这项工作得到了中国国家自然科学基金（31530045和YAZ 31371500，QCY 31401245），中国科学技术部（2014CB964800），中国科学院（XDB19000000〜YAZ）和中国科学院细胞生物学学会（QCY早期职业奖学金）。

实验程序由中国科学院上海生物化学与细胞生物学研究所动物保护与使用委员会批准，经批准协议SIBCB-NAF-15-002-S335-003批准。 从小鼠乳腺中分离VESCs <ol><li> 乳腺收获和组织消化。 <ol><li>使用8周龄的Actin-GFP成熟的原始记录小鼠，重量范围在20至25g之间，作为组织供体。 注意:源自供体的细胞可以通过GFP表达轻易追踪。 </li><li>在RPMI1640中制备由5％胎牛血清（FBS），1％pen / strep和25mM HEPES组成的组织消化基培养基。通过0.22μm过滤器过滤混合物，在水浴中灭菌并预热至37°C。 </li><li>在CO 2室中牺牲小鼠。通过首先制作垂直皮肤解剖第四对腹股沟乳腺使用手术刀（或手术剪刀）在下腹部中线切割。将皮肤轻轻剥去一侧，用镊子露出腹股沟乳腺。从两侧获得第四个腹股沟乳腺，并使用剪刀将其切成6厘米培养皿中的细小块。 <ol><li>测量切碎的组织的总重量并将其置于50mL管中。 注意:确保每件产生的组织碎片尺寸小于1 mm 3 。典型的产量是每只动物约5,000个VESCs。 </li></ol></li><li>以每1g组织切片以10 mL的体积在50 mL加盖的离心管中制备消化底物培养基。以每10毫升消化基培养基300个单位的浓度添加3型胶原酶，构成消化缓冲液。均匀混合，并将其加入组织切片。 </li><li>将50 mL离心管水平放置在摇摆平台上，将速度设置为100 rpm，温度为37°C。激荡乳腺组织2 h。每20分钟检查并手动摇动管子，以确保正确消化。 注意:FACS之前组织消化的目的是从固体组织制备单细胞溶液。因此，消化结束时，内容物应该是没有可见组织块的厚溶液。 </li></ol></li><li> 单细胞溶液制备。 <ol><li>消化后，用预先加热的无菌PBS将50mL离心管加满，并将试管均匀混合。以200×g离心管5分钟，得到细胞沉淀。 </li><li>取出上清液，轻轻摇动管底部，松开细胞沉淀。加入3 mL红细胞裂解缓冲液（见材料表），并在组织培养箱中室温（RT）孵育5 min，以消除红细胞。 </li><li>加入10mL无菌PBS，并将管反向混合均匀。将组织内容物以200 xg离心5分钟，然后离心上清。加入3mL预热的0.05％胰蛋白酶并将管储存在37℃5分钟以进一步消化剩余的组织。 </li><li>加入3mL预热的IMDM，然后加入100μLDNA酶I溶液（0.25mg在1mL PBS中稀释的DNA酶I）。将管储存在37℃以分解DNA细丝。将组织混合物通过70μm的细胞过滤器以获得细胞溶液。用2 mL PBS + 5％FBS冲洗过滤器两次，以中和酶消化。 </li><li>以200×g离心细胞混合物5分钟以沉淀细胞。弃去上清液，将细胞沉淀重悬于1 mL PBS + 5％FBS中进行抗体染色。 </li></ol></li><li> 基于FACS的Procr + VESC隔离。 <ol><li>将从供体乳腺组织获得的细胞混合物按公开的常规方案进行抗体染色8 。 注意:使用以下抗体:抗小鼠血清谱系FITC，抗小鼠CD31（PECAM1）-PECY7，抗小鼠CD105-APC（均以1μg/ mL浓度使用），抗小鼠CD201（Procr） - 生物素（浓度为2.5 μg/ mL）和链霉抗生物素-v450（以0.5μg/ mL的浓度使用）。 <ol><li>抗体染色后，用PBS + 5％FBS补充细胞，然后以200g离心5分钟。弃去上清液，并用1 mL PBS + 5％FBS重悬细胞。通过40μm的过滤器过滤。放在冰上直到FACS分选。 </li></ol></li><li>继续进行基于FACS的排序以分离Procr + VESCs。 <ol><li>分析未染色样品和每个单色控件以确定每种颜色的电压。计算补偿参数以避免自动荧光或背景染色。设置模板以进行适当的门控和排序，以获得所需的细胞群体。 </li><li>建立FACS分类的层次结构，首先消除碎片所有事件，然后通过触发宽度丢弃双峰或粘合细胞簇。