Name: isolating hair follicle stem cells and epidermal keratinocytes from dorsal mouse skin
Uploaded: 2016-04-29T15:00:00.000+00:00
Duration: 6 min 51 s
Description: Watch this Scientific Journal Video about Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin at JoVE.com

This work was supported in part by NIH grant RO1GM60124 (to H.S.). H.S. is an Investigator with the Howard Hughes Medical Institute. Y.F. is supported by the Deloro Career Advancement Chair and The German Israeli Foundation (I-2381-412.13/2015). D.S. is supported by the Coleman-Cohen post-doctoral fellowship.

本研究是在严格按照指南中列出了以色列卫生部实验动物的护理和使用的建议进行的。所有动物均按照批准的机构动物护理协议技术的以色列Technion工业研究所的IL-02302-2015处理。 1.实验准备<ol><li>螯合胎牛血清的制备 注意:上皮细胞对钙非常敏感，所以关键是要控制这些细胞对钙的曝光。为了确保细胞不分离过程螯合期间暴露于钙被用来从胎牛血清用于制备染色缓冲液29的除去任何残留的钙。以下协议准备用于制备FACS染色缓冲需要大约1升无钙胎牛血清的。 <ol><li>加入400克干螯合材料， 如中，CHELEX，成4升烧杯中，并加蒸馏水2 O至总体积为4L。封面和搅连续使用磁力搅拌器。 </li><li>用10 1N HCl溶液，同时搅拌调节pH到7.4。继续搅拌20分钟，并根据需要，直到pH值超过20分钟保持稳定重新调节pH值。 </li><li>在4℃过夜孵育烧杯以允许螯合材料，以形成一个紧凑的颗粒。小心吸H 2 O和补充新鲜蒸馏水2 O 4 L的总容积</li><li>重复pH调节如步骤1.1.2。 </li><li>放置在4℃下将烧杯1小时，以允许螯合材料，以形成一个紧凑的颗粒。小心吸H 2 O. </li><li>慢慢胎牛血清两个500ml瓶中加入螯合材料。在4℃下缓慢搅拌1小时确保速度设定不产生气泡。 </li><li>放置在4℃下将烧杯1小时，以允许螯合材料，以形成一个补偿行动沉淀。 </li><li>小心地将血清转移到1升玻璃瓶中，并通过在无菌条件下一个瓶顶过滤器的过滤。存储螯合血清在-20°C或立即使用。 </li></ol></li><li>染色缓冲液制备<ol><li>制备100ml的PBS螯合FBS 3％，无Ca2 +和Mg2 +和保持在冰上。 </li></ol></li></ol> 2.成人表皮毛囊的分离<ol><li>使用麻醉使用感应盒5％异氟醚的小鼠。这里描述的方案，用50-80日龄的小鼠是在HF周期的休止期进行了优化。 </li><li>根据标准实验室程序安乐死的小鼠。在这里，用安乐死的 CO 2过量的安乐死室老鼠。 </li><li>刮胡子使用电动推剪背部皮肤所有的头发。避免头部和四肢。保持快船尽可能接近皮肤。避免损伤皮肤和皮下组织层。 </l我&gt; <li>当所有的头发被删除，使用70％的乙醇，以消除任何残留的头发和消毒背部皮肤。 </li><li>放在解剖垫鼠标，然后使用镊子拉起靠近尾部的皮肤，用剪刀做一个小切口。通过仔细地切去皮肤和它从筋膜分离切成后 - 前方向的整个背部皮肤。避免损伤皮下组织层。 </li><li>地方，头发面朝下放在解剖垫皮肤，牵制皮肤的两个相邻边缘的地方来解决。轻轻刮去油脂，用钝的刀，直到真皮清晰整洁。 注意:当刮去油脂，用弯钳适用于皮肤温和的压力。取适量涂抹于使用镊子的弯曲侧皮肤的压力。这将防止皮肤撕裂。 </li><li>将皮肤，真皮侧倒，在100mM无菌培养皿中，并伸直的皮肤，以确保没有褶皱。