The authors wish to thank Shonna Johnston, Claire Cryer, Fiona Rossi and Will Ramsay at the University of Edinburgh and Alison Logar and Megan Blanchard at the University of Pittsburgh for their expert assistance with flow cytometry. We also wish to thank Anne Saunderson and Lindsay Mock for their help with obtaining human tissues. Human adult and fetal heart tissue samples were procured with full ethics permission of the NHS Scotland Tayside Committee on Medical Research Ethics and the NHS Lothian Research Ethics Committee (REC08/S1101/1) respectively. This work was supported by grants from the Medical Research Council (BP), British Heart Foundation (BP), Commonwealth of Pennsylvania (BP), Children's Hospital of Pittsburgh (BP), National Institute of Health R01AR49684 (JH) and R21HL083057 (BP), and the Henry J. Mankin Endowed Chair at University of Pittsburgh (JH). JEB was supported by a British Heart Foundation Centre of Research Excellence doctoral training award (RE/08/001/23904). WC was supported in part by an American Heart Association predoctoral fellowship (11PRE7490001).

1.人力心脏样品处理<ol><li>确保所有的液体，容器，仪器，和专用业务区域是无菌的。 </li><li>放置心脏组织样品含有20％胎牛血清（FBS），并在冰上1％青霉素 - 链霉素（P / S）冷冻Dulbecco改进的Eagle培养基（DMEM）的组成，在存储介质（由组织库或外科团队购买）交通3。 </li><li>从存储介质中删除心脏样品并用补充有2％FBS和1％P / S的磷酸盐缓冲盐水（PBS）组成的洗涤介质洗涤。当处理样品，用细尖镊子把握大血管或心包和减少心肌的破碎。 </li><li>淹没在洗涤介质中的样品在培养皿和描述3取出心包，心内膜及大血管用消毒剪刀虹膜和细尖镊子。 </li><li>切剩下的myocardium成约1mm 3小块使用单一侧刀片或一对锋利的虹膜剪的。注意:如果样品立即处理最高细胞产量将获得。如果延迟是不可避免的，在新鲜的存储介质的存储处理后的心脏组织的（&gt;5毫升每1cm 3组织）于4℃多达72小时是可能的，但是推测可能与在细胞中产量相关联的下降。 </li></ol> 2.细胞的组织和隔离消化<ol><li>新鲜弥补消解液含有1.5-2.4毫升每胶原酶I，II胶原酶，和胶原酶IV（均在DMEM 0.5毫克/毫升），并以温暖的37℃水浴。 注:音量和胶原酶的浓度是适宜的约1cm 3个样本。 </li><li>通过100微米滤网过滤悬浮组织碎片，用PBS洗两次。转移组织块到无菌30毫升容器一个密封的盖子。 注:短而宽的盆，而不是高的细管给出更好的结果。可替代地，把组织片段在无菌50ml管中，如果在30ml容器不可用水平放置。 </li><li>添加全部消化解决方案（4.5毫升总数）并放置一个密封的塑料袋内的容器设置为120转一个37℃摇动的水浴。另外，与封蜡膜和地方的容器中加热轨道摇床设置为120 RPM。 </li><li> 15分钟后，取出和更换再15分钟前三次颠倒了锅。除去和淬火用5ml的DMEM补充有20％FBS和1％P / S的胶原酶。 </li><li>用10毫升血清吸管，打破了任何组织团块轻轻吹打十到二十次磨碎的摘要。通过100微米过滤悬浮液顺序地，70μm和最后40微米的细胞滤网以除去未消化的物质，将获得单细胞悬浮液。 