门出血谱系细胞，然后使用CD31 +和CD105 +来定义内皮细胞群。 注意:与FMO对照相比，从CD31 + CD105 + ECs分离出Procr +细胞。代表性的FACS分析图如图1所示。 FACS分离的VESC群体应暂停在IMDM + 50％FBS中并储存在冰上直到进一步处理。 </li></ol></li></ol></li></ol> 2.使用乳腺脂肪垫进行体内 VESC血管形成测定<ol><li> 准备移植的主要ECs。 <ol><li>用10mL PBS + 5％FBS补充FACS分选的细胞，然后以200xg离心5分钟以将细胞沉降到底部。 </li><li>仔细吸取液体，使用血细胞计数器计数细胞数，并用基底膜基质混合物重悬细胞沉淀50％基底膜基质和含有20％FBS的PBS，补充有100ng / mL rmVEGF和60U / mL肝素）达到15,000个细胞/15μL的工作浓度。 注意:基底膜基质混合物应始终存放在冰上。 </li><li>连续稀释分选和计数的细胞，以达到1.5 mL微量管中所需的输入浓度，确保最终注射体积总计不超过15μL。在每个管中加入0.1％台盼蓝，以便在移植过程中更好地显示内容。彻底移液以混合。将管存放在冰上直到使用。 注意:可以使用至少100个VESCs来实现成功的血管生成。 </li></ol></li><li> 移植原发性ECs到乳腺脂肪垫。 <ol><li>消毒手术器械和台面。如果在多只动物上使用相同的仪器，则可以使用珠灭菌器进行消毒。 </li><li>麻醉3周龄的BALB / c裸麦克风e通过使用您机构的兽医和IACUC推荐的麻醉方案。将每只老鼠放在仰卧位，将其放回手术台上。用胶带固定肢体。 </li><li>将棉签浸入碘基杀菌溶液中。彻底擦拭鼠标的整个腹部区域，对手术前的皮肤表面进行消毒。 </li><li>用镊子握住并抬起鼠标的腹部皮肤。使用外科手术刀做一个小的，1厘米的垂直切口。做两个大约0.5 - 1厘米长的切口，每个朝向后腿，以实现倒置的Y形切口。 <ol><li>使用镊子和棉签轻轻地将皮肤从腹部分开，并将皮肤侧向穿过以暴露腹股沟乳腺。将鼠标放在立体镜下，并将放大倍数设置为2.0倍。确保调整焦点，使乳腺脂肪垫可以清晰地看到并操作。 </li></ol></li><li>放置一个27G 1/2"需要在冰上预冷。预先用基底膜基质混合物注入1-mL注射器以除去捕获在针附着区域中的可能的气泡。将15μL混合溶液吸出到微量离心管的盖上，然后将整个体积吸入注射针头的注射器中。 </li><li>握住乳腺脂肪垫在腹股沟淋巴结前面，使用细镊子，轻轻抬起以方便注射。将大约1/4的注射针插入脂肪垫。 <ol><li>从垫子上释放镊子，抓住并稳定注射针。缓慢注入溶液以防止液体渗漏。保持几秒钟，以确保细胞混合物固化，以尽量减少拔出针头时的渗漏。 </li></ol></li><li>用可吸收的缝合线关闭皮瓣。使用5-0非反应性单丝缝合缝合皮瓣，并用外科医生式结结封闭伤口，每个结近似相距0.3厘米。或者，用伤口缝合物关闭皮肤。 </li><li>将操作的小鼠放在单独的笼子中以进行恢复。将加热灯放在笼子上，以防止麻醉老鼠遭受低体温。将软纸和棉布放在笼子的底部，以保持老鼠在手术后温暖。 <ol><li>仔细观察小鼠，直到所有受试者完全清醒。在接下来的几天里，先将手术后止痛药应用于所有动物或按照设施兽医的指示。如果发生伤口感染，按照批准的动物方案应用抗生素。如果手术伤口用伤口钉关闭，则在手术后10天，当伤口完全关闭时，取下伤口夹。 </li></ol></li></ol></li></ol> 3.全套组织准备用于显微镜观察<ol><li> 组织收获 <ol><li>在植入后2-4周麻醉接受者BALB / c腹膜内注射2.5％麻醉剂的裸鼠，其剂量为每20g体重0.12mL，如步骤2.2.2所示。 注意:细胞移植后2〜4周可检测到阳性血管生长。 </li><li>用50μL/ 25g体重的尾静脉注射isolectin-647染料标记受体全身循环，重悬于50μL无菌PBS中。允许5 - 10分钟的休息时间。用CO 2牺牲小鼠。 </li><li>在步骤2.2.4之后解剖最初注射细胞的乳腺组织区域;使用手术剪刀。使用手术刀片，将组织切成6厘米培养皿中的大约3至4毫米3立方体。 </li></ol></li><li> 组织消化和抗体染色制备。 <ol><li>在装有10mL预先加热的消化基础培养基（步骤1.1.2）的15mL离心管中摘取组织片，补充有胶原酶III（300U / 10mL）。 </li><li>将15 mL离心管水平放置在摆动平台上，速度设置为100 rpm，温度设置为37°C约1 h，松开组织结构并除去过多的脂肪细胞组织。 注意:脂肪细胞倾向于产生自发荧光，这可能导致嘈杂的荧光背景。消化结束时，组织块应具有柔软，多云的外观。最佳消化时间取决于切碎的组织块的大小，因此应相应调整以获得最佳结果。由于组织脉管系统已经用荧光标记的Isolectin标记，因此所有以下程序都应进行防护。 </li><li>在室温下用PBS洗涤组织片10分钟。通过小心地将片放置在6厘米培养皿中，用4％PFA（pH 7.2 - 7.4）将其全部固定，以保持荧光。放置培养皿在摇摆平台上轻轻搅拌组织30分钟。 注意:PFA是致癌物质，应谨慎处理。 </li><li>取出剩余的PFA并用全固定洗涤缓冲液（PBS中0.1％吐温）洗涤固定组织三次，每次洗涤间隔10分钟。 </li></ol></li><li> 全身组织抗体染色。 <ol><li>在2 mL离心管中稀释抗体染色缓冲液中的一抗。在4℃下将组织与初级抗体孵育过夜。 注意:使用大鼠抗CD31（浓度:10μg/ mL）或大鼠抗VE-钙粘蛋白（CD144 / Cdh5;浓度:2.5μg/ mL）作为一抗。 CD31和VE-Cadherin都是用于识别内皮的细胞表面标记物。用500mM马来酸pH7.5制备5x抗体染色缓冲液; 750mM NaCl;和0.5％（v / v）吐温溶于PBS。在制备新鲜的1x工作溶液时，只加入适当的血清体积（5％）9 。 </li><li>取出抗体染色缓冲液，用洗涤缓冲液以10分钟的间隔洗涤整体组织三次。使用二级抗体（参见材料表 ）加上DAPI在摇床平台上培养4小时或在4℃过夜孵育组织。 注意:使用最终浓度为5μg/ mL的DAPI（4&#39;，6-二脒基-2-苯基吲哚）来显现核。 </li><li>用洗涤缓冲液洗涤整个安装的组织3次，每次10分钟。将组织块在80％甘油或80％蔗糖中孵育过夜，使组织脱水以更好地保存。 </li><li>将各个组织片放在玻璃载玻片上，并除去过量的脱水缓冲液。用安装介质覆盖组织，放置盖玻片，轻轻挤压组织进行设置。 </li><li>进行共聚焦显微镜图像采集。对于整体乳腺，采用40％的NA = 1.3石油对象获取图像五个。以1,024×1,024的光学分辨率和1.0-1.2μm的层间距获取Z-叠层的平铺扫描。 注意:为了捕获正确的荧光信号，滤波器分束器应设置为三分频（TD）488/553/638，增益设置为PMT增益600-800。应使用以下激发/发射源:mGFP，激发/发射最大值为488/509 nm;激发/发射最大值为532/588 nm的mTomato; Alexa-647，激发/发射最大值为594/665;和DAPI，激发/发射最大值为350/470。 </li></ol></li></ol>使用遗传修饰的鼠标模型进行谱系追踪<ol><li> 使用鼠标模型检测Procr + VESCs。 <ol><li>使用Procr CreERT2-IRES-tdTomato / + （简称Procr CreERT2 ）敲入小鼠毒株。 注意:在这个鼠标模型中，一个CreERT2-IRES-tdTomato磁带<s在Procr 11的第一个ATG密码子之后插入up class ="xref"&gt; 10。 <ol><li>交叉Procr CreERT2小鼠与Rosa26 mTtomatomGFP （简称R26 mTmG ）报道菌株，以追踪体内内源性Procr + ECs的命运11 。使用GFP表达鉴定所有标记的内源性Procr + ECs及其后代。 </li></ol></li><li>在青春期阶段（5周）以4mg / 25g体重的剂量腹膜内给予他莫昔芬溶液11 （10mg溶于1mL花生油+ 10％乙醇中的10mg乙醇以制备10mg / mL的储备溶液）旧）跟踪Procr + ECs的发展命运。 </li><li>通过在步骤3中概述的制备步骤后，通过全套免疫染色分析标记细胞的克隆形成效率。在短期后从处理的小鼠获得乳腺脂肪垫（2天）和延长的时间段（最多10个月）。参见图3 。 </li></ol></li></ol>