加入10毫升的PBS不含Ca 2+ </SUP&gt;和Mg 2+（PBS - ）到培养皿，并在需要伸直皮肤。 </li><li>吸出PBS和加入10 mL 0.25％胰蛋白酶。确保肌肤展开，并自由浮动。孵育一块每10ml 0.25％胰蛋白酶的皮肤。 </li><li>在37℃孵育样品30-120分钟或在4℃下过夜。 </li></ol> 3.从毛囊单细胞悬液的制备<ol><li>用刮弯钳和手术刀所有的头发皮肤，在从前到后方向。使用镊子的弯曲侧保持在适当位置的皮肤，并使用手术刀刮去毛。 注:刮去毛到在步骤2.8加入胰蛋白酶溶液。开始在尾部，然后按照头发的生长方向。刮擦头发生长的方向将导致细胞产量HFS的显着损失和减少。 HFS倾向于粘附到每个在一起，形成小块;为了降低第HF团块E尺寸尝试一次刮去一小块地方。 </li><li>转移无毛皮肤新的培养皿加入10毫升的PBS -防止其干燥。检查皮肤和刮去任何剩余毛发。 </li><li>分解用手术刀和镊子直至获得单一的HF悬浮在HFS。大力磨碎用10毫升吸管几分钟，打破了所有的头发团块HF停牌。 注意:执行所有以下步骤在冰上。 </li><li>在预标记的50ml管中传输的HF悬浮液。 </li><li>吸出PBS -从皮肤并用它来 ​​冲洗包含高频悬浮细胞培养皿;转移到50ml管中。 </li><li>洗涤<ol><li>通过穿过装配到50ml管70微米的细胞滤网过滤悬浮液。洗初始50ml管中，并用10毫升染色缓冲液的过滤器（PBS - 3％螯合FBS）。 </li><li>通过使过滤悬浮液通过40微米的细胞滤网装配到50ml的管中。用5-10毫升染色缓冲液洗管，以确保所有细胞已被转移到新的管中。保持该悬浮液在冰上，直到所有的动物都被处理。 </li><li>离心机在300 xg离心在4℃下15分钟。小心吸上清液没有在5毫升染色缓冲液的干扰颗粒和重新悬浮颗粒。 </li><li>离心在4℃下5分钟300 XG。小心吸上清，悬浮细胞沉淀在800微升染色缓冲液。 注意:上面指定的量是足够的染色从两个成年小鼠的细胞。 </li></ol></li><li>转移到暂停标记预流式细胞仪管。 <ol><li>为对照管，取25-50微升细胞悬浮液（或从细胞悬浮液的任何残余左）和补到300微升用染色缓冲液的总体积。 注:准备对照管（总体积300微升）的未染色（无抗体无DAPI）;仅DAPI; β1整合;整合α6;的Sca-1; CD34。 </li></ol></li><li>添加第一抗体和DAPI（参见适当稀释材料表）在所需的浓度到合适的管中。轻轻一抖管混合的细胞悬液。回到冰上，用铝箔覆盖避光。 </li><li>孵育在冰上30分钟，并轻轻振动小管，每10分钟，以确保细胞保持在悬浮液中。 </li><li>通过用染色缓冲液（大约4毫升，每管）填满各个FACS管洗​​涤细胞;在4℃下300 XG离心5分钟。 </li><li>小心吸上清，悬浮细胞沉淀在800微升染色缓冲液。 </li><li>转移细胞悬液至FACS管中的细胞过滤帽，以确保单细胞悬液。 </li></ol> 4.流式细胞仪分析<ol><li>继续使用细胞流与Signa的适当的过滤器立即流式细胞分选升检测。使用405纳米紫光（对于DAPI测量），488纳米的蓝色（对FSC，SSC，PE，PE-Cy7的和FITC的测量）和633纳米的红色激光（APC的激励） 注意:确保仪器从探测器记录所有三个荧光通道。 </li><li>基于未染色样品和补偿光谱重叠使用单一的彩色控制设置荧光门。 注意:使用单一​​染色控制用于​​调整信道间的补偿，以消除在荧光信号的任何重叠。薪酬调整将取决于所使用的流式细胞分选机。 </li><li>基于DAPI设置主门以排除死细胞。 </li><li>调整散点图（SSC-A对FSC-A），选择单事件。 </li><li>建立FSC和SSC的高度和宽度参数来消除双峰和单事件的歧视。 </li><li>门细胞具有高α6和β1的表达，并从这一人群在CD门细胞34高表达或SCA1高表达。 </li><li>排序细胞进入预标记的FACS管。 注:细胞可以分离的两个普通人群; α6+ / +β1/ CD34 + /的Sca-1 -和α6+ / +β1/的Sca-1 + / CD34 - 。排序细胞应在任何时候都保持在冰上，直到它们被用于下游应用，如细胞培养或RNA分离</li></ol>

