作者要感谢贝克基金会和皇家儿童少年基金会“女科学家奖学金”支持Zerina Lokmic的。安德鲁G. Elefanty和爱德华G.士丹利NHMRC资深研究员的研究。在他们的实验室工作是由NHMRC和干细胞澳大利亚支持。

伦理声明：伦理学批准征收的淋巴管畸形及包皮组织从人类研究伦理委员会获得皇家儿童医院，墨尔本，澳大利亚。签名同意从患者的父母接受手术前。组织样本从确诊患者与LMS进行外科手术作为临床管理的一部分，并择期包皮环切收集。所有实验均符合国家卫生和医学研究理事会，澳大利亚的指引执行。 从包皮和淋巴管畸形组织1.准备细胞悬液<ol><li>缓冲器和媒体准备。 <ol><li>使用市售的EGM-2中压子弹套件温热的EGM-2培养基，轻轻解冻试剂盒组分在37℃水浴中制备完整的内皮细胞的介质。在II级生物安全柜，无菌每个组件添加到EGM-2介质。一旦所有的组件添加到媒体，请参阅此媒体称为“完整的内皮细胞培养基”。使用无菌50毫升吸管混合使用前的内容。 注 ：所有涉及的细胞培养步骤是在II类生物危害柜进行。所有解决方案都存储在4℃按制造商的说明，并加热至在使用前室温。完整的内皮细胞培养基，细胞培养缓冲液和酶溶液加热至37℃，使用前20分钟。 </li><li>补充完整内皮细胞培养基用50ng / ml的VEGF-C。分装补充内皮细胞培养基到50ml无菌管中并储存于4℃直至使用。储存在4℃下，当媒体是稳定至少4周。 </li><li>通过溶解8.752克氯化钠，1.416克的Na 2 HPO制备钙和镁的磷酸盐缓冲盐水（PBS）4 .2H 2 O和0.395克KH 2 PO 4在1,000ml水中。调节pH至7.4。使用0.22微米的过滤器，并储存在4℃过滤消毒PBS。在使用之前，添加抗生素/抗真菌溶液（在1中使用：100）。 </li><li>为了制备人纤连蛋白，溶解在10毫升无菌水中1毫克纤维连接蛋白。商店重新溶解在100μl的等分溶液在-20℃下。上使用的当天无菌PBS加入10 mL至100μl纤连蛋白等分试样（以得到最终工作的10微克/毫升浓度）。 </li><li>用于酶介质，制备含有0.04％分散酶II，0.25％胶原酶II和0.01％DNA酶I在无菌PBS中的溶液。首先，称量分散酶和胶原酶，放置在无菌的50ml管中，加入的PBS所需的体积和30分钟孵育振荡在37℃下使其溶解。一旦溶解，在生物安全罩过滤消毒（0.22微米过滤器）分散酶/胶原酶溶液。无菌在37℃下加入DNA酶I.商店酶溶液直到使用。 注 ：抗酶是一种商业细胞培养级试剂可作为无菌冻干粉。 </li></ol></li></ol>从包皮和淋巴管畸形2.准备细胞悬液<ol><li>称量50ml管含有10毫升的DMEM / 2％抗生素 - 抗真菌溶液。该管将用于组织采集。 </li><li>下列手术切除淋巴管畸形和包皮的组织，在无菌条件下在冰上添加组织标本到管并转移到细胞培养实验室。 </li><li>称量装有组织的管，并计算组织重量。使用该权重来计算的消化所述组织所需的酶溶液的体积。 </li><li>在II级生物安全柜，用无菌镊子来组织和媒体转移到100毫米组织培养皿。用无菌剪刀精细剁碎组织成约1 立方毫米件。 </li><li>转移组织糜混合媒体我NTO 50ml管含有额外的10毫升无菌DMEM / 2％抗生素 - 抗真菌溶液。通过颠倒混合到重悬浮组织。离心样品在300×g离心5分钟。 </li><li>去除上清。基于组织重量，加入1 ml预热（37℃）胶原酶II每100mg组织糜的/ DNA酶I /分散酶II消化溶液。通常，使用将10ml酶溶液对约1g组织糜的。 </li><li>孵育在37℃培养箱的组织为20-90分钟，恒定200rpm振荡。在这一时期结束时，确保几乎所有的组织的被消化成细碎片。 注 ：在隔离的从主组织影响细胞存活的时间曝光的细胞胶原酶和分散酶的。我们已经发现根据经验，多数新生儿包皮样品最优地需要消化20-30分钟，而纤维化淋巴管畸形样品可能需要消化60-90分钟。 LM LEC产量S被纤维化的存在量的影响，更纤维组织产生较少的细胞，这可能是由于长期暴露的有害影响，胶原酶II和分散酶II。 </li><li>消化后，将管转移到生物安全柜，并通过一个70微米的过滤器放入一个无菌50ml管中通过消化的组织方案。使用3个毫升注射器活塞与柱塞橡胶，磨下来的剩余组织，直到细胞外基质的只有很小的痕迹观察。 </li><li>与卷的内皮细胞培养基相当于解离酶培养基以回收细胞粘到筛和使酶失活的体积洗细胞过滤网。例如，对于2毫升解离酶介质消化的组织糜的，内皮细胞培养基中加入2毫升至使酶失活。 </li><li>离心在300×g离心5分钟将细胞溶液和吸出上清液。 R&gt; E-暂停在10毫升PBS的细胞。离心细胞在300 XG5分钟后，除去上清液，然后重复细胞洗涤两次。 &gt;重悬细胞沉淀在20ml内皮细胞培养基中。使用台盼蓝然后种子计数细胞2×10&gt; 6个细胞在150厘米&gt; 2烧瓶中20ml的内皮细胞培养基的终体积预涂覆有纤连蛋白。培养在37℃，在5％CO&gt; 2的湿润空气培养箱中。&gt; 注 ：预计有核细胞的75％-90％的存活组织消化估算由锥虫蓝排除。初始细胞计数反映所有有核细胞中的细胞悬浮液（ 即角质形成细胞，白细胞，巨噬细胞，结缔组织细胞和血管细胞）。在5-7天的细胞计数反映已附加，存活和细胞培养过程中增殖的细胞。涂层组织培养瓶中用纤连蛋白也提高了随后的LEC和LM LEC生存。 </li><li>过夜孵育后，在洗涤三次以P洗去未结合的细胞BS / 1％的抗生素，抗真菌的解决方案，并添加新鲜的内皮细胞培养基。进行三次洗涤，有效地除去未结合的细胞和红血细胞存在于烧瓶中。改变媒体每隔一天。细胞将是​​〜80％汇合后5-7天。 </li></ol>细胞淋巴管内皮细胞表面标志物流式细胞仪的3抗体染色<ol><li>后的细胞达到80％汇合时，吸出介质并漂洗细胞，用PBS。 </li><li>通过孵育细胞用7ml的细胞脱附溶液分离贴壁细胞如的Accutase每150cm 2烧瓶中5-7分钟，在37℃。 </li><li>收获分离的细胞，通过加入3倍体积的内皮细胞培养基的灭活细胞脱离溶液和细胞悬液转移到一个新的无菌试管中。 </li><li>离心细胞，在300×g离心5分钟。 </li><li>吸去上清液并重新悬浮在5ml PBS中的细胞。离心细胞，在300×g离心5分钟，除去的S然后重复upernatant洗细胞。重新悬浮在5ml PBS中的细胞。 </li><li>计数活细胞的数量。混合10微升细胞悬浮液与90微升0.4％台盼蓝。转移10微升此悬浮液至血细胞计数计数。 注 ：最终细胞产率将取决于处理的样本大小。每150cm 2烧瓶范围通常的细胞产量从7×10 5〜1.2的活细胞×10 6。 </li><li>制备用于细胞染色的抗体溶液。 注意 ：下面的稀释液进行滴定染色1×10 6中的100微升的染色体积。 <ol><li>稀PE偶联的小鼠抗人VEGFR-3（1:50）; PE-Cy7的缀合的小鼠抗人缀合的CD34（1：200）; APC缀合的小鼠抗人CD31（1：100）和Alexa 488缀合的鼠抗人PODOPLANIN（1：200）中的100微升每组织样品无菌5％FBS / PBS溶液总体积。下面的准备抗体溶液，保持在冰上直到使用。同样准备稀释的同型对照抗体的混合物，以方便流式细胞仪门的设置。 </li></ol></li><li>为了减少非特异性抗体结合，离心细胞，在300×g离心2分钟，除去上清液，然后悬浮细胞在100μl灭菌的5％FBS /每个样品PBS溶液进行染色。孵育细胞在冰上20分钟，在4℃。 </li><li>离心细胞在300 XG 5分钟，弃上清然后重新暂停细胞中的共轭抗体的鸡尾酒。还染色在100μl同种型对照抗体混合物的细胞（1×10 5）的更小的等分试样。孵育细胞在冰上进行20分钟。 </li><li>以除去未结合的抗体，加入2毫升的2％FBS / PBS溶液和离心机将细胞在300×g离心5分钟。吸出上清液，重复清洗。 </li><li>重悬细胞沉淀于同种型对照和抗体染色样品中加入300微升的0.5毫克/毫升丙锭io的要排序啶/ 2％FBS / PBS溶液。放在冰管，直到排序。 </li></ol> 4.细胞分选<ol><li>使用以下材料来设置仪器;对准珠如市售的荧光颗粒，磷酸盐缓冲的基于盐水鞘流体和下降延迟珠，如Accudrop荧光珠。 </li><li>隔离在一个多参数荧光激活细胞分选仪纯化的人淋巴管内皮细胞。设置仪器和对齐根据制造商的建议。此外，运行单染补偿控制与每个排序以生成补偿矩阵，并通过荧光的发射，以从而消除实质泄放如PE进入PE-CY通道。 注 ：FACS分选的细胞比例较高生存的隔离，如果一个100微米的喷嘴被使用，细胞分类成内皮细胞培养基中。 </li></ol> 5.细胞培养后FACS排序ING <ol><li>以下排序，离心细胞，在300×g离心5分钟。吸出上清液并将细胞重新悬浮在5-10毫升的培养基（取决于所使用的种子的细胞在烧瓶）。如果低于50,000元分类中，细胞是在纤连蛋白涂覆为25cm 2烧瓶中培养。 </li><li>否则培养在5％的CO 2，空气增湿培养箱将细胞在75cm 2的纤连蛋白涂覆的烧瓶在37℃。术后第2天更换介质。 注 ：细胞培养基改变每隔一天，因为延长介质变化的时间似乎有利于非内皮细胞的存活。 PROX-1，VEGFR-3，Podoplanin，CD34和CD31，当细胞是第一分割为进一步扩大15：我们使用免疫组织化学标记的检测验证的淋巴管内皮细胞的表型。 </li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualisation+and+stereological+assessment+of+blood+and+lymphatic+vessels.">Visualisation and stereological assessment of blood and lymphatic vessels.</a> Histol Histopathol. 26 (6), 781-796 (2011).</li><li>Hagerling, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+multistep+mechanism+for+initial+lymphangiogenesis+in+mouse+embryos+based+on+ultramicroscopy.">A novel multistep mechanism for initial lymphangiogenesis in mouse embryos based on ultramicroscopy.</a> EMBO J. 32 (5), 629-644 (2013).</li><li>Pollmann, C., Hagerling, R., Kiefer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+lymphatic+vessel+development,+growth,+and+function.">Visualization of lymphatic vessel development, growth, and function.</a> Adv Anat Embryol Cell Biol. 214, 167-186 (2014).</li><li>Breiteneder-Geleff, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiosarcomas+express+mixed+endothelial+phenotypes+of+blood+and+lymphatic+capillaries:+podoplanin+as+a+specific+marker+for+lymphatic+endothelium.">Angiosarcomas express mixed endothelial phenotypes of blood and lymphatic capillaries: podoplanin as a specific marker for lymphatic endothelium.</a> Am J Pathol. 154 (2), 385-394 (1999).</li><li>Kaipainen, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+fms-like+tyrosine+kinase+4+gene+becomes+restricted+to+lymphatic+endothelium+during+development.">Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development.</a> Proc Natl Acad Sci U S A. 92 (8), 3566-3570 (1995).</li><li>Schacht, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T1alpha/podoplanin+deficiency+disrupts+normal+lymphatic+vasculature+formation+and+causes+lymphedema.">T1alpha/podoplanin deficiency disrupts normal lymphatic vasculature formation and causes lymphedema.</a> EMBO J. 22 (14), 3546-3556 (2003).</li><li>Kriehuber, E., et al. <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+characterization+of+dermal+lymphatic+and+blood+endothelial+cells+reveal+stable+and+functionally+specialized+cell+lineages.">Isolation and characterization of dermal lymphatic and blood endothelial cells reveal stable and functionally specialized cell lineages.</a> J Exp Med. 194 (6), 797-808 (2001).</li><li>Florez-Vargas, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+D2-40+and+LYVE-1+immunostaiing+in+lymphatic+malformations.">Comparative analysis of D2-40 and LYVE-1 immunostaiing in lymphatic malformations.</a> Lymphology. 41 (3), 103-110 (2008).</li><li>Makinen, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDZ+interaction+site+in+ephrinB2+is+required+for+the+remodeling+of+lymphatic+vasculature.">PDZ interaction site in ephrinB2 is required for the remodeling of lymphatic vasculature.</a> Genes Dev. 19 (3), 397-410 (2005).</li><li>Connell, F. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+coding+regions+of+VEGFR3+and+VEGFRC+in+Milroy+disese+and+other+primary+lymphoedemas.">Analysis of coding regions of VEGFR3 and VEGFRC in Milroy disese and other primary lymphoedemas.</a> Hum Genet. 124 (6), 625-631 (2009).</li></ol>