注意:不细胞滤网之间冲洗，以避免被酶消化过程debrisgenerated细胞。 </li><li>离心机在200×g离心4分钟的细胞悬浮液，小心地倒出上清液并用4ml洗涤介质的稀释前重新悬浮在1ml红细胞裂解缓冲液，在室温下2分钟的沉淀。 </li><li>重复离心步骤和重新悬浮在1ml洗涤培养基的沉淀。拌匀取10微升细胞悬浮液为细胞计数。 </li><li>混合10微升细胞悬浮液与10微升台盼蓝染色，并放置10微升该混合物的对细胞计数标准血细胞计数器。算上明亮，圆形细胞存在于四角方块（每个细分成十六小方块）的数量除以4，让每角方的平均值。乘以2的平均值以帐户为污渍稀释因子，然后通过10 4，得到每1ml样品中的细胞的总数。 </li></ol><ol><li>加入50μl的小鼠血清对细胞悬浮液，并在4℃下孵育20分钟以阻断非特异性抗体结合。 </li><li>离心机在200×g离心4分钟小鼠血清的细胞悬浮液，取出上清液，并在洗涤介质中以每100微升1×10 6个细胞的浓度重悬。 </li><li>等分每个细胞悬浮液的同种型和未染色对照的50微升成两个聚苯乙烯圆底流式细胞管然后将剩余体积为第三管为多色染色。 </li><li> CD34-PE，CD45-APC-Cy7的，CD56-PE-Cy7的，CD144-PerCP-经Cy5.5，和CD146 - AF647抗体添加（总共1:100）的单细胞悬浮液用于多色染色。对于同种型对照，加入PE-，APC-Cy7-，PE-Cy7-，PerCP-Cy5.5-和AF647共轭合同种型抗体的等效容积。轻轻移液管混合与细胞抗体，并在4℃下孵育所有试管20分在黑暗中。 </li><li>通过在五个聚苯乙烯圆底流式细胞仪管加入正珠一滴100微升洗涤介质的准备补偿控制珠。添加的CD34-PE，CD45-APC-Cy7的，CD56-PE-Cy7的，CD144-PerCP-经Cy5.5，和CD146 - AF647抗体（所有1:100）分别到每个5管子。孵育所有试管于4℃在黑暗中20分钟。 </li><li>在温育，制备各用500μl内皮生长培养基-2在4℃（EGM-2）培养基和地点的细胞收集管中。 </li><li>以下抗体孵育，添加5毫升洗涤介质洗涤的细胞和小珠以除去未结合的抗体。离心机在200 XG 4分钟都管。重复洗涤和离心步骤。小心倒出上清，轻轻吸管重新悬浮细胞时。 </li><li>在洗涤介质中以每250微升约1×10 6个细胞的浓度重悬。重悬在100μl的洗涤微米珠edium。 </li><li>交通建设在暗处的冰所有细胞和珠悬浮液的细胞分选仪。负珠1滴添加到正珠补偿控制管，并使用这些设置荧光补偿。 </li><li>运行，根据制造商的说明来建立背景荧光和电压设置为以下未染色的对照细胞:FSC 150V; SSC 290V; V450 / 50 350V; V710 / 50 620V; B530 / 30 400V; B710 / 50 530V; YG780 / 60 460; R670 / 30 480V; R780 / 60 460V。根据制造商的说明运行同种型对照以建立与非特异性结合背景荧光阈值。运行多色染色样品和收集周细胞和外膜细胞群进入收集管（ 图1）。 注:最佳可行的细胞产量是周细胞的总活细胞解离的大约3％和外膜细胞的4％。 </li></ol> 4.细胞培养<ol><li>根据FACS分选的结果，选择合适的尺寸培养板中。加入100微升无菌0.2％的明胶溶液的每生长面积的平方厘米和手动搅动涂覆整个孔中。在4℃下进行10分钟孵育板和完全除去明胶溶液。保持收集的细胞在冰上而制备培养板。 注:当在30,000到40,000细胞/ cm 2的明胶包被培养表面的密度接种新鲜整理周细胞外膜和细胞生长良好。 </li><li>离心新鲜采集的细胞以200×g离心4分钟，并在EGM-2.Seed适量在明胶包被的平板的细胞轻轻重悬浮细胞沉淀。 注意:每孔和待添加的EGM-2的量被接种的细胞的实际数量取决于板的选择。例如加入0.5ml和1ml EGM-2则分别需要每孔为48和24孔板中。 </li><li>交换EGM-2为血管周围细胞生长培养基（的DMEM补充有20％FBS和1％P / S）为周细胞和外膜细胞一次细胞已（后至少72小时）定居并粘附到板上。从此改变介质每72小时。进行初次和所有后续的孵育在5％CO 2，并在37℃。 </li><li>使用0.05％胰蛋白酶的EDTA当周细胞和外膜细胞达到80〜90％汇合解离的细胞。用在PBS中的20％的FBS，离心机在200×g离心4分钟淬火，以1重悬浮在血管周围细胞的生长培养基中，然后通过将细胞:3的比例到未涂覆聚苯乙烯培养板中。从通道2开始，传代细胞以1:5的比例（或在约7000名细胞/ cm 2），以未涂覆的聚苯乙烯培养板或烧瓶中。 </li></ol>