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J., 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=Intestinal+crypt+homeostasis+results+from+neutral+competition+between+symmetrically+dividing+Lgr5+stem+cells.">Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells.</a> Cell. 143 (1), 134-144 (2010).</li><li>Soriano, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generalized+lacZ+expression+with+the+ROSA26+Cre+reporter+strain.">Generalized lacZ expression with the ROSA26 Cre reporter strain.</a> Nat Genet. 21 (1), 70-71 (1999).</li><li>Barker, N., 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=Lgr5(+ve)+stem+cells+drive+self-renewal+in+the+stomach+and+build+long-lived+gastric+units+in+vitro.">Lgr5(+ve) stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro.</a> Cell Stem Cell. 6 (1), 25-36 (2010).</li><li>Solar, M., 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=Pancreatic+exocrine+duct+cells+give+rise+to+insulin-producing+beta+cells+during+embryogenesis+but+not+after+birth.">Pancreatic exocrine duct cells give rise to insulin-producing beta cells during embryogenesis but not after birth.</a> Dev Cell. 17 (6), 849-860 (2009).</li></ol>

作者宣称他们没有竞争的经济利益。

血管生成测定法是研究血管动力学的一个很好的实验方法。已经被证明是研究血管生成的有吸引力的模型12 ，鼠后视网膜脉管系统。尽管它相对易于使用，但在视网膜血管床内的实际操作是相当困难的。到目前为止，最佳描述的体内移植是插塞测定法，其将细胞包围在基底膜基质块内，并在接受者小鼠的侧翼皮下手术植入/注射。这种血管生成测定在测试嵌入细胞的再生潜...

在发育和体内平衡过程中，根据器官生长和修复，血管生长和重塑真实地发生。血管生成描述了从先前存在的血管产生新血管，并被认为是介导这些动态血管变化的主要力量。每个血管内衬一层内皮细胞​​（ECs），它们似乎是血管结构的基础。长期以来，在稳态期间补充EC池的机制尚不清楚，并且提出血管周转是否是成熟的EC增殖的结果，还是血管干/祖细胞活性的贡献。由于缺乏直接的生理学证据，血管内皮细胞（VESCs）的存在和细胞同一性也是有争议的。 用于验证干细胞行为的最常用方法之一是通过transplan将推定的干细胞置于受体小鼠中。该方法测量候选干细胞在体内的干细胞潜能。移植首先应用于骨髓干细胞研究1 ，有助于建立造血系统的分层特征2 。在内皮区域，皮下插入侧腹皮下的基底膜基质（ 例如，基质胶）栓塞已经是用于解决移植的ECs的血管形成能力的标准体内血管生成测定法。多种实验方法，包括在3D培养系统和移植的集落形成，都建议潜在EC祖细胞/ VESC群3，4，5，6。然而，由于嵌入基底膜基质中的ECs相对分开周围组织，这不能提供充分探索移植细胞的血管生成潜力所需的最佳利基环境。结果，在矩阵塞内形成的血管主要是毛细血管样的，并且在功能上是不可测量的。 乳腺发育成熟，其中最强劲的生长发生在青春期和怀孕期间。在青春期阶段，乳腺上皮经历快速扩张，占据整个乳腺脂肪垫，伴随着周围血管结构的有效重塑。因此，乳腺为血管生成研究提供了极好的模型。它从生长的乳腺上皮提供空间，基质和丰富的血管生成刺激因素，因此是评估外源细胞血管生成潜力的理想的嫁接部位。此外，乳腺脂肪垫允许形成的外源血管与宿主循环系统整合，进一步促进f手术评估，代表皮下移植的优势。 虽然体外培养和移植测定作为研究细胞群的再生特性的有效方法，但是已知当细胞从其天然栖息地中取出时，这种测定可以刺激可塑性，并且当细胞从他们的生理环境7 。因此，获得细胞命运的直接体内证据是推进目前对内皮细胞群体行为的理解的关键方法。 遗传命运映射（ 即，体内谱系追踪）对于鉴定VESCs和调查其在体系中的性质是至关重要的，因为它可以在其生理环境中揭示体内干细胞行为，并且实际的干性可以是评估。沿袭traci提供了候选VESCs的长期持续性（ 即自我更新）及其产生组织的细胞类型（ 即分化能力）的能力的直接证据。 该方案描述了一种新型乳腺脂肪移植技术和谱系追踪方法来观察VESCs的血管生成能力。这些技术克服了当前可用测定的缺点，并提供了一种新的方法来最佳评估VESCs的干细胞特性。这些方法是可用于评估内皮群体的行为和血管形成性质以及确定病理环境中的血管细胞效力改变的有效工具。