The authors have nothing to disclose.

淋巴管内皮细胞在维持流体稳态，对外来抗原的吸收和某些营养素转运免疫应答中起重要作用。 LEC动态平衡可受疾病过程，如细菌感染和肿瘤转移，但淋巴管内皮细胞还可以开发导致形成的不正常的淋巴管和显著发病为受影响的患者的体细胞突变。要通过植入体内和体外实验获得的淋巴管畸形的病因更多的理解，并发现新的治疗选择，我们首先开发了LEC分离方法的基础上使用CD34 CD31...

淋巴血管系统的一个主要功能是吸收淋巴液，含有脂质，蛋白质和细胞成分的过量的组织液，并进行到血液静脉系统。毛细淋巴管的一个网络指示淋巴在那里它筛选外源抗原，在免疫监视和部署的白血细胞中和外来抗原的重要过程的存在的淋巴结。 单向淋巴系统开始在组织中与初始毛细淋巴管，一个独特的结构，薄壁扁平的内皮细胞的一个连续单层与专门的细胞连接，允许淋巴条目1,2。这些毛细管经由锚定纤丝以防止在增加间质的压力3存在容器塌陷附连到相邻的结缔组织基质。最初的毛细淋巴管直接排入收集毛细淋巴管这凝聚成较大的淋巴管静脉或。在比较初始淋巴毛细血管，收集淋巴管具有较厚的血管壁，配对淋巴阀和由不连续的基底膜，其中几个平滑肌细胞包埋4被装入。平滑肌细胞协调开口和淋巴阀门关闭和收缩有助于流淋巴结3。在人体中，从身体的各个区域淋巴静脉连接形成淋巴中继线其合并以形成两个淋巴管：胸导管和右淋巴管。胸导管排出淋巴液从身体的左侧和从右侧胸部下方而从右侧臂的头，颈和胸部的和右侧的右淋巴管下水道淋巴。两管道在颈部5进行淋巴进入锁骨下静脉。 淋巴系统疾病，大致分为获取的第二先天性（ 表1）。收购条件的例子是淋巴管炎和继发性淋巴水肿。淋巴管炎是一种淋巴管的炎症因细菌感染。受影响的淋巴管扩张并充满渗出液含有多形核细胞。在皮肤中，这些淋巴管是作为红，痛苦皮下条纹常伴相关引流淋巴结（淋巴结）6的放大可见。继发性淋巴水肿出现损坏或阻塞的淋巴管或淋巴结阻塞的后果。这导致慢性进行性肿大，由于淋巴结远端积累的损坏或阻塞。在发达国家中，继发性淋巴水肿是最常见的恶性肿瘤，其中转移性肿瘤妨碍淋巴管或区域淋巴结，或作为抗癌治疗的结果在手术切除的淋巴结，辐照后纤维化和炎症后血栓的相关bosis和疤痕7。在世界的其他地方，继发性淋巴水肿可继发于引起寄生蠕虫淋巴阻塞如班氏丝虫 6。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td colspan="4"> 淋巴血管系统疾病 </td></tr><tr><td> 后天 </td><td colspan="3"> 先天性 </td></tr><tr><td rowspan="3">淋巴结炎 继发性淋巴水肿</td><td> 原发性淋巴水肿10 </td><td> 零星的淋巴管畸形13 </td><td> 淋巴管畸形伴有证13 </td></tr><tr><td rowspan="2"> 例如： 米尔罗伊综合征 美格综合征</td><td> 简单 ： 淋巴管畸形</td><td rowspan="2"> 例如： 畸形骨Tranaunay综合征 公园Weber综合征 斯特奇 - 韦伯综合征</td></tr><tr><td> 结合 ： 毛细淋巴管畸形 毛细淋巴管静脉畸形 毛细淋巴管，动静脉畸形 毛细淋巴管静脉动静脉畸形</td></tr></tbody></table> 表1概述淋巴血管系统的疾病。 淋巴系统的先天性病症包括初级（特发性）淋巴水肿被认为是由基因突变，淋巴管扩张和淋巴系统8,9的异常所引起的。初级淋巴水肿可能是零星的推测是通过从头突变引起，或继承。淋巴病症也可以是分离的或包括一个更一般化综合征10的一部分。在儿科人群中，97淋巴结％水肿是零星的，在淋巴管结构异常的影响区域淋巴结引流11。米尔罗伊疾病是由突变的VEGFR-3的基因明显在出生时或不久后12初级淋巴水肿的一个例子。虽然大多家族条件下，米尔罗伊疾病，也可在婴儿识别未经米尔罗伊疾病32的家族史。任何淋巴水肿的严重程度是依赖于淋巴生产和运输淋巴回静脉循环6能力的量。 根据临床表现和原位内皮细胞增殖，淋巴系统的异常被分类为淋巴肿瘤或淋巴管畸形13。 Kaposiform淋巴血管瘤病是一个LEC肿瘤14的一个例子。淋巴管畸形被认为是胚胎发育过程中产生和成长的比例给孩子15,16。他们很少倒退，但可以remaiñ无症状直到外伤或感染沉淀的快速增长，导致临床并发症。淋巴管网和淋巴传导的有序结构，从组织到上述静脉循环受到干扰的淋巴管畸形其中包括充满淋巴液不正常的囊性结构的局部集合。虽然没有临床或实验证据表明，这些囊状容器被连接​​到淋巴循环或它们包含官能淋巴阀，其淋巴同一性是通过淋巴细胞标记物如PODOPLANIN，CD31，淋巴管内皮受体1范围表达证实（LYVE-1），普洛斯彼罗同源异型盒蛋白1（PROX-1）和VEGFR-3 15,17,18。这些囊性结构可以是小的（微囊）或大（macrocystic），但大多数淋巴管畸形同时包含微囊和macrocystic组件（ 图1）16。手术后，注射液ñ硬化和/或射频消融治疗的淋巴管畸形往往重演。 <img alt="图1" src="/files/ftp_upload/52691/52691fig1highres.jpg" width="700" /> 图1.形态人体淋巴血管和淋巴管畸形。正常人体淋巴（A）和淋巴管畸形血管（B和C）标记抗体PODOPLANIN（棕色标签，箭头）。人类淋巴管畸形血管的特征是显着的扩张和管腔大小相当大的变化。这些局部异常囊性结构可以是小的（微囊，*）（B）或大（macrocystic，＃）（℃）。多数淋巴管畸形同时含有微囊和macrocystic组件。 <a href="/files/ftp_upload/52691/52691fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> &lt;/ P&gt; 一些研究者已经提出，淋巴管畸形代表淋巴脉管系统的发育障碍，其中所述淋巴管内皮细胞不具有异常生长潜力，而是未能连接到正常循环19。