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The authors have no conflict to disclose.

越来越多的证据支持成人心脏损伤后有限的再生能力。负责受伤的心如再生反应本土前体细胞的识别和鉴定是相关的机制和信号通路双方的理解和制定办法来治疗利用这些细胞的关键。 先前协议已经从人骨骼肌25中所述的血管周围的前体细胞亚群的分离。然而，这些技术对心脏组织的直接应用经常导致非常差的细胞产量。因此，我们以丰富血管周围的前体细胞，即周细胞和外膜细胞，从人心肌活组织检查的两个分立的亚群并促进两个子集的同时纯化作出协议重大调整。从S两种细胞群的分离AME样品不仅优化了使用珍贵的心脏组织活检也可以让不同的血管周细胞亚群的直接比较。 为了获得细胞的最大产率，组织样品的新鲜度是至关重要的。对于周细胞和外膜细胞最大活细胞产量是大约3％和总活细胞解离的4％。产率可以从每子集约30,000至400,000细胞变化很大，并且高度依赖于许多因素，包括量和起始组织的质量和保存期限。周围细胞都比较细腻;自溶显示出变化的证据样本FACS后总是产生较低的细胞数量。类似地，血管周围细胞对苛刻消化技术敏感，和过消化也将导致降低活细胞的产率。的消化时间，搅拌速度是定制化调整可能是由individua必要升实验室。最后，供体的年龄和样本大小也会对细胞产量有重要影响。 我们广泛特征在于以这种方式3中获得的心脏周细胞。这些细胞表现在文化同质性，没有内皮细胞污染。群体倍增通道3和12之间约60小时的时间与两个共同周细胞标记物的一致的表达指出（碱性磷酸酶，NG2和α-SMA）和在此期间的经典的MSC标记（CD44，CD73，CD90和CD105）。有趣的是，我们发现脱甲基化后，心脏周细胞的一小部分表示的心肌转录因子的Nkx2.5和GATA4。当共同培养与新生大鼠心肌细胞，脱甲基化的心脏周细胞的一小部分所表达的肌节蛋白标志物的α-辅肌动蛋白和肌钙蛋白T，与未成年组进一步呈现自发胞质钙通量。两者合计，这些结果suggeST该心脏周细胞的亚群具有心肌潜力，因此可能在固有心肌修复过程中发挥作用。在与周细胞相比，心外膜细胞在很大程度上仍然未鉴定。我们未公布的初步数据显示，心外膜细胞表达一些周细胞标记物如PDGFR-β，NG2，尽管在较低的水平比心脏周细胞，而在文化失去CD34。这些细胞也表达经典MSC标记和显示成骨与成脂分化潜能。心外膜细胞，包括血管和亲心源性性能进一步的研究正在进行中。 这里所描述的协议使心脏和周细胞外膜细胞的同时，前瞻性纯化一个人类心脏组织样本。这些细胞代表与潜在心肌性能新颖心肌前体细胞群，可能被证明是合适的candidatES为未来的细胞疗法。