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A., Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+epithelial+stem+cells.">Isolation and culture of epithelial stem cells.</a> Methods Mol Biol. 482, 215-232 (2009).</li></ol>

The authors have nothing to declare

这里所描述的协议是公认的用于从成年小鼠的背部皮肤隔离HFSCs但可以同样地适用于高频结构内其他人群的隔离，基于标记2,16,23,28,29的选择。这种方法是在细胞分离的其他方法，如组织离解特别有利的，在一个特定的细胞类型，可以选择并从异质细胞群体的混合物收获。此外，这里所描述的方法是快速，可靠和可用​​于高达隔离到基于所使用的标记物的差异表达水平四个不同HFSC种群。它可以用于浓缩和HFSCs用于进一步分子分析，如RNA测序或质谱的隔离，而且对于随后的细胞培养和扩展和体内移植。 高品质的制备中，单细胞悬液是SUC的一个基本组成部分cessful FACS 29。这种协议的效率还取决于用于高频隔离的起始原料。为了最大限度地减少在原料护理非表皮组织的量，必须采取剃和解剖背部皮肤时时。这提高了纯度和表皮细胞的产率。为了确保最佳的单细胞悬液，HFS应以头至尾方向和在一个时间少量确保所有毛发团块在较小的部分打破使用解剖刀并能获得一个单一的HF悬浮液刮掉。此外，细胞应小心处理在任何时候和悬浮液应保持在冰上，以保证细胞活力。最后，无菌技术对于特别是如果细胞是用于细胞培养污染的风险最小化是至关重要的。 在这里所描述的协议中，细胞的不同群体的隔离是通过使用四种细胞表面标志物来实现的。一个对于成功的细胞分选关键的一步是FACS，使用单染色控制和门控参数的等级相应的赔偿适当的初始设置。成功的细胞分选也需要碎片和消除歧视有效地从双峰/聚集单事件。汗衫从基于FSC和SSC width和height参数双峰区别。用于细胞分选的参数需要根据分拣机类型，但也给使用的荧光染料缀合物进行调节。在这里，用一四十二分之五百八十五过滤收集的PE荧光，FITC和PE-Cy7的荧光分别使用530/30和六十〇分之七百八十零过滤，收集并用六十零分之七百八十〇过滤收集APC荧光。 细胞分选是选通基于低DAPI表达和前向散射活细胞首先进行。单峰事件最初通过调整分散栅极选择并双峰被选通一个消除<html>抗拒着向前汗衫其次是散单峰。两整抗体，α6和β1被用来选择用于表皮细胞以保证富集对所有凸起旺。 Blanpain 等 ，表现坠胀HFSCs的两个群体表达或高或低的水平整合α6但CD34阳性2的存在。因此，细胞同时表达β1 高 / 低 α6β1和高 / 高 α6前应门控CD34选择。然后根据具体的隆起旺，CD34标记，并选择表皮角质形成细胞的标志物，的Sca-1 23的表达选择两个群体。的Sca-1与在漏斗和IFE的整合素α6共定位，但在HF隆起不表达。相比之下，坠胀HFSCs表达CD34和整合素α623，允许两个不同群体的差异隔离; HFSCs与表皮角质形成细胞。 e_content"&gt;这里讨论稳健的方法已被用于许多下游应用， 例如 ，HFSCs细胞培养23,28的隔离，接枝23,24和分子分析17。另外，选择特定的标志物的组合，或使用不同的报告小鼠允许亚群的隔离高频结构内， 例如 ，针对Lgr6 21，LGR5 25，LRIG1 24，并 ​​允许在这些细胞中发生的分子变化直接比较。此外，这种方法可用于比较在HFSC数目改变其它应用程序之间在野生型与基因敲除动物，一旦伤口施加某种检查28中的SC数量的变化，因此在SC和皮肤研究的宝贵工具。