然而，我们已经发现，在LM淋巴管内皮细胞增殖速度更快，对细胞凋亡比包皮的LEC 15提示有在LM淋巴管内皮细胞的主缺陷更有抵抗力。当LM晶状体上皮细胞植入小鼠异种移植模型中，他们形成的结构让人想起淋巴管畸形15。这支持一个假说，即淋巴管畸形可能由胎儿发育过程中的LM淋巴管内皮细胞产生的一种或多种体细胞突变引起的。事实上，最近的报告已经确定这样的一个突变在磷酸肌醇-3-激酶（PIK3CA）基因20的p110α催化亚基。 鉴于DNA测序技术，相关的突变c中的进步乌尔德在孤立的LM晶状体上皮细胞更容易识别，引导这些条件进一步研究。可行的淋巴管内皮细胞的分离将促进试验，例如迁移，增殖，管形成能力和存活异常和正常淋巴管内皮细胞之间的比较响应于降低的养分可用性或促凋亡剂15。孤立的LEC将进一步使我们能够进行细胞特异性基因表达和蛋白质组研究中，描绘新的LEC亚群，并发现适合淋巴管畸形的临床管理新的药理学试剂。 之前我们已经发布了基于晶状体上皮细胞从新生儿包皮和淋巴管畸形15磁珠分离LEC隔离法。我们报道从基于缺乏CD34的表达的，接着进行的CD34 负片细胞级分，以正选择为CD31血管内皮细胞的分离正常和患病的淋巴管内皮细胞的一个策略。然而，这种方法受到阻碍残留非内皮细胞的存在。这是独立前的后续结缔组织消化去除表皮。这些污染物通常增殖得更快，从而最终茂密生长尽管后续尝试重复LEC隔离的内皮细胞培养物。的确，非内皮细胞低至2％至5％的初始污染足以压倒LEC群体15。这促使我们探索荧光激活细胞分选法作为一种选择，以改善LEC细胞产率和纯度。此外，我们使用了多参数排序以增强LEC群体的特异性，增加VEGFR-3和PODOPLANIN到选择标记，以确定CD34 低 CD31 方位 VEGFR-3的方位 PODOPLANIN 方位淋巴管内皮细胞。 选择这些标记的基本原理是根据报告，虽然晶状体上皮细胞和血液血管内皮CELLS有许多共同的细胞表面标志物，例如CD31，淋巴管内皮细胞表现出在它们的CD34，PODOPLANIN和VEGFR-3的细胞表面标志物的表达表型变异时相比，血液血管内皮细胞21-23。 CD31是一种130 kDa的跨膜糖蛋白也被称为血小板内皮细胞粘附分子-1（PECAM-1）。它被认为是一个泛内皮细胞标记物，因为它表达于所有类型的血液和淋巴管21,24,25。 CD34是存在于大多数造血祖110-kDa的跨膜糖蛋白和干细胞，血管内皮细胞和某些淋巴管26。 VEGFR-3，受体对血管内皮细胞生长因子C和D，是最初存在于在小鼠胚胎显影静脉，但以下淋巴规范由转录调控因子SRY相关的HMG-盒（SOX）-18 角 hicken Øvalbuminüpstrea米p romoter 牛逼ranscription˚F演员2（COUPTF-II）和PROX-1，VEGFR-3静脉表达丢失，它成为限制胚胎的LEC 25,27。 PODOPLANIN，一个38 kDa的膜粘蛋白，首先注意对小鼠胚胎发育28淋巴管在胚胎大约11天（〜E11.0），并同时强烈微血管淋巴管，PODOPLANIN表达在淋巴管畸形macrocystic淋巴管内皮细胞表达更变15。流式细胞术实验表明，至少一些的CD34 高 CD31 方位内皮细胞表达淋巴标记PODOPLANIN 29。虽然LYVE-1和PODOPLANIN染色在人淋巴管畸形系统的评估表明，两者都是在染色淋巴管畸形内皮30有效，在正常组织中，LYVE-1据报道，强烈存在于初始淋巴毛细血管内皮但降低，即使在收集淋巴管内皮31不存在。因为我们的目标是隔离两个我们已选择的初始和收集淋巴管内皮细胞不使用LYVE-1作为我们的小区选择策略的一部分。最后，聘请这些标记的决定也是基于抗体的诊断被用于为显微成像标记淋巴管的可用性，这将允许流式细胞仪及免疫研究之间相关性的特征。 本文将描述该组织消化法，细胞染色和FACS设置所需的CD34 低 CD31 波什VEGFR-3 POS PODOPLANIN 波什晶状体上皮细胞的成功分离，以及来自包皮和淋巴管畸形高 ​​CD34 CD31 波什VEGFR-3 POS PODOPLANIN 波什内皮细胞组织。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Name of Material/Equipment</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Description</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Life Technologies (Gibco)</td>
			<td>11965-092</td>
			<td>Used to collect tissue samples from the operating theatre.</td>
		</tr>
		<tr>
			<td>EGM-2 MV Bullet Kit</td>
			<td>Lonza</td>
			<td>CC-3202</td>
			<td>EGM-2 MV Bullet Kit contains 500 ml of EBM-2 media and human EGF, hydrocortisone, gentamycin (GA-1000), fetal bovine serum (FBS), VEGF, human FGF-b, R3-IGF-1 and ascorbic acid.</td>
		</tr>
		<tr>
			<td>VEGF-C</td>
			<td>R&#38;D Systems</td>
			<td>2179-VC-025</td>
			<td>Complete endothelial cell media is supplemented with 50 ng/mL VEGF-C</td>
		</tr>
		<tr>
			<td>Antibiotic-antimycotic solution (100x)</td>
			<td>Life Technologies (Gibco)</td>