该心脏一直被认为是一个有丝分裂后的器官。然而，最近的研究已经证明有限心肌周转的成年人的心脏1的存在。原生干/祖细胞，心肌细胞分化的潜能也被认定为成人啮齿动物和人的心，其中的Sca-1 +，C-KIT +，cardiosphere形成心肌内，以及最近的血管周围前体细胞2,3。这些细胞代表了旨在通过细胞移植或原位增殖的刺激增强心脏修复/再生疗法具有吸引力的候选者。 间充质干细胞/基质细胞（MSC）已经从几乎每个人组织4,5的MSC的治疗应用的临床试验已进行了多种病理条件下分离如心血管修补6，移植物抗宿主病7 </sup&gt;和肝硬化8。有益的作用已被归因于MSCs向的能力:家里炎症9的部位;分化成不同细胞类型10;分泌促修复分子11;和调节宿主的免疫反应12。干细胞的分离历来依靠其优惠的坚持塑料基板。然而，所得到的细胞群是典型地显着异质13。通过使用荧光激活细胞分选（FACS）用的关键血管周围细胞标记物的结合，我们已经能够分离和纯化多能的MSC样前体群体（CD146 + / CD31 - / CD34 - / CD45 - / CD56 - ）从多种人体组织包括成人骨骼肌和白色脂肪14。 在各种非心脏组织的血管周围细胞群已被显示出具有干/祖细胞特性的第二正在研究在心血管设置临床使用。周细胞，最知名的血管周围细胞亚群之一，是一个异质人口玩几个病理生理作用，包括在新船15的发展，血压16和血管完整性17,18的维护管理。由于在多种组织所示，周细胞的特定子集本身表达MSC抗原和FACS纯化后14维持在原代培养的MSC样表型。此外，这些细胞稳定地维持培养于他们的长期的表型，并表现出多谱系分化潜能，类似的MSCs 19,20。这些结果表明，周细胞是难以捉摸的MSC 14的起源地之一。周细胞的治疗潜力已经证明在心肌疤痕的减小和增强移植后的心脏功能到缺血性损伤心21。最近，我们成功地从人类心肌纯化周细胞和无骨骼肌肉发生3展示了他们MSC样表型和多能（脂肪形成，软骨和骨）。此外，心肌当周细胞与其他器官纯化的同行相比表现差心肌潜力和血管生成能力。 多能血管周围干/祖细胞，外膜细胞的第二群体，已经从人隐静脉分离阳性CD34的表达22的基础上的。静脉外膜细胞已被证明具有克隆的潜力，中胚层分化能力和体外促血管生成潜力。这些细胞注入小鼠的缺血性损伤的心移植导致间质纤维化的减少，增加血管生成和心肌血流量，减少心室稀通货膨胀，而增加心脏射血分数23。有趣的是，脂肪外膜细胞已被证明失去CD34的表达和上调响应于血管生成素II治疗中培养CD146表达，提示通过一个周细胞表型与刺激24。内的心脏，然而，外膜细胞群体尚未前瞻性通过FACS和/或良好表征纯化。利用在以下各节中描述的细胞分离方法，我们目前正在表征心肌外膜细胞和调查其对再生的应用潜力。 这里我们描述了分离和从人胎儿或成年心肌纯化血管周围干/祖细胞的两个亚群的方法。本前瞻性细胞分离方法将使研究人员能够从人的心脏活检的比较研究和furthe获得等基因血管周围干/祖细胞亚群- [R探讨其在各种心脏病理状态的治疗潜力。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AbC Anti-mouse Bead Kit</td>
			<td>Molecular Probes</td>
			<td>A-10344</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase I</td>
			<td>Gibco</td>
			<td>17100-017</td>
			<td rowspan="3">Reconstitute powder as required and filter sterilise</td>
		</tr>
		<tr>
			<td>Collagenase II</td>
			<td>Gibco</td>
			<td>17101-015</td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Gibco</td>
			<td>17104-019</td>
		</tr>
		<tr>
			<td>anti-human CD34-PE</td>
			<td>BD Pharmingen</td>
			<td>555822</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>anti-human CD45-APC-Cy7</td>
			<td>BD Pharmingen</td>
			<td>557833</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>anti-human CD56-PE-Cy7</td>
			<td>BD Pharmingen</td>
			<td>557747</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>anti-human CD144-PerCP-Cy5.5</td>
			<td>BD Pharmingen</td>
			<td>561566</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>anti-human CD146-AF647</td>
			<td>AbD Serotec</td>
			<td>MCA2141A647</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>EGM2-BulletKit</td>
			<td>Lonza</td>
			<td>CC-3162</td>
			<td>For collection of cells and culture until adhered</td>
		</tr>
		<tr>
			<td>DMEM, high glucose, GlutaMAX without sodium pyruvate</td>
			<td>ThermoFischer Scientific</td>
			<td>10566-016</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>ThermoFischer Scientific</td>
			<td>10500-064</td>
			<td>Freeze in aliquots and keep sterile</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma Aldrich</td>
			<td>G1393</td>
			<td>Dilute with sterile water</td>
		</tr>
		<tr>
			<td>IgG1k-PE</td>
			<td>BD Pharmingen</td>
			<td>559320</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>IgG1k-APC-Cy7</td>
			<td>BD Pharmingen</td>
			<td>557873</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>IgG1k-PE-Cy7</td>
			<td>BD Pharmingen</td>
			<td>557872</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>IgG1k-PerCP-Cy5.5</td>
			<td>BD Pharmingen</td>
			<td>561566</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>IgG1k-647</td>
			<td>AbD Serotec</td>
			<td>MCA1209A647</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>Mouse serum</td>
			<td>Sigma Aldrich</td>
			<td>M5905</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>Paraffin Film - Parafilm M</td>
			<td>Sigma Aldrich</td>
			<td>P7793</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15979-063</td>
			<td>Freeze in aliquots and keep sterile</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline pH 7.4</td>
			<td>ThermoFischer Scientific</td>
			<td>10010-023</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>Red Blood Cell Lysing Buffer Hybri-Max</td>
			<td>Sigma Aldrich</td>
			<td>R7757</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution</td>
			<td>Sigma Aldrich</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.5%(10X)</td>
			<td>Invitrogen</td>
			<td>15400-054</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;FACSARIA FUSION</td>
			<td>BD Pharmingen</td>
			<td></td>
			<td>Fluorescence Activated Cell Sorter</td>
		</tr>
	</tbody>
</table>