<table><tbody><tr><td>0.05% Trypsin-Ethylene Diamine Tetraacetic Acid(EDTA) (1x)</td><td>Gibco (Life Technologies)</td><td>25300-062</td><td>0.05% Trypsin</td></tr><tr><td>0.22 &amp;micro;m Filter Unit</td><td>Merck Millipore Ltd.</td><td>SLGP033RB</td><td>0.22 &amp;micro;m Filter</td></tr><tr><td>2-Methylbutanol-2, ReagentPlus</td><td>Sigma-Aldrich</td><td>24, 048-6</td><td>Tert Amyl Alcohol</td></tr><tr><td>222, Tribromethanol</td><td>Sigma-Aldrich</td><td>T48402-25g</td><td>222, Tribromethanol</td></tr><tr><td>4&#039;,6-diamidino-2-phenylindole(DAPI)</td><td>Invitrogen</td><td>D1306</td><td>DAPI</td></tr><tr><td>70 &amp;micro;m Cell Strainer</td><td>BD FAL</td><td>352350</td><td>70 &amp;micro;m Cell Strainer</td></tr><tr><td>Adhesion Microscope Slides</td><td>CITOGLAS</td><td>188105</td><td>Glass slides</td></tr><tr><td>Anti-mouse CD105 APC</td><td>eBioscience</td><td>17-1051-82, clone MJ7/18</td><td>for FACS analusis use at 1 &amp;micro;g/mL</td></tr><tr><td>Anti-mouse CD201 Biotin</td><td>eBioscience</td><td>13-2012-82</td><td>for FACS analusis use at 2.5 &amp;micro;g/mL</td></tr><tr><td>Anti-mouse CD31 PE-Cyanine 7</td><td>eBioscience</td><td>25-0311-82, clone 390</td><td>for FACS analusis use at 1 &amp;micro;g/mL</td></tr><tr><td>BD FACSJazz Cell Sorter</td><td>BD Biosciences</td><td>655489</td><td>FACS</td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>100190332</td><td>BSA</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810R</td><td>Centrifuge</td></tr><tr><td>Collagenase type 3</td><td>Worthington-Biochem</td><td>#LS004183</td><td>Collagenase III</td></tr><tr><td>Confocal microscopy</td><td>Leica</td><td>sp8</td><td>Confocal</td></tr><tr><td>Deoxyribonuclease I from bovine pancreas</td><td>Sigma</td><td>D2463-5VL</td><td>Dnase I</td></tr><tr><td>Donkey anti-Rat Cy3</td><td>Jackson ImmunResearch</td><td>712-165-150</td><td>Secondary antibody, use at 0.5 &amp;micro;g/mL</td></tr><tr><td>Dumont Forceps</td><td>WPI</td><td>500342</td><td>Froceps</td></tr><tr><td>Dumont Forceps - Micro-Blunted Tips</td><td>FST</td><td>11253-20</td><td>Forceps</td></tr><tr><td>Fetal Bovine Serum</td><td>Hyclone</td><td>SH30396.03</td><td>FBS</td></tr><tr><td>FITC Ms CD3/Gr1/CD11b/CD45R(B220)/Ter-119</td><td>BioLegend</td><td>78022</td><td>Blood lineage cocktail for FACS analysis, use at 25 &amp;micro;L per test</td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G6279</td><td>Glycerol</td></tr><tr><td>Iscov&#039;s Modified Dulbecco&#039;s Medium</td><td>Gibco (Life Technologies)</td><td>12440-053</td><td>IMDM</td></tr><tr><td>Isolectin GS-IB4 from Griffonia Simplicifolia, Alexa Fluora&amp;nbsp; 647</td><td>Invitrogen</td><td>Z32450</td><td>Isolectin-647</td></tr><tr><td>Matrigel&amp;nbsp; Matrix (growth factor reduced)</td><td>BD</td><td>356231</td><td>Matrigel</td></tr><tr><td>Mouse strain (Actin-GFP)</td><td>Jax Laboratories</td><td>3773</td><td>Actin-GFP</td></tr><tr><td>Mouse strain (C57BL/6)</td><td>SLAC</td><td>C57BL/6</td><td>C57BL/6</td></tr><tr><td>Mouse strain&amp;nbsp; (BALB/c nude)</td><td>SLAC</td><td>BALB/c nude</td><td>Nude mice</td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148</td><td>PFA</td></tr><tr><td>Penicilin Streptomycin</td><td>Gibco (Life Technologies)</td><td>5140-122</td><td>Pen/Strep</td></tr><tr><td>Phosphate Buffered Saline (pH7.2) 1x</td><td>Gibco (Life Technologies)</td><td>c20012500BT</td><td>PBS</td></tr><tr><td>PRIM1640</td><td>Gibco (Life Technologies)</td><td>c11875500CP</td><td>PRIM1640</td></tr><tr><td>ProLong Gold antifade reagent</td><td>Life Technologies</td><td>P36934</td><td>Mounting Medium</td></tr><tr><td>Rat anti CD144</td><td>BD Biosciences</td><td>550548</td><td>for whole-mount analysis, anti-VE-Cadherin / Cdh5 antibdy for endothelial tight junction, use at 2.5 &amp;micro;g/mL</td></tr><tr><td>Rat anti CD31-biotin</td><td>BD Biosciences</td><td>553371</td><td>for whole-mount analusis, anti-CD31/PECAM1 antibody for endothelial surface adhesion molecule, use at 10 &amp;micro;g/mL</td></tr><tr><td>Red Cell Lysis Buffer</td><td>Sigma</td><td>R7767-100ML</td><td>Red blood cell lysing buffer</td></tr><tr><td>Straight/Sharp-Blunt/10cm</td><td>FST</td><td>14028-10</td><td>Fine Scissors</td></tr><tr><td>Streptavidin eFluor 450</td><td>eBioscience</td><td>48-4317-82</td><td>for FACS analysis use at 0.5 &amp;micro;g/mL</td></tr><tr><td>Tamoxifen</td><td>Sigma</td><td>101551374</td><td>TAM</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787-250ML</td><td>TritonX</td></tr><tr><td>Wax Coated Braided Silk (Size 5-0 USP (1 Metric), 18 inches (45 cm) BLACK on C-1 Needle)</td><td>COVIDIEN</td><td>S1173</td><td>Suture&amp;nbsp;</td></tr><tr><td>Sterile Disposable Scaplels</td><td>Swann Morton</td><td>#10</td><td>Scalpel</td></tr><tr><td>Betadine</td><td>Yifeng Medical</td><td>20160101</td><td>Antiseptic solution</td></tr></tbody></table>