			<td>15240-062</td>
			<td>This solution contains 10,000U/ml of penicillin, 10,000 mg/ml of streptomycin and 25 mg/ml of Fungizone antimycotic. Use at 1:100 dilution.</td>
		</tr>
		<tr>
			<td>Fibronectin from human plasma</td>
			<td>Sigma-Aldrich</td>
			<td>F2006</td>
			<td>Used at 10 mg/ml concentrations. It can be re-used for up to 1 month if kept at 4 degrees and maintained sterile. Use 7 mL to coat 150 cm2 flask.</td>
		</tr>
		<tr>
			<td>StemPro Accutase Cell dissociation reagent</td>
			<td>Life Technologies (Gibco)</td>
			<td>A11105-01</td>
			<td>StemPro&#174;Accutase&#174; is used at 0.05 ml per cm2 to cover the entire surface area of the flask. Using lesser volumes may result in incomplete cell detachment.</td>
		</tr>
		<tr>
			<td>0.4% Trypan Blue dye</td>
			<td>Life Technologies (Gibco)</td>
			<td>15250-061</td>
			<td>Used as a viability stain.</td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Roche Applied Biosciences</td>
			<td>4942078001</td>
			<td>Used at 0.04%</td>
		</tr>
		<tr>
			<td>Collagenase II</td>
			<td>Worthington Lab</td>
			<td>4176</td>
			<td>Used at 0.25%</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Roche Applied Biosciences</td>
			<td>11284932001</td>
			<td>Used at 0.01%</td>
		</tr>
		<tr>
			<td>70 mm nylon cell strainers</td>
			<td>BD Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-conjugated VEGFR-3 clone 9D9F9, clone WM59</td>
			<td>BioLegend</td>
			<td>356204</td>
			<td>Used at 1:50 dilution</td>
		</tr>
		<tr>
			<td>PE-Cy7-conjugated CD34, clone 581</td>
			<td>BioLegend</td>
			<td>343516</td>
			<td>Used at 1:200 dilution</td>
		</tr>
		<tr>
			<td>APC-conjugated mouse anti human CD31, clone WM59</td>
			<td>BioLegend</td>
			<td>303116,</td>
			<td>Used at 1:100 dilution</td>
		</tr>
		<tr>
			<td>Alexa 488-conjugated rat anti human PODOPLANIN, clone NC-80.</td>
			<td>BioLegend</td>
			<td>337006</td>
			<td>Used at 1:200 dilution</td>
		</tr>
		<tr>
			<td>PE-conjugated mouse IgG1, k isotype, clone MOPC-21</td>
			<td>BioLegend</td>
			<td>400112</td>
			<td>Used at 1:50 dilution</td>
		</tr>
		<tr>
			<td>PE-Cy7-conjugated mouse IgG1, k isotype, clone MOPC-21</td>
			<td>BioLegend</td>
			<td>400126</td>
			<td>Used at 1:200 dilution</td>
		</tr>
		<tr>
			<td>APC-conjugated mouse IgG1, k isotype, clone MOPC-21</td>
			<td>BioLegend</td>
			<td>400120</td>
			<td>Used at 1:100 dilution</td>
		</tr>
		<tr>
			<td>Alexa 488-conjugated mouse IgG2a, k isotype, clone RATK2758</td>
			<td>BioLegend</td>
			<td>400525</td>
			<td>Used at 1:200 dilution</td>
		</tr>
	</tbody>
</table>