isolation of perivascular multipotent precursor cell populations from human cardiac tissue

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34- pericytes and vascular CD34+CD146- adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.

单细胞从碎片和双峰正向和侧向散射分布的基础上区别。活细胞被他们未能采取了DAPI染料标识。选通策略被选择这个活，全心脏细胞的解离（ 图1）的同种型对照标记的基础上。从活细胞，CD45 +细胞被第一选通出来，接着CD56 +细胞。 CD144 +内皮细胞然后从CD56取出-级分。从这个最后的人口，CD146 + / CD34 -被选择/ CD34 +外膜细胞和随后收集（ 图2） -周细胞和CD146。细胞收集后立即，每个亚群的一个小样本（500-1000细胞）是通过分选器重新运行，以确认细胞分布为记录在初始排序。 FACS纯化心肌佩里奇<html> ytes和外膜细胞都显示出主轴星状在培养细胞的形态（ 图3）。 <img alt="图1" src="/files/ftp_upload/54252/54252fig1.jpg" /> 图 1:标记同型对照的FACS门控策略代表点地块分离人类心脏细胞说明的那种闸门的定位.. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54252/54252fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54252/54252fig2.jpg" /> 图2: 心肌血管周围细胞的FACS纯化从单个人类心肌样品心肌血管周围细胞前体细胞的两个亚群的代表的排序。ES / ftp_upload / 54252 / 54252fig2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/54252/54252fig3.jpg" /> 图 3: 血管周围细胞形态的文化中的两次心脏血管周围的前体细胞群，周细胞（左图）和外膜细胞（右）通过FACS纯化分拣至同质性，并进一步在培养物（展开在通道3中，比例尺=50μm的）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54252/54252fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue at JoVE.com

