尤德博士是EndGenitor科技公司的顾问和Rimedion科技公司的董事会成员

试剂和溶液肌电图-2媒体（龙沙，猫。编号：CC-3162包含的EBM-2培养基和临时股东大会-2 SingleQuot的套件补充，生长因子） EBM-2（龙沙，货号CC-3156），辅以整个SingleQuot套件补充和生长因子（龙沙，猫。CC-4176号），胎牛血清（FBS）和10％（V / V） 1％（V / V），青霉素（1万U /毫升）/链霉素（10,000微克/毫升）/两性霉素（25微克/毫升）。在4°C存储长达1个月建议的临时股东大会，2册，使用ECFC文化500μL/ 24孔板，2毫升/ 6孔板，5ml/25-cm 2瓶，10 ml/75-cm 2瓶，除非中另有规定的协议。 I型胶原溶液在495毫升无菌蒸馏水（0.02列印终浓度）稀释的冰醋酸（17.4N）0.575毫升。无菌过滤器的稀醋酸，用0.22微米VACuum过滤系统。添加25毫克鼠尾I型胶原的稀醋酸至终浓度为50微克/毫升。胶原蛋白的添加量会有所不同，具体取决于对胶原股票浓度。在4°C储存长达一个月 I型胶原涂层的组织培养表面的制备 I型胶原在每一个6孔培养板（使用300μL/ 24孔板，3ml/25-cm 2瓶，和8 ml/75-cm的2瓶）以及解决方案的地方1毫升。隔夜孵育1小时，在37°C。删除我的胶原蛋白溶液洗表面2次，每次用PBS（μL/ 24孔板，2毫升/孔以及使用6孔板，5ml/25-cm 2瓶，10 ml/75 500厘米2瓶）。立即用板，细胞培养。 流式细胞仪染色缓冲区磷酸盐缓冲液（PBS）的2％（V / V）胎牛血清（FBS），补充。商店üp到2个星期，在4°C。 A. ECFC的产物，克隆和扩展<ol><li>收集与肝素（10 U肝素/毫升血液）或EDTA作为一种抗凝血剂在室温下的实验室和运输的CB，并立即（分娩婴儿的2小时内）过程中，样品为单核细胞（MNC）的隔离。 </li><li> CB到每50毫升锥形离心管分装15毫升每个管的CB和移液器多次混合，并添加20毫升的PBS。到20毫升注射器吸取15毫升-Paque Ficoll分离和附加20G针或混合套管。将混合稀释血液的管底部套管的尖端，仔细与其本身15毫升Ficoll分离Pague。离心管，740 XG 30分钟，在室温下，没有减速刹车设置。 </li><li>使用移液管，删除低密度跨国公司朦胧的Paque的Ficoll分离和稀释血浆之间的接口层。免除跨国公司到50毫升锥形管续毫升10 aining的临时股东大会-2介质。离心515 XG跨国公司10分钟，在室温下，具有很高的减速刹车设置。 </li><li>小心吸弃上清。 EGM-2的两倍（10分钟）515 XG洗颗粒细胞和重新挂起跨国公司在EGM-2在1.25×10 7细胞/毫升。准备胶原蛋白，我涂的6孔细胞培养板，加入1毫升鼠尾I型胶原（50微克/毫升），每口井和孵化板一夜之间。吸取4毫升，这个跨国公司悬挂到每口井的细胞，并将其放置在37℃，5％ 二氧化碳培养箱孵化。 A.1.5）（1天）24小时后，慢慢地从井的花中（请勿打扰松散贴壁细胞），用吸管，慢慢加入4毫升EGM-2井，并返回板的孵化器。第2天，刷新慢慢用吸管以及从中期（请勿打扰松散贴壁细胞）和EGM-2 4毫升，每孔加入培养基。重复中的变化每天联合国此后直到第7天，隔日出现克隆直到ECFC殖民地。典型的殖民地之间出现5天，14天7天（ 图1）。 </li><li>可视化与鹅卵石形态的典型使用倒置显微镜ECFC殖民地（放大倍数10倍）和井底部的标志殖民地边界标记，以指示各殖民地的地位。 </li><li>第14天，这些主要的菌落洗2次，PBS和收获个别殖民地使用克隆气瓶和刚够TrypLE快递覆盖汽缸内的细胞。吸气的PBS最后一次洗涤后，每个菌落周围放置无菌凝胶涂层克隆缸，按坚决反对使用无菌镊子板。加入2-3滴温暖的TrypLE Express到每个克隆缸和孵育3-5分钟，直到汽缸内的细胞开始分离。添加到气缸中心约250μL临时股东大会，2个中等和移液器上下到generate单细胞悬液。单细胞悬液，分别从每个克隆缸转移到一个单独的微离心管。 </li><li>面积约250μL临时股东大会，2个中等收集所有剩余的细胞，并从每个气缸转移到各自的微离心管洗洗净气缸内的2-3倍。台式离心机上离心5分钟的高速（≤300 XG）的细胞。 </li><li>去除上清，重新悬浮细胞沉淀在1.5毫升新鲜临时股东大会，2个中等。转移到一个24孔培养板，用500μL的鼠尾胶原蛋白I（50微克/毫升）预涂从每管细胞悬液（含从个别殖民地的所有细胞）。 </li><li>扩大与媒体的孵化器内的地方改变隔日。 </li><li>当细胞接近80-90％汇合，删除用过的介质，然后洗细胞2次，用PBS，每孔加500μLTrypLE快递中等24孔培养板。放置3-5分钟，直到细胞开始围捕和分离板的孵化器内。每孔加入1毫升EGM-2（定义10％胎牛血清）经洗涤收集细胞，并将它们传送到15毫升管。 1毫升临时股东大会2中收集所有剩余的细胞，从每口井转移到各自的15毫升管洗，洗井。离心5分钟300 XG的细胞。 </li><li>删除1毫升新鲜的临时股东大会-2中定义的10％胎牛血清培养基重新悬浮细胞沉淀。获得活细胞计数的使用血球和台盼蓝排斥等分。 </li><li>扩大与媒体改变隔日播种到我预涂胶原组织在临时股东大会，2媒体文化表面大约5000个细胞/厘米2的细胞，直至细胞再次接近80-90％汇合前子培养。 </li></ol> B. 在-体外 ECFCs表型特征：内皮细胞表面抗原表达D为单细胞克隆增殖潜能检测<ol><li>为内皮细胞表面抗原的表达，分离TrypLE快递的使用为ECFC克隆和扩展描述的方法收集细胞的细胞。 </li><li>在流式细胞仪染色缓冲区重悬细胞（10×10 6细胞/1毫升流式细胞仪染色缓冲），然后加入Fc受体阻断剂（20μL/ 10×10 6个细胞），混合冰轻轻地点为10分钟。 </li><li>等分每个内皮细胞表面抗原和同型对照这个微型离心管中的细胞悬液100μL。添加适量的荧光标记单克隆抗体，承认人类内皮细胞抗原（CD31，CD144，CD146和CD105）或造血抗原（CD45和CD14的），避光30分钟离开冰注意：1×10 6细胞不需要为每个测试。作者通常准备0.5至 1×10 5细胞/管，获得2×10 4分析d个事件。此外， 每个测试使用的抗体量要遵循制造商的建议。可能需要一些抗体滴定，以确定最佳的抗体浓度。所有的作者都进行染色是单一颜色的染色。 </li><li>冰上孵育30分钟后，高速离心5分钟的台式离心机上每个试管中，然后取出上清，重新悬浮颗粒细胞在流式细胞仪缓冲。 </li><li>流式细胞仪分析的样本，以确定积极或消极的，每个抗原染色细胞的百分比。 ECFCs染色均匀CD31，CD144，CD146的阳性，但CD45和CD14阴性相比，相应的同型对照。 </li><li>研究克隆的增殖潜能的单细胞检测，分离与TrypLE快递早日通过（2-3）细胞，收集细胞，使用方法类似，为ECFC克隆和扩展。 </li><li>感染这些细胞与增强型绿色荧光蛋白（EGFP）的表达慢病毒16和收集细胞表达EGFP的荧光仪注意：慢工作被认为是生物危害，并应遵守所有的生物安全防范措施。 </li><li>文化，他们形容为培养ECFC节ECFC扩张。 </li><li>准备我（50微克/毫升），每孔加入50μL的鼠尾型胶原Ⅰ型胶原涂层的96孔板和孵育过夜板块。收集使用TrypLE快递的绿色荧光蛋白+ ECFCs和悬浮在EGM-2培养基终浓度〜10 6细胞/毫升。 </li><li> 20个细胞/秒的低流量使用流式细胞仪华帝（流式细胞迪克森）或其他类似的分拣排序一个单一的绿色荧光蛋白+ ECFC每一个96孔板和调整终体积200μLEGM-2％。使用倒置荧光显微镜，以确保每口井只有一个细胞。 吴国良伊利，单细胞可以排序基于FSC和SSC和Sytox核染色法可用于细胞评分和定量的殖民地。 </li><li>两家媒体上的第5天，10天进行的变化（200μL临时股东大会的2使用多通道移液器）在一个为期两周的孵育板。文化第14天，用100μLPBS固定细胞，用100微升4％多聚甲醛30分钟，每口井。对于那些不表达EGFP的ECFCs，添加Sytox试剂，绿色荧光核染料，多聚甲醛固定，并在4°C过夜孵育。 </li><li>使用荧光显微镜检查每口井的定量扩大，从一个单细胞培养14天的内皮细胞的数量。对于定量分析，井与2个或更多的内皮细胞为阳性（增殖）的得分和总内皮细胞计数检查他们进一步用荧光显微镜视觉计数。 </li><li>为了显示t他获得数据，井与内皮细胞数目：2到50 51-2000年和2001年或以上，被视为：内皮细胞簇，低增殖潜力（LPP）ECFCs，高增殖潜力（HPP） - ECFCs分别。 ECFCs表现在单细胞水平，培养14天，引起内皮集群，LPPS，和高潜质完整克隆增殖潜能的层次。 </li></ol> C. 在体内的功能特性ECFCs： 在体内血管形成实验检查的ECFC的血管生成的潜力<ol><li> cellularized凝胶植入准备通过计算总体积（ml）（括号中的终浓度）以下的凝胶材料，每年需要投1毫升凝胶（稍后将平分产生2种植体）：胎牛血清（10 ％）的EBM-2 10:1（调整最终体积为1毫升），碳酸氢钠（1.5毫克/毫升），氢氧化钠（调节pH值至7.4），肝素钠（25毫米），fibronectiN（100微克/毫升），I型胶原（1.5毫克/毫升）。 </li><li>凝胶材料的计算后，分离TrypLE快递细胞，收集细胞，使用方法类似，为ECFC克隆和扩展。取得的等分使用血球和台盼蓝活细胞计数。 50毫升锥形管和颗粒细胞在515 XG，室温离心转移到2.4万个活细胞。同时，准备加入冰冷的50毫升锥形管（在他们的相同顺序列出）肝素钠的计算量，碳酸氢钠的EBM-2 10:1，血糖，纤维连接蛋白，胶原蛋白凝胶基质的解决方案。调匀，并同时保持对冰的解决方案，用NaOH调整pH值至7.4。 </li><li>丢弃从离心后的细胞上清液重新悬浮在360μL温暖的临时股东大会2沉淀，并加入840μl凝胶基质溶液的pH值调整，使最终体积凝胶植入1毫升。凝胶基质溶液混合成细胞缓慢，直到胶溶液中的细胞被彻底暂停。 </li><li>这1的毫升cellularized凝胶暂停转让的12孔培养板孵育20-30分钟，直到凝胶聚合。轻轻覆盖2毫升温水临时股东大会，2凝胶和孵育过夜。 </li><li>立即植入前，平分到两个相等的块，用虹膜剪刀过夜凝胶的凝胶块，并返回相同的文化，以及含有EGM-2中等。 </li><li>由行政异氟醚麻醉使用动物的手术设施，以稳重的动物（5-6周龄NOD / SCID小鼠）。剃腹部下部，并彻底清洗手术部位用酒精垫和优碘或其他防腐剂。然后用窗帘隔离区域为的IACUC和体制的指引。 </li><li>使用无菌尖锐的虹膜剪到约5毫米的切口在腹部下腹，露出皮肤和腹部肌肉之间的皮下空间。