成体干细胞（SCS）是用于通过替换死亡细胞维持组织稳态和在损伤修复受损的组织是至关重要的。这些雪旺由它们经受持续自我更新和分化成各种细胞谱系1-3能力来定义。最好研究的系统，这取决于他们的补给成人的SC，包括造血系统，肠和皮肤1,2,4。 在胚胎发生期间，皮肤开始作为表皮细胞的一个单层。当间质细胞填充皮肤，形成一个基本的胶原真皮5毛囊（HF）的形态开始。专业间充质细胞，后来构成真皮乳头（DP），直接组织表皮层下方，刺激上皮以形成开始向下6长出头发placodes。高度增殖基质细胞，位于在HF的底部，信封这些间充质细胞，并形成毛球，而内层开始分化成同心圆柱体，以形成毛干（HS）及周边内根鞘（IRS）2,3。 在出生后皮肤的表皮是由三个室:所述滤泡表皮（IFE），皮脂腺（SG）和HF。与此相反的IFE和SG这是在稳态的恒定状态时，HF是一个动态的微型器官而经历生长的连续周期（生长期），破坏（退化期）和其余（休止期）4,7。毛囊干细胞（HFSCs），该燃料这个永久循环，驻留在HF内的一个专门利基称为凸起4。在从DP生长期的HFSCs退出隆起，下面的启动信号，开始增殖并下降向下从而形成称为外根鞘细胞的长直线步道（ORS）8-10。基质细胞，该包围的DP在HF，快速循环的基础和迁移向上进行从而产生在HS和IRS 10（ 图1）终末分化。生长期的持续时间决定了头发的长度和依赖于基质细胞6的增殖和分化的能力。当高频进入退行期，在灯泡停止过境放大基质细胞增殖，凋亡和完全消退，而向上拉动DP直到它到达的高频8,11非循环部分。在此回缩HF形成被称为上皮链，这是退行期的特征的临时结构，并且包含许多凋亡细胞。在小鼠中，退行期持续天3-4之间，并在第一毛发周期高度同步。当HF到达休止所有HFSCs变得静止。高频周期的不同阶段的特征还在于，由于到m在小鼠的皮肤的颜色变化elanin生产。从黑退行期期间生长期为深灰色期间皮肤的变化休止期6,7,12,13时为粉红色。 <img alt="图1" src="/files/ftp_upload/53931/53931fig1.jpg" /> 图1:毛囊周期 。在HF由永久上部，并且经历了快速增长（生长期），破坏（退行期）和相对静止阶段或休息（静止期）的连续周期不断降低重塑，单车部分。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53931/53931fig1large.jpg" target="_blank">请点击此处查看大图版本这个数字。</a> 维持HF南海初步确定使用追踪实验，以氚化胸苷，即透露，在HF的永久居住区就在SG 14下方慢骑自行车的标签滞留细胞（LRC）的人口。在HFSC进展表征揭示少量可用于从HF利基15鉴定和分离特定的SC标记。也许HFSCs富集的最佳标记是CD34，细胞表面标记也被确定为在人16造血SC标记。在这个CD34 +人口两个不同的群体也被隔离的基础上整合α6表达式2。另一个标志物是角蛋白15（K15），其在所述凸出区域中高度表达，具有CD34的表达共定位和K15启动子被用于定位和在转基因动物15,17-19隔离HFSCs。在过去的十年中HFSCs和祖细胞的其它几个不同的种群也有报道驻留在HF 17,20-27内。 HFSCs的另一个令人兴奋的是他们的皮肤修复的贡献。在正常情况下HFSCs补充HF和不参加IFE平衡的一部分。豪版本，以应对伤人，这些细胞退出他们的SC利基，并有助于重新填充IFE 9。我们最近表明，删除小鼠促凋亡Sept4 /艺术基因显示CD34，K15和Sox9的+ HFSCs，这表明凋亡电阻的数量增加。 HFSCs从Sept4 /文理分离- / -利用背皮荧光激活细胞分选（FACS），并有在CD34 +和K15 + HFSCs的数目多于一个两倍。这些Sept4 /文理- / - HFSCs 体外扩增和不仅引起了更多的菌落，但也能够承受更苛刻的条件下与对照相比28。 作为具有HFSCs数目增加的结果，Sept4 /文理- / -小鼠显著更快响应于皮肤切除损伤愈合。引人注目的是，Sept4 /艺术- / -小鼠displayeDA大量从伤口床再生HFS和显著较小的疤痕。此外，对于XIAP（细胞凋亡的X连锁抑制 ​​剂），的文理生化靶，缺失的小鼠表现出受损的愈合28。 我们的结果及其他实验室进行工作已经表明，HFSCs作为研究成人的SC的生物学和功能的理想模型。在这里，我们描述了用于浓缩和HFSCs并基于四种标志物的表达表皮角化细胞的分离方法:整合α6;整合β1;的Sca-1（对于表皮角化细胞的标记）和CD34。也可以使用K15-GFP报告鼠标19执行K15 + HFSCs的类似的隔离。