isolation of human lymphatic endothelial cells by multi-parameter fluorescence-activated cell sorting

淋巴系统疾病如原发性淋巴水肿，淋巴管畸形和淋巴管瘤是导致发病率显著但鲜为人知的是，他们的生物学罕见的情况。隔离患病和健康组织高纯度的人淋巴管内皮细胞（晶状体上皮细胞）将促进淋巴管内皮细胞在基因，分子和细胞水平的研究。可以预料，这些调查可能揭示目标为新的疗法可能改变这些条件的临床管理。描写人包皮淋巴管内皮细胞和淋巴管畸形淋巴管内皮细胞（LM的LEC）的隔离协议提出。以获得单细胞悬液组织切碎并用分散酶II和胶原酶II酶处理。将所得的单细胞悬浮液，然后标记抗体的分化（CD）标志物CD34，CD31，血管内皮生长因子-3（VEGFR-3）和PODOPLANIN群集。 STAI奈德的活细胞进行分选上的荧光激活细胞分选（FACS）对CD34 低 CD31 方位 VEGFR-3的方位 PODOPLANIN 方位的LM LEC人口从其他内皮细胞和非内皮细胞中分离出来。排序LM淋巴管内皮细胞培养和扩增的纤维连接蛋白包瓶中进一步的实验中使用。