通过腹部肌肉的真皮层15-20毫米宽的口袋里，上级领导到上腹部象限执行钝性解剖。重复类似的程序来建立类似的另一面相同的鼠标腹部开放口袋。 </li><li>起重只是尾切口真皮层凝胶腹袋的一侧半片和另一半凝胶片插入另一侧腹部口袋。 </li><li>凝胶插入后，关闭每个切口2-3用5-0聚丙烯缝线切割针针。适当的标记笼卡和执行体制和协议的要求，为手术后的监测和镇痛，并允许在这些植入凝胶14天的细胞来生成新的血管。 </li><li>最后，收获凝胶植入物通常植入后14天内完成后实施安乐死的鼠标。 </li><li>擦拭腹部和用酒精垫原切口线切开腹部皮肤尾。仔细，解剖切除皮瓣的皮肤尾鳍可能位置凝胶植入。海关植入切割圆周周围的凝胶，然后在锌固定液的地方（BD Biosciences公司，以获得最佳效果按照制造商的建议），并允许他们在室温下1-2小时内修复。 </li><li>准备石蜡包埋凝胶，使用标准的组织化学协议，并准备执行苏木精和曙红，抗人CD31的或抗鼠CD31的可视化血管内凝胶染色玻片上的5微米的部分。 ECFCs接受从头血管生成人性化的船只 在cellularized凝胶植入物插入到免疫缺陷小鼠14天，4,8,11。 </li></ol> D.代表结果使用这ECFC推导技术，我们已经看到了增长的主要殖民地形式ATION作为早在5天（ 图1）。出增长ECFC殖民地表现出典型的鹅卵石的外观，并引起了克隆后殖民地皮卡长期扩张后&gt; 40人口倍增。扩大殖民地表示内皮细胞的抗原，但不表达造血抗原（ 图2）。重要的是，他们在单细胞水平（ 图3）显示完整的克隆增殖潜能的层次。此外，ECFCs形成人性化的血液灌注时，移植到免疫缺陷小鼠4，6，8，11（ 图4）与宿主红细胞的血管。 <img alt="图1。" src="/files/ftp_upload/3872/3872fig1.jpg" /> 图1。从脐带血内皮细胞集落生长的单核细胞（MNCs）的隔离形成 Ficoll分离Pague密度梯度分离脐血细胞在细胞培养跨国公司。跨国公司的形式白膜层（ECFCs）。文化跨国公司在鼠尾胶原分离白膜层涂层板我在ECFC殖民地的产物，在5至14天的结果。的产物ECFC的殖民地（由箭头头表示）显示鹅卵石形态7。 <img alt="图2。" src="/files/ftp_upload/3872/3872fig2.jpg" /> 图2。 在体外代表内皮细胞和造血干细胞表面抗原表达的表型评估。脊髓血源性ECFCs的免疫透露，ECFCs表示内皮细胞抗原CD31的表达CD34，CD144，CD146的，FLT-1，FLK-1，FLT-4，NRP2不表达造血抗原，CD45，CD14，CD11b的cKit，CXCR4的或AC133 7，8。 <img alt="图3。" src="/files/ftp_upload/3872/3872fig3.jpg" /> 图3。代表在体外克隆和增殖潜能的CB衍生ECFCs定量 。 （一）脐带血衍生ECFCs显示了从殖民地&gt; 2001年2-50细胞群的菌落层次克隆增殖潜能。 （二）殖民地（殖民地，Sytox，质量更好的图片核绿色荧光染料染色）的层次结构的显微照片后脐血衍生ECFCs在14天的培养，单细胞水平。比例尺表示100μm的7，8。 <img alt="图4。" src="/files/ftp_upload/3872/3872fig4.jpg" /> 图4。 代表体内脐血源性ECFCs的功能特性 。一）H＆E染色脊髓血源性ECFC含有cellularized凝胶植入物显示微血管（充满主机红细胞的形成）在胶原纤维连接蛋白凝胶植入后14天。 b）抗人CD31的染色（褐色的染色），进一步证实了这些船只的人类起源。