<table><tbody><tr><td>Isoflurane&amp;nbsp;</td><td>Primal Critical Care</td><td>&amp;nbsp;66794-017-10</td><td></td></tr><tr><td>Carbon dioxide</td><td>-</td><td>-</td><td></td></tr><tr><td>Electro Shaver</td><td>Oster&amp;nbsp;</td><td>Golden A5</td><td>Shaver from any other company could be used</td></tr><tr><td>70% ethanol</td><td>Gadot Lab</td><td>830000051</td><td>96% ehtanol diluted with distilled water&amp;nbsp;</td></tr><tr><td>Dissection mat</td><td></td><td></td><td>Dissection tools from any provider can be used</td></tr><tr><td>Forceps</td><td>Dumont</td><td>11251-10</td><td>Foreceps from any other company could be used</td></tr><tr><td>Scissors&amp;nbsp;</td><td>Dumont</td><td>14094-11</td><td>Scissors from any other company could be used</td></tr><tr><td>Needles/Pins</td><td>-</td><td>-</td><td></td></tr><tr><td>Scalpel</td><td>Albion</td><td>10</td><td>Ensure that the scalpel has a blunt end</td></tr><tr><td>Tissue culture dish 60 mm x 15 mm</td><td>Sigma-Aldrich</td><td>CLS430166</td><td></td></tr><tr><td>PBS</td><td>-</td><td></td><td>In house PBS without Calcium and Magnesium</td></tr><tr><td>0.25% Trypsin/EDTA</td><td>Biological Industries</td><td>03-050-1A</td><td>Trypsin obtained from a different company might have a different activity and duration of the trypsin digest has to be adjusted accordingly</td></tr><tr><td>Pipettes 10 ml</td><td>Sigma-Aldrich</td><td>Corning, 4488</td><td></td></tr><tr><td>Ice</td><td>-</td><td>-</td><td></td></tr><tr><td>50 ml sterile centrifuge tubes</td><td>Minplast Ein-shemer</td><td>35050-43</td><td></td></tr><tr><td>70 &amp;micro;m Cell strainer</td><td>Fisher</td><td>22362548</td><td></td></tr><tr><td>40 &amp;micro;m Cell strainer</td><td>Fisher</td><td>22362549</td><td></td></tr><tr><td>Staining buffer</td><td>-</td><td></td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf 5804 R</td><td>5805 000.017</td><td></td></tr><tr><td>FACS tubes with Cell strainer caps&amp;nbsp;</td><td>Falcon&amp;nbsp;</td><td>352235</td><td></td></tr><tr><td>FACS tubes&amp;nbsp;</td><td>Falcon&amp;nbsp;</td><td>352063</td><td></td></tr><tr><td>Integrin &amp;beta;1</td><td>eBioscience</td><td>25-0291</td><td>1:400</td></tr><tr><td>Integrin &amp;alpha;6</td><td>eBioscience</td><td>15-0495</td><td>1:600</td></tr><tr><td>Sca I</td><td>eBioscience</td><td>11-5981</td><td>1:200</td></tr><tr><td>CD34</td><td>eBioscience</td><td>9011-0349</td><td>1:300</td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9542</td><td>50 ng/ml</td></tr><tr><td>Dry Chelex</td><td>BioRad</td><td>142-2842</td><td></td></tr><tr><td>Beaker&amp;nbsp;</td><td>Pyrex</td><td>-</td><td></td></tr><tr><td>Distilled H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;</td><td>-</td><td>-</td><td></td></tr><tr><td>Stir bar</td><td>-</td><td>-</td><td></td></tr><tr><td>NHCl</td><td>BioLab</td><td>1903059</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Beit Haemek Biological Industries</td><td>400718</td><td>FBS obtained from a different company can be used</td></tr><tr><td>1 L glass bottle</td><td>Ilmabor</td><td>Boro 3.3</td><td></td></tr><tr><td>Bottle top filter</td><td>Autofil</td><td>1102-RLS</td><td></td></tr></tbody></table>