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

人体心脏组织窝藏多能血管周围的前体细胞群可能适合于心肌再生。这里描述的技术允许与天然血管， 即 CD146 + CD34相关联的两个多能基质干细胞群的同时分离和纯化-周细胞和CD34 + CD146 -外膜细胞，从人的心肌。

从人类心脏组织血管周围多能前体细胞群的分离

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their ...

isolation-perivascular-multipotent-precursor-cell-populations-from

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

7.3K 次观看.  University of Edinburgh. 人体心脏组织窝藏多能血管周围的前体细胞群可能适合于心肌再生。这里描述的技术允许与天然血管， 即 CD146 + CD34相关联的两个多能基质干细胞群的同时分离和纯化-周细胞和CD34 + CD146 -外膜细胞，从人的心肌。 

视频: Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

从人类骨骼肌成肌原细胞和成纤维细胞的分离和定量免疫细胞化学表征

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

科研

发育生物学

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila melanogaster, inductive microenvironments known as larval Hematopoietic Pockets (HPs) have been identified as anatomical sites for the development and regulation of blood cells (hemocytes), in particular of the self-renewing macrophage lineage.&#160;HPs are segmentally repeated pockets between the epidermis and muscle layers of the larva, which also comprise sensory neurons of the peripheral nervous system. In the larva, resident (sessile) hemocytes are exposed to anti-apoptotic, adhesive and proliferative cues from these sensory neurons and potentially other components of the HPs, such as the lining muscle and epithelial layers. During normal development, gradual release of resident hemocytes from the HPs fuels the population of circulating hemocytes, which culminates in the release of most of the resident hemocytes at the beginning of metamorphosis. Immune assaults, physical injury or mechanical disturbance trigger the premature release of resident hemocytes into circulation. The switch of larval hemocytes between resident locations and circulation raises the need for a common standard/procedure to selectively isolate and quantify these two populations of blood cells from single Drosophila larvae. Accordingly, this protocol describes an automated method to release and quantify the resident and circulating hemocytes from single larvae. The method facilitates ex vivo approaches, and may be adapted to serve a variety of developmental stages of Drosophila and other invertebrate organisms.

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Drosophila blood cells, or hemocytes, cycle between resident sites and circulation. In the larva, resident (sessile) hemocytes localize to inductive microenvironments, the Hematopoietic Pockets, while circulating hemocytes move freely in the hemolymph. The goal of this protocol is the standardized isolation and quantification of these two, behaviorally distinct but interchanging, hemocyte populations.

的测定血细胞种群<em&gt;果蝇</em&gt;幼虫

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila ...

Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human development and investigate the pathophysiology of disease. By employing site-specific nucleases (SSNs) for genome editing, the rapid derivation of new hPSC lines harboring specific genetic alterations in an otherwise isogenic setting becomes possible. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 are the most commonly used SSNs. All of these nucleases function by introducing a double stranded DNA break at a specified site, thereby promoting precise gene editing at a genomic locus. SSN-meditated genome editing exploits two of the cell&#39;s endogenous DNA repair mechanisms, non-homologous end joining (NHEJ) and homology directed repair (HDR), to either introduce insertion/deletion mutations or alter the genome using a homologous repair template at the site of the double stranded break. Electroporation of hPSCs is an efficient means of transfecting SSNs and repair templates that incorporate transgenes such as fluorescent reporters and antibiotic resistance cassettes. After electroporation, it is possible to isolate only those hPSCs that incorporated the repair construct by selecting for antibiotic resistance. Mechanically separating hPSC colonies and confirming proper integration at the target site through genotyping allows for the isolation of correctly targeted and genetically homogeneous cell lines. The validity of this protocol is demonstrated here by using all three SSN platforms to incorporate EGFP and a puromycin resistance construct into the AAVS1 safe harbor locus in human pluripotent&#160;stem cells.