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vessel+wall-derived+endothelial+cells+rapidly+proliferate+because+they+contain+a+complete+hierarchy+of+endothelial+progenitor+cells.">Vessel wall-derived endothelial cells rapidly proliferate because they contain a complete hierarchy of endothelial progenitor cells.</a> Blood. 105, 2783-276 (2005).</li><li>Ingram, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+novel+hierarchy+of+endothelial+progenitor+cells+using+human+peripheral+and+umbilical+cord+blood.">Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood.</a> Blood. 104, 2752-2760 (2004).</li><li>Yoder, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redefining+endothelial+progenitor+cells+via+clonal+analysis+and+hematopoietic+stem/progenitor+cell+principals.">Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals.</a> Blood. 109, 1801-1809 (2007).</li><li>Reinisch, A., Strunk, D. <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+Animal+Serum+Free+Expansion+of+Human+Umbilical+Cord+Derived+Mesenchymal+Stromal+Cells+(MSCs)+and+Endothelial+Colony+Forming+Progenitor+Cells+(ECFCs.">Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs.</a> J. Vis. Exp. (32), e1525 (2009).</li><li>Au, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+in+vivo+potential+of+endothelial+progenitor+cells+from+human+umbilical+cord+blood+and+adult+peripheral+blood+to+form+functional+long-lasting+vessels.">Differential in vivo potential of endothelial progenitor cells from human umbilical cord blood and adult peripheral blood to form functional long-lasting vessels.</a> Blood. 111, 1302-135 (2008).</li><li>Critser, P. J., Kreger, S. T., Voytik-Harbin, S. L., Yoder, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+matrix+physical+properties+modulate+endothelial+colony+forming+cell-derived+vessels+in+vivo.">Collagen matrix physical properties modulate endothelial colony forming cell-derived vessels in vivo.</a> Microvasc. Res. 80, 23-30 (2010).</li><li>Melero-Martin, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+robust+and+functional+vascular+networks+in+vivo+with+human+adult+and+cord+blood-derived+progenitor+cells.">Engineering robust and functional vascular networks in vivo with human adult and cord blood-derived progenitor cells.</a> Circ. Res. 103, 194-202 (2008).</li><li>Melero-Martin, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+vasculogenic+potential+of+human+blood-derived+endothelial+progenitor+cells.">In vivo vasculogenic potential of human blood-derived endothelial progenitor cells.</a> Blood. 109, 4761-4768 (2007).</li><li>Hofmann, N. A., Reinisch, A., Strunk, D. <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+Large+Scale+Expansion+of+Adult+Human+Endothelial+Colony+Forming+Progenitor+Cells.">Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells.</a> J. Vis. Exp. (32), e1524 (2009).</li><li>Lin, Y., Weisdorf, D. J., Solovey, A., Hebbel, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origins+of+circulating+endothelial+cells+and+endothelial+outgrowth+from+blood.">Origins of circulating endothelial cells and endothelial outgrowth from blood.</a> J. Clin. Invest. 105, 71-77 (2000).</li><li>Witting, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+Large+Volume+Lentiviral+Vector+Production+Using+Flow+Electroporation.">Efficient Large Volume Lentiviral Vector Production Using Flow Electroporation.</a> Hum. Gene. Ther. , (2011).</li><li>Yoon, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+neovascularization+by+mixed+transplantation+of+early+endothelial+progenitor+cells+and+late+outgrowth+endothelial+cells:+the+role+of+angiogenic+cytokines+and+matrix+metalloproteinases.">Synergistic neovascularization by mixed transplantation of early endothelial progenitor cells and late outgrowth endothelial cells: the role of angiogenic cytokines and matrix metalloproteinases.</a> Circulation. , 112-1618 (2005).</li><li>Dubois, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+progenitor+cell+populations+on+left+ventricular+remodeling+and+myocardial+neovascularization+after+myocardial+infarction.">Differential effects of progenitor cell populations on left ventricular remodeling and myocardial neovascularization after myocardial infarction.</a> J. Am. Coll. Cardiol. 55, 2232-2243 (2010).</li><li>Medina, R. J., O'Neill, C. L., Humphreys, M. W., Gardiner, T. A., Stitt, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outgrowth+endothelial+cells:+characterization+and+their+potential+for+reversing+ischemic+retinopathy.">Outgrowth endothelial cells: characterization and their potential for reversing ischemic retinopathy.</a> Invest. Ophthalmol. Vis. Sci. 51, 5906-5913 (2010).</li></ol>