isolating hair follicle stem cells and epidermal keratinocytes from dorsal mouse skin

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by distinct pools of SCs, which are located in the permanent portion of the HF, a region known as the bulge. These multipotent bulge SCs were initially identified as slow cycling label retaining cells; however, their isolation has been made feasible after identification of specific cell markers, such as CD34 and keratin 15 (K15). Here, we describe a robust method for isolating bulge SCs and epidermal keratinocytes from mouse HFs utilizing fluorescence activated cell-sorting (FACS) technology. Isolated hair follicle SCs (HFSCs) can be utilized in various in vivo grafting models and are a valuable in vitro model for studying the mechanisms that govern multipotency, quiescence and activation.

该协议详细描述了充实和两类人群的隔离:隆起种姓和表皮角质形成图2说明了协议的主要步骤。利用皮肤从背垫层8周龄小鼠的去除，我们富集使用CD34标志物，这是只在HFSCs表示隆起旺;和的Sca-1，其标签表皮角化细胞。 图3示出了皮肤上皮细胞的α6+ /β1+群体内的CD34和的Sca-1的表达的不同的模式。细胞根据整合素α6的表达第一选通 和整合素β1，其在图3指定为P1，与阳性α6细胞群/β1expression然后根据CD34和的Sca-1细胞中的表达门控。细胞的两种不同的人群都出现在CD34 VS的Sca-1地块。 α6+ /β1 <suP&gt; + / CD34 + /的Sca-1 -表示HFSCs和α6+ /β1+ /的Sca-1 + / CD34（ 图3中指定为P2） -代表表皮角化细胞（ 图3中指定为P3）。每α6+β1+池的动物大约150-500×10 3细胞CD34 + HFSCs。 <img alt="图2" src="/files/ftp_upload/53931/53931fig2.jpg" /> 图2:HFSC和角化细胞的分离背皮肤从小鼠中取出。脂肪刮下与皮肤在胰蛋白酶温育。 HFS进行从皮肤刮下来，并通过70微米和40微米的细胞滤网过滤悬浮液。与表面抗体的细胞标记之后用流式细胞仪分类和细胞的两个群体是基于相对表达收集:CD34 + </suP&gt; /的Sca-1 -和CD34 - /的Sca-1 +。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53931/53931fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53931/53931fig3.jpg" /> 图3: 分选的细胞的仿形背侧皮肤评估从小鼠分离HFSCs的百分比的FACS分析。 FACS纯化α6+β1+细胞分选的CD34 +的Sca-1 - （粉红色，α6+β1+ CD34 +的Sca-1 - ;蓝色，α6+β1+ CD34 -的Sca-1 +;橙色，α6+β1+ CD34 -的Sca-1 - ）。显示的数据是从四个结合小鼠和分类在一起。在野生型小鼠α6+β1+池约4-6％（指定P1）是CD34 + /的Sca-1 - HFSCs（P2） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53931/53931fig3large.jpg" target="_blank">点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin at JoVE.com

Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin

An ideal model for studying adult stem cell biology is the mouse hair follicle. Here we present a protocol for isolating different populations of hair follicles stem cells and epidermal keratinocytes, employing enzymatic digestion of mouse dorsal skin followed by FACS analysis.

从背小鼠皮肤分离毛囊干细胞和表皮角质形成细胞