以下初始组织消化，在24小时中未分离的样品培养之后，不同的内皮细胞集落可以观察到（ 图2A）一起成纤维细胞样细胞和平滑肌细胞。以下排序和24小时细胞培养物后，将CD34 低 CD31 方位 VEGFR-3的方位 Podoplanin 方位细胞附着并显示典型鹅卵石形态（ 图2B和C）。使用上述流式细胞仪的方法，我们能够从CD34 高 CD31 方?...

Watch this Scientific Journal Video about Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting at JoVE.com

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

这个协议的目标是隔离淋巴管内皮细胞衬里使用荧光激活细胞分选（FACS）人淋巴管畸形囊肿状容器和包皮。随后的细胞培养和扩大这些细胞的实验允许为复杂遗传，蛋白组学，功能和细胞分化研究的一个新的水平。

人淋巴管内皮细胞中的多参​​数荧光激活细胞分选分离

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

isolation-human-lymphatic-endothelial-cells-multi-parameter

Research

JoVE Journal

Medicine

14.2K 次观看.  The Royal Children&#146;s Hospital. 这个协议的目标是隔离淋巴管内皮细胞衬里使用荧光激活细胞分选（FACS）人淋巴管畸形囊肿状容器和包皮。随后的细胞培养和扩大这些细胞的实验允许为复杂遗传，蛋白组学，功能和细胞分化研究的一个新的水平。 

视频: Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of endothelial junctions and finally invasion and migration of tumor cells through the endothelial layer to enter the bloodstream as a means of transport to distant sites in the host1-3. Once in the circulatory system, cancer cells adhere to capillary walls and extravasate to the surrounding tissue to form metastatic tumors4,5. The various components of tumor cell-endothelial cell interaction can be replicated in vitro by challenging a monolayer of human umbilical vein endothelial cells (HUVEC) with cancer cells. Studies performed with electron and phase-contrast microscopy suggest that the in vitro sequence of events fairly represent the in vivo metastatic process6. Here, we describe an electrical-impedance based technique that monitors and quantifies in real-time the invasion of endothelial cells by malignant tumor cells.
Giaever and Keese first described a technique for measuring fluctuations in impedance when a population of cells grow on the surface of electrodes7,8. The xCELLigence instrument, manufactured by Roche, utilizes a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance9-12. The impedance is displayed as a dimensionless parameter termed cell-index, which is directly proportional to the total area of tissue-culture well that is covered by cells. Hence, the cell-index can be used to monitor cell adhesion, spreading, morphology and cell density.
The invasion assay described in this article is based on changes in electrical impedance at the electrode/cell interphase, as a population of malignant cells invade through a HUVEC monolayer (Figure 1). The disruption of endothelial junctions, retraction of endothelial monolayer and replacement by tumor cells lead to large changes in impedance. These changes directly correlate with the invasive capacity of tumor cells, i.e., invasion by highly aggressive cells lead to large changes in cell impedance and vice versa. This technique provides a two-fold advantage over existing methods of measuring invasion, such as boyden chamber and matrigel assays: 1) the endothelial cell-tumor cell interaction more closely mimics the in vivo process, and 2) the data is obtained in real-time and is more easily quantifiable, as opposed to end-point analysis for other methods.

This article describes an in vitro technique for monitoring cancer cells invading through a monolayer of endothelial cells. The data is acquired in real-time as a function of changes in impedance on the surface of electrodes at the well bottom.

一个实时的基于电阻抗技术测量癌细胞入侵内皮细胞单层

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of ...

科研

医学

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

人牙髓干细胞的分离，鉴定和比较性恒牙使用两种不同的方法，得出

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human cancer cells in mitosis. The phenanthridine PJ-34 was the most potent molecule. Declustering of extra-centrosomes causes mitotic failure and cell death in multi-centrosomal cells. Most solid human cancers have high occurrence of extra-centrosomes. The activity of PJ-34 was documented in real-time by confocal imaging of live human breast cancer MDA-MB-231 cells transfected with vectors encoding for fluorescent &gamma;-tubulin, which is highly abundant in the centrosomes and for fluorescent histone H2b present in the chromosomes. Aberrant chromosomes arrangements and de-clustered &gamma;-tubulin foci representing declustered centrosomes were detected in the transfected MDA-MB-231 cells after treatment with PJ-34. Un-clustered extra-centrosomes in the two spindle poles preceded their cell death. These results linked for the first time the recently detected exclusive cytotoxic activity of PJ-34 in human cancer cells with extra-centrosomes de-clustering in mitosis, and mitotic failure leading to cell death. According to previous findings observed by confocal imaging of fixed cells, PJ-34 exclusively eradicated cancer cells with multi-centrosomes without impairing normal cells undergoing mitosis with two centrosomes and bi-focal spindles. This cytotoxic activity of PJ-34 was not shared by other potent PARP1 inhibitors, and was observed in PARP1 deficient MEF harboring extracentrosomes, suggesting its independency of PARP1 inhibition. Live confocal imaging offered a useful tool for identifying new molecules eradicating cells during mitosis.