没有利益冲突的声明。

假定内皮祖细胞的表型和功能特性，是重要的，以确定是否真正是能够克隆，并连续在文化再镀的的善意 ECFCs和耐用和功能， 在体内植入血管引起。脐血丰富与ECFCs这些循环细胞老化或疾病的10下降浓度。最近的研究表明，ECFC可能在血管修复或再生血管损伤，心肌梗死，或视网膜病变17-19的情况下发挥重要作用。 在这里，我们描述了简单?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>肝素钠注射液，USP </td><td> APP的制药</td><td> 504031 </td></tr><tr><td> Ficoll分离，Pague </td><td> Amersham Biosciences公司</td><td> 17-1440-03 </td></tr><tr><td>混合套管</td><td>马士基医学</td><td> 500.11.012 </td></tr><tr><td> EGM-2 </td><td>龙沙</td><td> CC-3162 </td></tr><tr><td>定义胎牛血清</td><td>胎牛血清</td><td> SH30070.03 </td></tr><tr><td> TrypLE快递</td><td> Gibco公司</td><td> 12605 </td></tr><tr><td>大鼠I型胶原</td><td> BD公司</td><td> 354236 </td></tr><tr><td>基底膜</td><td> BD公司</td><td> 356234 </td></tr><tr><td> FCR座</td><td>美天旎生物技术</td><td> 130-059-901 </td></tr><tr><td> hCD31，FITC标记conjuga特德</td><td>屋宇署Pharmingen公司</td><td> 555445 </td></tr><tr><td> hCD45，FITC标记</td><td>屋宇署Pharmingen公司</td><td> 555482 </td></tr><tr><td> hCD14，FITC标记</td><td>屋宇署Pharmingen公司</td><td> 555397 </td></tr><tr><td> hCD144，体育共轭</td><td> eBioscience </td><td> 12-1449-80 </td></tr><tr><td> hCD146，体育共轭</td><td>屋宇署Pharmingen公司</td><td> 550315 </td></tr><tr><td> hCD105，体育共轭</td><td> Invitrogen公司</td><td> MHCD10504 </td></tr><tr><td> 1，K抗体，IgG的女士FITC标记</td><td>屋宇署Pharmingen公司</td><td> 555748 </td></tr><tr><td> 1，K抗体，IgG的女士聚乙烯共轭</td><td>屋宇署Pharmingen公司</td><td> 559320 </td></tr><tr><td>女士抗体2A，K抗体，FITC标记</td><td>屋宇署Pharmingen公司</td><td> 555573 </td></tr><tr><td>抗人类CD31的</td><td> Dako公司</td><td>克隆JC70 / </td></tr><tr><td>抗-谅解备忘录éCD31的</td><td>屋宇署Pharmingen公司</td><td> 553370 </td></tr><tr><td> 0.22微米的真空过滤系统</td><td> Millipore公司</td><td> SCGPU05RE </td></tr><tr><td>冰醋酸，17.4N </td><td>费舍尔</td><td> A38-500 </td></tr><tr><td>抗生素，抗霉菌</td><td> Invitrogen公司</td><td> 15240-062 </td></tr><tr><td>胎牛血清（FBS）， </td><td>胎牛血清</td><td> SH30070.03 </td></tr><tr><td>免疫组化锌定影液</td><td> BD公司</td><td> 550523 </td></tr><tr><td> sytox绿色试剂</td><td> Invitrogen公司</td><td> S33025 </td></tr><tr><td>克隆缸，无菌</td><td> Fisher Scientific则</td><td> 07-907-10 </td></tr></tbody></table>