a novel mammary fat pad transplantation technique to visualize the vessel generation of vascular endothelial stem cells

内皮细胞（ECs）是血管结构的基本组成部分，并介导血管生长和重建，以确保适当的血管发育和体内平衡。然而，由于缺乏获取途径的工具以及直接评价其在体内的行为，对内皮谱系等级的研究仍然难以捉摸。为了解决这个缺点，已经开发了一种使用乳腺脂肪垫研究血管生成的新组织模型。乳腺主要发生在产后阶段，包括青春期和怀孕，在此期间，强壮的上皮细胞增殖伴随着广泛的血管重塑。乳腺脂肪垫从生长的乳腺上皮提供空间，基质和丰富的血管生成刺激。此外，乳腺脂肪垫位于腹腔外部，使得它们是用于评估外源性细胞的血管生成潜力的易于接近的移植部位。这项工作也描述了一种效率使用荧光报告小鼠在体内特异性标记血管内皮干细胞（VESCs）的目标群体的nt追踪方法。这种谱系追踪方法与随后的组织全固定显微镜相结合，能够直接观察目标细胞及其后代，从而可以量化扩增能力，分化承诺可以命运映射。使用这些方法，最近在多个血管系统中鉴定了表达VESCs的双能蛋白C受体（Procr）群体。 Procr + VESCs，引起新的EC和周细胞，积极促进血管生成发育，体内平衡和损伤修复。总的来说，这份手稿描述了一种新的乳腺脂肪移植和体内谱系追踪技术，可用于评估VESCs的干细胞特性。

乳腺VESCs分离: 为了分离内皮细胞以进行随后的移植测定，收获成熟（8周龄）的原始小鼠乳腺作为供体材料。使用基于抗体的细胞分选技术分离内皮细胞。在8周龄的C57BL / 6乳腺EC中，VESC群体的FACS分析的代表性图示于图1 （图中已经允许从Yu 等 8获得）。 <p class="jove_step" fo:keep-togeth...

Watch this Scientific Journal Video about A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells at JoVE.com

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

这项工作展示了一种新的方法来评估通过乳腺脂肪移植移植血管内皮细胞（VESCs）的增殖，分化和血管形成潜力，然后进行全面的组织制备用于显微镜观察。还提出了研究VESCs 体内行为的谱系追踪策略。

新型乳腺脂肪移植技术可视化血管内皮干细胞的血管生成

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

a-novel-mammary-fat-pad-transplantation-technique-to-visualize-vessel

Research

JoVE Journal

Developmental Biology

9.2K 次观看.  Chinese Academy of Sciences. 这项工作展示了一种新的方法来评估通过乳腺脂肪移植移植血管内皮细胞（VESCs）的增殖，分化和血管形成潜力，然后进行全面的组织制备用于显微镜观察。还提出了研究VESCs 体内行为的谱系追踪策略。 