Genome editing of human pluripotent stem cells (hPSCs) can be done quickly and efficiently. Presented here is a robust experimental procedure to genetically engineer hPSCs as exemplified by editing the AAVS1 safe harbor locus to express EGFP and introduce antibiotic resistance.

建立基因组编辑的人多能干细胞系:从定位到隔离

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human ...

Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells

Use of human induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC) for cell replacement therapies holds great promise. Several limitations including low yields and heterogeneous populations of differentiated cells hinder the progress of stem cell therapies. A fate restricted immortalized multipotent otic progenitor (iMOP) cell line was generated to facilitate efficient differentiation of large numbers of functional hair cells and spiral ganglion neurons (SGN) for inner ear cell replacement therapies. Starting from dissociated cultures of single iMOP cells, protocols that promote cell cycle exit and differentiation by basic fibroblast growth factor (bFGF) withdrawal were described. A significant decrease in proliferating cells after bFGF withdrawal was confirmed using an EdU cell proliferation assay. Concomitant with a decrease in proliferation, successful differentiation resulted in expression of molecular markers and morphological changes. Immunostaining of Cdkn1b (p27KIP) and Cdh1 (E-cadherin) in iMOP-derived otospheres was used as an indicator for differentiation into inner ear sensory epithelia while immunostaining of Cdkn1b and Tubb3 (neuronal &#946;-tubulin) was used to identify iMOP-derived neurons. Use of iMOP cells provides an important tool for understanding cell fate decisions made by inner ear neurosensory progenitors and will help develop protocols for generating large numbers of iPSC or ESC-derived hair cells and SGNs. These methods will accelerate efforts for generating otic cells for replacement therapies.

The current protocols to maintain immortalized multipotent otic progenitor (iMOP) cells and otic differentiation are described. Culture conditions and molecular markers that indicate differentiation into sensory epithelia and spiral ganglion neurons (SGN) are highlighted.

在永生多能耳科内皮祖细胞分化启动

Use of human induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC) for cell replacement therapies holds great promise. Several limitations ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

从人胎盘组织源分离和间充质干扩张/基质细胞

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

从人类脐带和胎盘胎儿分离和鉴定间充质干细胞

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

Expansion and Adipogenesis Induction of Adipocyte Progenitors from Perivascular Adipose Tissue Isolated by Magnetic Activated Cell Sorting

Expansion of Perivascular Adipose Tissue (PVAT), a major regulator of vascular function through paracrine signaling, is directly related to the development of hypertension during obesity. The extent of hypertrophy and hyperplasia depends on depot location, sex, and the type of Adipocyte Progenitor Cell (APC) phenotypes present. Techniques used for APC and preadipocytes isolation in the last 10 years have drastically improved the accuracy at which individual cells can be identified based on specific cell surface markers. However, isolation of APC and adipocytes can be a challenge due to the fragility of the cell, especially if the intact cell must be retained for cell culture applications.
Magnetic-activated Cell Sorting (MCS) provides a method of isolating greater number of viable APC per weight unit of adipose tissue. APC harvested by MCS can be used for in vitro protocols to expand preadipocytes and differentiate them into adipocytes through use of growth factor cocktails allowing for analysis of the prolific and adipogenic potential retained by the cells. This experiment focused on the aortic and mesenteric PVAT depots, which play key roles in the development of cardiovascular disease during expansion. These protocols describe methods to isolate, expand, and differentiate a defined population of APC. This MCS protocol allows isolation to be used in any experiment where cell sorting is needed with minimal equipment or training. These techniques can aid further experiments to determine the functionality of specific cell populations based on the presence of cell surface markers.