phenotypic and functional characterization of endothelial colony forming cells derived from human umbilical cord blood

新的血液通过血管生成，血管，小动脉血管的形成长期的看法，最近已检讨1。循环内皮祖细胞（EPCs）的存在，首次发现麻原等。成人外周血中，在1997年2将注入新的假说和血管再生和修复策略。内皮祖细胞是罕见的，但正常的血液循环的组成部分，血管形成或血管重塑的网站，并在很大程度上促进通过旁分泌刺激现有的血管壁细胞中的3任产后的血管生成，血管生成，或小动脉。没有特定的标记，以确定一个EPC已经确定，目前该领域的国家是理解，许多类型的细胞，包括血管形成的造血干细胞和祖细胞，循环血管细胞，Tie2的+单核细胞 ，髓系祖CELLS，肿瘤相关巨噬细胞和M2激活巨噬细胞在多种临床前动物模型系统和人类受试者在许多疾病状态4，5，刺激血管生成过程的参与。内皮细胞集落形成细胞（ECFCs）是可行的罕见循环内皮细胞，特点是强大的克隆增殖潜能，二级和三级集落形成能力后replating，为6-8免疫缺陷小鼠在移植后体内血管形成的内在能力。虽然ECFCs已成功地从外周血中分离的健康成人受试者，健康新生儿脐带血（CB）的，和血管壁无数人的动脉和静脉血管6-9，CB拥有ECFCs 7的频率最高，显示最强大的克隆增殖潜能和耐用，功能血管的形式在体内 8，10-13。而推导从成人外周血ECFC已提交14，15，在这里，我们描述的推导方法，克隆，扩展，并在体外以及在从人脐CB ECFCs 体内表征。

Watch this Scientific Journal Video about Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood at JoVE.com

Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood

具有强大的克隆增殖潜能内皮细胞集落形成细胞（ECFCs）循环内皮细胞，显示内在<em&gt;在体内</em&gt;血管形成能力。会CB衍生内皮细胞生长的表型和功能特性，是重要的识别和分离<em&gt;善意</em&gt; ECFCs修复受损的组织，为潜在的临床应用。

来自人类脐带血内皮细胞集落形成细胞的表型和功能特性

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of ...

phenotypic-functional-characterization-endothelial-colony-forming

Research

JoVE Journal

Biology

16.2K 次观看.  Indiana University School of Medicine. 具有强大的克隆增殖潜能内皮细胞集落形成细胞（ECFCs）循环内皮细胞，显示内在在体内血管形成能力。会CB衍生内皮细胞生长的表型和功能特性，是重要的识别和分离善意 ECFCs修复受损的组织，为潜在的临床应用。

视频: Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood

Isolation of Human Umbilical Vein Endothelial Cells (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, lumen formation, anastomosis, and formation of a new basement membrane. Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels often do not resemble vessels in vivo. Here we demonstrate an optimized in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts. This model recapitulates all of the key early stages of angiogenesis, and importantly the vessels display patent intercellular lumens surrounded by polarized EC. Vessels can be easily observed by phase-contrast and time-lapse microscopy, and recovered in pure form for downstream applications.