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by ...

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Unit 1

Stem Cell Biology And Renewal in Epithelial Tissue

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20.9K 次观看.  Technion Israel Institute of Technology. An ideal model for studying adult stem cell biology is the mouse hair follicle. Here we present a protocol for isolating different populations of hair follicles stem cells and epidermal keratinocytes, employing enzymatic digestion of mouse dorsal skin followed by FACS analysis.

视频: Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin

Murine Dermal Fibroblast Isolation by FACS

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast physiology and pathology underlie a spectrum of clinical entities, including fibroses in multiple organs, hypertrophic scarring following burns, loss of cardiac function following ischemia, and the formation of cancer stroma. However, fibroblasts remain a poorly characterized type of cell, largely due to their inherent heterogeneity. Existing methods for the isolation of fibroblasts require time in cell culture that profoundly influences cell phenotype and behavior. Consequently, many studies investigating fibroblast biology rely upon in vitro manipulation and do not accurately capture fibroblast behavior in vivo. To overcome this problem, we developed a FACS-based protocol for the isolation of fibroblasts from the dorsal skin of adult mice that does not require cell culture, thereby preserving the physiologic transcriptional and proteomic profile of each cell. Our strategy allows for exclusion of non-mesenchymal lineages via a lineage negative gate (Lin-) rather than a positive selection strategy to avoid pre-selection or enrichment of a subpopulation of fibroblasts expressing specific surface markers and be as inclusive as possible across this heterogeneous cell type.

Fibroblast behavior underlies a spectrum of clinical entities, but they remain poorly characterized, largely due to their inherent heterogeneity. Traditional fibroblast research relies upon in vitro manipulation, masking in vivo fibroblast behavior. We describe a FACS-based protocol for the isolation of mouse skin fibroblasts that does not require cell culture.

小鼠皮肤成纤维隔离用流式细胞仪

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast ...