视频: A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal mammary epithelial cell (NAMEC)-derived extracellular vesicles can be isolated from NAMEC medium via differential ultracentrifugation. Based on their density, EVs can be purified via ultracentrifugation at 110,000 x g. The EV preparation from ultracentrifugation can be further separated using a continuous density gradient to prevent contamination with soluble proteins. The purified EVs can then be further evaluated using nanoparticle-tracking analysis, which measures the size and number of vesicles in the preparation. The extracellular vesicles with a size ranging from 50 to 150 nm are exosomes. The NAMEC-derived EVs/exosomes can be ingested by mammary epithelial cells, which can be measured by flow cytometry and confocal microscopy. Some mammary stem cell properties (e.g., mammary gland-forming ability) can be transferred from the stem-like NAMECs to mammary epithelial cells via the NAMEC-derived EVs/exosomes. Isolated primary EpCAMhi/CD49flo luminal mammary epithelial cells cannot form mammary glands after being transplanted into mouse fat pads, while EpCAMlo/CD49fhi basal mammary epithelial cells form mammary glands after transplantation. Uptake of NAMEC-derived EVs/exosomes by EpCAMhi/CD49flo luminal mammary epithelial cells allows them to generate mammary glands after being transplanted into fat pads. The EVs/exosomes derived from stem-like mammary epithelial cells transfer mammary gland-forming ability to EpCAMhi/CD49flo luminal mammary epithelial cells.

This protocol describes methods for purifying, quantitating, and characterizing extracellular vesicles (EVs)/exosomes from non-adherent/mesenchymal mammary epithelial cells and for using them to transfer mammary gland-forming ability to luminal mammary epithelial cells. EVs/exosomes derived from stem-like mammary epithelial cells can transfer this cell property to cells that ingest the EVs/exosomes.

通过细胞外囊泡/外来体转移乳腺基质上皮细胞和乳腺腔细胞之间的乳腺形成能力

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal ...

科研

发育生物学

Rat Mammary Epithelial Cell Transplantation into the Interscapular White Fat Pad

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary epithelium using transplantation techniques takes advantage of the hormonal environment provided by the adolescent rodent host. These studies are ideally suited to explore the impact of various biological manipulations on mammary gland development and dissect many aspects of mammary gland biology. A common, but limiting, feature is that transplanted epithelial cells are strongly influenced by the surrounding stroma and outcompeted by endogenous epithelium; to utilize native mammary tissue, the abdominal-inguinal white fat pad must be cleared to remove host mammary epithelium prior to the transplantation. A major obstacle when using the rat model organism is that clearing the developing mammary tree in post-weaned rats is not efficient. When transplanted into gland-free fat pads, donor epithelial cells can repopulate the cleared host fat pad and form a functional mammary gland. The interscapular fat pad is an alternative location for these grafts. A major advantage is that it lacks ductal structures yet provides the normal stroma that is necessary to promote epithelial outgrowth and is easily accessible in the rat. Another major advantage of this technique is that it is minimally invasive, because it eliminates the need to cauterize and remove the growing endogenous mammary tree. Additionally, the interscapular fat pad contains a medial blood vessel that can be used to separate sites for grafting. Because the endogenous glands remain intact, this technique can also be used for studies comparing the endogenous mammary gland to the transplanted gland. This paper describes the method of mammary epithelial cell transplantation into the interscapular white fat pad of rats.

This article describes a transplantation method to graft donor rat mammary epithelial cells into the interscapular white fat pad of recipient animals. This method can be used to examine host and/or donor effects on mammary epithelium development and eliminates the need for pre-clearing, thereby extending the usefulness of this technique.

大鼠乳腺内皮细胞移植到间膜白脂肪垫

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary ...

癌症研究

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

组织工程血管在小鼠模型的生成和嫁接

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

生物工程

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

在组织工程由内在血管化<em&gt;在体内</em&gt;组织工程商会

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

制备基于微流体的血管化人类微肿瘤模型

Establishing a Physiologic Human Vascularized Micro-Tumor Model for Cancer Research

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical cancer research and therapeutic development.&#160;To overcome this problem, we have developed the vascularized microtumor (VMT), or tumor chip, a microphysiological system that realistically models the complex human tumor microenvironment. The VMT forms de novo within a microfluidic platform by co-culture of multiple human cell types under dynamic, physiological flow conditions. This tissue-engineered micro-tumor construct incorporates a living perfused vascular network that supports the growing tumor mass just as newly formed vessels do in vivo. Importantly, drugs and immune cells must cross the endothelial layer to reach the tumor, modeling in vivo physiological barriers to therapeutic delivery and efficacy. Since the VMT platform is optically transparent, high-resolution imaging of dynamic processes such as immune cell extravasation and metastasis can be achieved with direct visualization of fluorescently labeled cells within the tissue. Further, the VMT retains in vivo tumor heterogeneity, gene expression signatures, and drug responses. Virtually any tumor type can be adapted to the platform, and primary cells from fresh surgical tissues grow and respond to drug treatment in the VMT, paving the way toward truly personalized medicine. Here, the methods for establishing the VMT and utilizing it for oncology research are outlined. This innovative approach opens new possibilities for studying tumors and drug responses, providing researchers with a powerful tool to advance cancer research.

作者聚焦：创建人类血管化微肿瘤作为转化癌症研究的模型 

This protocol presents a physiologically relevant tumor-on-a-chip model to perform high-throughput basic and translational human cancer research, advancing drug screening, disease modeling, and personalized medicine approaches with a description of loading, maintenance, and evaluation procedures.