Isolation of Human Umbilical Vein Endothelial Cells HUVEC

This video protocol illustrates the isolation and culture of human umbilical vein endothelial cells (HUVEC) from human umbilical cord. Once isolated these cells can be used for in vitro angiogenesis assays like the Optimized Fibrin Gel Bead Assay also demonstrated by the Hughes lab.

分离人脐静脉内皮细胞（HUVEC）

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps ...

科研

生物学

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

成人内皮细胞集落形成祖细胞的分离和大型规模扩张

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

隔离和动物血清自由膨胀的人脐血源性间质基质细胞（MSCs），内皮细胞集落形成祖细胞（ECFCs）

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Isolation and Enrichment of Rat Mesenchymal Stem Cells (MSCs) and Separation of Single-colony Derived MSCs

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and can be easily separated from the hematopoietic stem cells (HSCs) due to their plastic adherence. This protocol describes how to isolate MSCs from rat femurs and tibias. The isolated cells were further enriched against two MSCs surface markers CD54 and CD90 by magnetic cell sorting. Expression of surface markers CD54 and CD90 were then confirmed by flow cytometry analysis. HSC marker CD45 was also included to check if the sorted MSCs were depleted of HSCs. MSCs are naturally quite heterogeneous. There are subpopulations of cells that have different shapes, proliferation and differentiation abilities. These subpopulations all express the known MSCs markers and no unique marker has yet been identified for the different subpopulations. Therefore, an alternative approach to separate out the different subpopulations is using cloning cylinders to separate out single-colony derived cells. The cells derived from the single-colonies can then be cultured and evaluated separately.

Isolation and Enrichment of Rat Mesenchymal Stem Cells MSCs and Separation of Single-colony Derived MSCs

Rat MSCs were isolated from femurs and tibias and then enriched by magnetic cell sorting. Sorted cells were confirmed for the expression of surface markers by flow cytometry. These cells were also cultured at clonal density to form single colonies and then these colonies were separated by cloning cylinders.

分离和富集大鼠间质干细胞（MSCS）及衍生的干细胞分离单细胞集落

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and ...

Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded and their collection does not imply any added risk to the newborn or mother s health. Moreover no ethical concerns are raised. The UC is composed by one vein and two arteries from which both endothelial cells (ECs) 1 and smooth muscle cells (SMCs) 2, two of the main cellular components of blood vessels, can be isolated. In this project the SMCs were obtained after enzymatic treatment of the UC arteries accordingly the experimental procedure previously described by Jaffe et al 3. After cell isolation they were kept in t-flash with DMEM-F12 supplemented with 5% of fetal bovine serum and were cultured for several passages. Cells maintained their morphological and other phenotypic characteristics in the different generations. The aim of this study was to isolate smooth muscle cells in order to use them as models for future assays with constrictor drugs, isolate and structurally characterize L-type calcium channels, to study cellular and molecular aspects of the vascular function 4 and to use them in tissue engineering.

Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC

The umbilical cords are used to isolate smooth muscle cells by different ways. In this work we used the enzymatic treatment to isolated smooth muscle cells.

人脐动脉平滑肌细胞的分离（HUASMC）

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

从新生小鼠的肺血管内皮细胞的分离和培养

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

一个定量测定胰岛素表达细胞集落形成先觉

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

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

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

从人类Lipoaspirates脂肪来源干细胞的手动隔离

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

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

从人骨骼肌隔离血管源性多能前体

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

细胞内钙的双光子成像<sup&gt; 2+</sup&gt;的离体大鼠主动脉的处理和一氧化氮的内皮细胞和平滑肌细胞

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...