科研

发育生物学

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

小鼠表皮神经嵴干细胞（EPI的非公务员合约雇员）文化

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

生物学

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

一种简便有效的成人皮肤组织中人角质形成细胞的分离方法

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

Isolation and Staining of Mouse Skin Keratinocytes for Cell Cycle Specific Analysis of Cellular Protein Expression by Mass Cytometry

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the epidermis of mouse skin. There are seven important steps: separation of the epidermis from the dermis, digestion of the epidermis, staining of the epidermal cell populations with cisplatin, sample barcoding, staining with metal tagged antibodies for cell cycle markers and proteins of interest, detection of metal-tagged antibodies by mass cytometry, and the analysis of expression in the various cell cycle phases. The advantage of this approach over histological methods is the potential to assay the expression pattern of &#62;40 different markers in a single cell at different phases of the cell cycle. This approach also allows for the multivariate correlation analysis of protein expression that is more quantifiable than histological/imaging methods. The disadvantage of this protocol is that a suspension of single cells is needed, which results in the loss of location information provided by the staining of tissue sections. This approach may also require the inclusion of additional markers to identify different cell types in crude cell suspensions. The application of this protocol is evident in the analysis of hyperplastic skin disease models. Moreover, this protocol can be adapted for the analysis of specific sub-type of cells (e.g., stem cells) by the addition of lineage-specific antibodies. This protocol can also be adapted for the analysis of skin cells in other experimental species.

This protocol describes how to isolate skin keratinocytes from mouse models, to stain with metal-tagged antibodies, and to analyze stained cells by mass cytometry in order to profile the expression pattern of proteins of interest in the different cell cycle phases.

小鼠皮肤角质细胞的分离和染色,用于细胞周期特定分析细胞蛋白表达的量度细胞测定

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the ...

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 ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

在毛发再生的上皮 - 间充质信号的快速遗传分析

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Isolation, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid enzymatic dissociation of skin. Primary keratinocytes, fibroblasts, and Schwann cells are all harvested from the human newborn foreskin, which is available following standard of care procedures. The removed skin is disinfected, and the subcutaneous fat and muscle are removed using a scalpel. The method consists of enzymatic and mechanical separation of epidermal and dermal layers, followed by additional enzymatic digestion to obtain single-cell suspensions from each of these skin layers. Finally, single cells are grown in appropriate cell culture media following standard cell culture protocols to maintain growth and viability over weeks. Together, this simple protocol allows isolation, culturing, and characterization of all three cell types from a single piece of skin for in vitro evaluation of skin-nerve models. Additionally, these cells can be used together in co-cultures to gauge their effects on each other and their responses to in vitro trauma in the form of scratches performed robotically in the culture associated with wound healing.

The study of wound healing associated with musculoskeletal injury often requires the assessment of in vitro interactions between Schwann cells (SCs), keratinocytes, and fibroblasts. This protocol describes the isolation, culturing, and characterization of these primary cells from the human foreskin.

从人包皮中分离、培养和表征原代施旺细胞、角质形成细胞和成纤维细胞

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid ...

神经科学

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

来自新生儿和成年鼠皮肤的原代小鼠角化细胞的分离和培养

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 &#177; 2 days old) yielding approximately 30 x 106 viable cells per mouse. Primary adult mouse keratinocytes are harvested from the dorsal skin of female mice. Male mice (~6 weeks old) can be used for keratinocyte harvesting depending on the requirements of the experiment. Euthanized mice are shaved and sterilized with serial washes in povidone iodine and ethanol solutions (70% alcohol). After disinfecting the mice, the dorsal skin is removed and the subcutaneous fat and muscle are removed with a scalpel and discarded. The skins are cut into small pieces and treated with a mild, low temperature trypsinization to detach the lower dermis from the epidermis. The scraped epidermises are stirred at low speed, filtered to remove the hairs, counted, and re-suspended in culture medium. This method provides an excellent single cell suspension of highly culturable cells for many downstream applications.

The goal of this protocol is to isolate epidermal keratinocytes from the dorsal skin of adult mice for a variety of downstream applications such as molecular biology, biochemistry, fluorescence activated cell sorting, and primary in vitro uses (e.g., clonogenic keratinocytes).

小鼠表皮角质细胞的分离及其体外克隆培养物

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 &#177; 2 days old) yielding approximately ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

从成人小鼠腭中分离和培养原代口腔角质形成细胞

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...