的细胞表面抗原的表达（CD44 + CD24低/ - ）2 。从那时起，社区体育会已确定在其他人类恶性肿瘤，使用CD44和CD24以及其他表面抗原的数量越来越多。生理特性，包括醛脱氢酶（ALDH）的活性，也被用来隔离恶性组织3-5个社区体育会。 最近，我们和其他人发现胰腺癌社区体育会的基础上乙醛脱氢酶的活性和表达的细胞表面抗原CD44和CD24和CD133的6-8。这些高致瘤性的人口可能会或可能不会重叠和显示等功能。我们发现，ALDH +和CD44 + CD24 +胰腺癌社区体育会是同样的致瘤性，但ALDH +细胞是相对更侵入8。在这个协议中，我们描述了从低通过人类异种移植的方法来隔离可行的胰腺癌社区体育。从小鼠移植瘤肿瘤收获，并制作成单细胞悬液。组织碎片和死细胞是从活细胞分离，然后使用抗CD44和CD24抗体和使用ALDEFLUOR试剂，乙醛脱氢酶 10的荧光底物染色。社区体育会是由荧光激活细胞分选分离。隔离的社区体育会，然后可以用于活细胞检测分析或功能的要求。

Watch this Scientific Journal Video about Isolation of Stem Cells from Human Pancreatic Cancer Xenografts at JoVE.com

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

癌症干细胞（CSCS）已确定在一些恶性肿瘤。在这个协议中，我们描述了一个流式细胞仪方法，利用醛脱氢酶活性和CD44和CD24的表达，从人胰腺癌移植瘤的社区体育会隔离。然后可以使用这些活细胞功能和分析研究。

人胰腺癌移植瘤的干细胞的分离

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

isolation-of-stem-cells-from-human-pancreatic-cancer-xenografts

Research

JoVE Journal

Biology

22.2K 次观看.  Johns Hopkins University School of Medicine. 癌症干细胞（CSCS）已确定在一些恶性肿瘤。在这个协议中，我们描述了一个流式细胞仪方法，利用醛脱氢酶活性和CD44和CD24的表达，从人胰腺癌移植瘤的社区体育会隔离。然后可以使用这些活细胞功能和分析研究。

视频: Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

从胎盘中的造血干细胞的分离和分析

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

科研

生物学

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

从人类胚胎干细胞的染色质免疫沉淀

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristic, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we have developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004, and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. Briefly, after cleaning the pancreas from the surrounding tissue, it was perfused with enzyme solution (Serva Collagenase + Neutral Protease or Sigma V enzyme). The distended pancreas was then transferred to the Ricordi digestion chamber, connected to a modified, closed circulation tubing system, and warmed up to 37&deg;C. During the digestion, the chamber was shaken gently. Samples were taken continuously to monitor the digestion progress. Once free islets were detected under the microscope, the digestion was stopped by flushing cold (4&deg;C) RPMI dilution solution (Mediatech, Herndon, VA) into the circulation system to dilute the enzyme. After being collected and washed in M199 media supplemented with human albumin, the tissue was sampled for pre-purification count and incubated with UW solution before purification. Purification process will be described in Part II: Purification and Culture of Human Islets.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part I: digestion and collection of pancreatic tissue) using a modified automated method.

人类胰岛的分离：第一部分：收集和胰腺组织消化

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

成人从人体皮肤的上皮干细胞的分离和培养

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

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 ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

从原发肿瘤的免疫细胞的分离

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

天真成人干细胞的分离从初级人类成纤维细胞培养

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

从人类Lipoaspirates脂肪来源干细胞的手动隔离

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

从成年小鼠切牙牙源性上皮干细胞的分离和培养

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

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 ...