The cytotoxic activity of the phenanthridine PJ-34 in cancer cells undergoing mitosis was documented in real time by live confocal imaging. PJ-34 eradicated human breast cancer MDA-MB-231 cells harboring extra-centrosomes in mitosis. Unlike normal bi-focal mitosis, the extra-centrosomes were not clustered in the two spindle poles in the presence of PJ-34.

共聚焦成像在肝癌细胞中观察到的异常有丝分裂细胞死亡相关

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

分离，培养和成像胎儿胰腺细胞集群

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

的肿瘤特异性内皮细胞的分离与培养扩展

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

分离培养犬从动脉和静脉主血管内皮细胞

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the neovascularization of ischemic tissues. The origin, classification and characterization of EPCs are complex; notwithstanding, two prominent sub-types of EPCs have been established: so-called &#34;early&#34; EPCs (subsequently referred to as early-EPCs) and late-outgrowth EPCs (late-EPCs). They can be classified by biological properties as well as by their appearance during in vitro culture. While &#34;early&#34; EPCs appear in less than a week after culture of peripheral blood-derived mononuclear cells in EC-specific media, late-outgrowth EPCs can be found after 2-3 weeks. Late-outgrowth EPCs have been recognized to be directly involved in neovascularization, mainly through their ability to differentiate into mature endothelial cells, whereas &#34;early&#34; EPCs express various angiogenic factors as endogenous cargo to promote angiogenesis in a paracrine manner. During myocardial ischemia/reperfusion (I/R), various factors control the homing of EPCs to regions of blood vessel formation.
Macrophage migration inhibitory factor (MIF) is a chemokine-like pro-inflammatory and ubiquitously expressed cytokine and was recently described to function as key regulator of EPCs migration at physiological concentrations1. Interestingly, MIF is stored in intracellular pools and can rapidly be released into the blood stream after several stimuli (e.g. myocardial infarction). 
This protocol describes a method for the reliable isolation and culture of early-EPCs from adult human peripheral blood based on CD34-positive selection with subsequent culture in medium containing endothelial growth factors on fibronectin-coated plates for use in in vitro migration assays against serum samples of cardiac surgical patients. Furthermore, the migratory influence of MIF on chemotaxis of EPCs compared to other well-known angiogenesis-stimulating cytokines is demonstrated.

Endothelial progenitor cells (EPCs) are crucially involved in the neovascularization of ischemic tissues. This method describes the isolation of human EPCs from peripheral blood, as well as the identification of their migratory potential against serum samples of cardiac surgical patients.

从健康志愿者和它们的迁徙可能通过血清中的影响心脏手术后的内皮祖细胞的分离

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

制备和<em&gt;体外</em&gt;磁化的表征的miR-改性内皮细胞

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

Isolation and Profiling of Human Primary Mesenteric Arterial Endothelial Cells at the Transcriptome Level

Endothelial cells (ECs) are crucial for vascular and whole-body function through their dynamic response to environmental cues. Elucidating the transcriptome and epigenome of ECs is paramount to understanding their roles in development, health, and disease, but is limited in the availability of isolated primary cells. Recent technologies have enabled the high-throughput profiling of EC transcriptome and epigenome, leading to the identification of previously unknown EC cell subpopulations and developmental trajectories. While EC cultures are a useful tool in the exploration of EC function and dysfunction, the culture conditions and multiple passages can introduce external variables that alter the properties of native EC, including morphology, epigenetic state, and gene expression program. To overcome this limitation, the present paper demonstrates a method of isolating human primary ECs from donor mesenteric arteries aiming to capture their native state. ECs in the intimal layer are dissociated mechanically and biochemically with the use of particular enzymes. The resultant cells can be directly used for bulk RNA or single-cell RNA-sequencing or plated for culture. In addition, a workflow is described for the preparation of human arterial tissue for spatial transcriptomics, specifically for a commercially available platform, although this method is also suitable for other spatial transcriptome profiling techniques. This methodology can be applied to different vessels collected from a variety of donors in health or disease states to gain insights into EC transcriptional and epigenetic regulation, a pivotal aspect of endothelial cell biology.

The protocol describes the isolation, culture, and profiling of endothelial cells from human mesenteric artery. Additionally, a method is provided to prepare human artery for spatial transcriptomics. Proteomics, transcriptomics, and functional assays can be performed on isolated cells. This protocol can be repurposed for any medium- or large-size artery.

人淋巴管内皮细胞中的多参​​数荧光激活细胞分选分离

人淋巴管内皮细胞中的多参数荧光激活细胞分选分离