Here we report a method for isolation of Adipocyte Progenitor Cell (APC) populations from Perivascular Adipose Tissue (PVAT) using Magnetic-activated Cell Sorting (MCS). This method allows for an increased isolation of APC per gram of adipose tissue when compared to Fluorescence-Activated Cell Sorting (FACS).

通过磁性活化细胞分选分离的血管周围脂肪组织的脂肪细胞祖细胞的扩增和脂肪生成诱导

Expansion of Perivascular Adipose Tissue (PVAT), a major regulator of vascular function through paracrine signaling, is directly related to the ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

小鼠胚胎心肌内祖群的定量全贴免疫荧光分析

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs)

Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and neurite outgrowth; and later stages characterized by axon/dendrite outgrowth and synapse formation. In neurodevelopmental disorders, often one or more of these processes are disrupted, leading to abnormalities in brain formation and function. With the advent of human induced pluripotent stem cell (hiPSC) technology, researchers now have an abundant supply of human cells that can be differentiated into virtually any cell type, including neurons. These cells can be used to study both normal brain development and disease pathogenesis. A number of protocols using hiPSCs to model neuropsychiatric disease use terminally differentiated neurons or use 3D culture systems termed organoids. While these methods have proven invaluable in studying human disease pathogenesis, there are some drawbacks. Differentiation of hiPSCs into neurons and generation of organoids are lengthy and costly processes that can impact the number of experiments and variables that can be assessed. In addition, while post-mitotic neurons and organoids allow the study of disease-related processes, including dendrite outgrowth and synaptogenesis, they preclude the study of earlier processes like proliferation and migration. In neurodevelopmental disorders, such as autism, abundant genetic and post-mortem evidence indicates defects in early developmental processes. Neural precursor cells (NPCs), a highly proliferative cell population, may be a suitable model in which to ask questions about ontogenetic processes and disease initiation. We now extend methodologies learned from studying development in mouse and rat cortical cultures to human NPCs. The use of NPCs allows us to investigate disease-related phenotypes and define how different variables (e.g., growth factors, drugs) impact developmental processes including proliferation, migration, and differentiation in only a few days. Ultimately, this toolset can be used in a reproducible and high-throughput manner to identify disease-specific mechanisms and phenotypes in neurodevelopmental disorders.

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells NPCs

Neurodevelopmental processes such as proliferation, migration, and neurite outgrowth are often perturbed in neuropsychiatric diseases. Thus, we present protocols to rapidly and reproducibly assess these neurodevelopmental processes in human iPSC-derived NPCs. These protocols also allow the assessment of the effects of relevant growth factors and therapeutics on NPC development.

人脑神经前体细胞神经发育表型的快速检测 (npc)

Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and ...

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of the heart after ischemic injury. When activated, epicardial cells undergo a process known as epithelial to mesenchymal transition (EMT) to provide cells to the regenerating myocardium. Furthermore, the epicardium contributes via secretion of essential paracrine factors. To fully appreciate the regenerative potential of the epicardium, a human cell model is required. Here we outline a novel cell culture model to derive primary epicardial derived cells (EPDCs) from human adult and fetal cardiac tissue. To isolate EPDCs, the epicardium is dissected from the outside of the heart specimen and processed into a single cell suspension. Next, EPDCs are plated and cultured in EPDC medium containing the ALK 5-kinase inhibitor SB431542 to maintain their epithelial phenotype. EMT is induced by stimulation with TGF&#946;. This method enables, for the first time, the study of the process of human epicardial EMT in a controlled setting, and facilitates gaining more insight in the secretome of EPDCs that may aid heart regeneration. Furthermore, this uniform approach allows for direct comparison of human adult and fetal epicardial behavior.

The epicardium plays a crucial role in the development and repair of the heart by providing cells and growth factors to the myocardial wall. Here, we describe a method to culture human primary epicardial cells that enables the study and comparison of their developmental and adult characteristics.

人成体和胎心标本中原发性心外膜细胞的分离培养

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...