建立用于癌症研究的生理性人血管化微肿瘤模型

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical ...

Mammary Epithelial Transplant Procedure

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, followed by the mammary epithelial transplant procedure. The mammary transplant procedure is widely used by mammary biologists because it takes advantage of the fact that significant development of the mammary epithelium doesn't occur until after puberty. At 3 weeks of age, growth of the mammary epithelial tree is confined to the vicinity of the nipple and the fat pad is largely devoid of mammary epithelium, but by 7 weeks of age the epithelial ductal tree extends throughout the entire fat pad. Therefore, if this small portion of the fat pad containing epithelium, the region between the nipple and the lymph node, is removed at 3 weeks of age, the endogenous epithelium will never populate the mammary fat pad and the fat pad is described as "cleared". At this time, mammary epithelium from another source can be transplanted in the cleared fat pad where it has the potential to extend mammary ductal trees through out the fat pad. This procedure has been utilized in many experimental models including the examination of tumor phenotype in transgenic mammary epithelial tissue without the confounding effects of genotype on the entire animal3, in the identification of mammary stem cells by transplanting cells in limited dilution4,5, determining if hyperplastic nodules proceed to mammary tumors6, and to assess the effect of prior hormone exposure on the behavior of the mammary epithelium7,8.
Three week old host mice are anesthetized, cleaned and restrained on a surgical stage. A mid-sagittal incision is made through the skin, but not the peritoneum, extending from the pubis to the sternum. Oblique cuts are made through the skin from the mid-sagittal incision across the pelvis toward each leg. The skin is pulled away from the peritoneum to expose the 4th inguinal mammary gland. The fat pad is cleared by removing the fat pad tissue anterior to the lymph node. Epithelium fragments or epithelial cells are transplanted into the remaining cleared fat pad and the mouse is closed.

This article demonstrates the procedure developed by DeOme KB et al. (1959) and the sparing procedure developed by Brill B et al. (2008) for clearing the 4th inguinal mammary fat pad of a pubescent mouse in preparation for transplantation of mammary fragments, mammary epithelial cells, or mammary tumor cells.

乳腺上皮移植手术

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, ...

生物学

Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several angiogenic and prevascularization strategies have been established. However, most cell-based approaches include time-consuming in vitro steps for the formation of a microvascular network. Hence, they are not suitable for intraoperative one-step procedures. Adipose tissue-derived microvascular fragments (ad-MVF) represent promising vascularization units. They can be easily isolated from fat tissue and exhibit a functional microvessel morphology. Moreover, they rapidly reassemble into new microvascular networks after in vivo implantation. In addition, ad-MVF have been shown to induce lymphangiogenesis. Finally, they are a rich source of mesenchymal stem cells, which may further contribute to their high vascularization potential. In previous studies we have demonstrated the remarkable vascularization capacity of ad-MVF in engineered bone and skin substitutes. In the present study, we report on a standardized protocol for the enzymatic isolation of ad-MVF from murine fat tissue.

We present a protocol to isolate adipose tissue-derived microvascular fragments that represent promising vascularization units. They can be rapidly isolated, do not require in vitro processing and, thus, may be used for one-step prevascularization in different fields of tissue engineering.

小鼠脂肪组织来源的微血管片段作为血管化单元的分离组织工程

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several ...

Transplantation Into the Mouse Ovarian Fat Pad

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic transplantation of ovarian surface and tubal epithelia include intraperitoneal and intrabursal administration of cells. The respective limitations of these methods include poorly defined location of injected cells and limited space volume. Furthermore, they are poorly suited for long-term structural preservation of transplanted organs. To address these challenges, we have developed an alternative approach, which is based on the introduction of cells and tissue fragments into the mouse fat pad. The mouse ovarian fat pad is located in the immediate vicinity of the ovary and uterine tube (aka oviduct, fallopian tube), and provides a familiar microenvironment for cells and tissues of these organs. In our approach fluorescence-labeled mouse and human cells, and fragments of the uterine tube are engrafted by using minimally traumatic dorsal incision surgery. Transplanted cells and their outgrowths are easily located in the ovarian fat pad for over 40 days. Long-term transplantation of the entire uterine tube allows correct preservation of all principle tissue components, and does not result in adverse side effects, such as fibrosis and inflammation. Our approach should be uniquely applicable for answering important biological questions such as differentiation, regenerative and neoplastic potential of specific cell populations. Furthermore, it should be suitable for studies of microenvironmental factors in normal development and cancer.

We describe an ovarian fad pad transplantation assay suitable for studies of normal and transformed epithelia of the female reproductive tract. The mouse fat pad allows transplantation of large tissue fragments, is easily accessible for surgery and imaging, and provides the most favorable native environment for tissues of M&#252;llerian origin.

移植到小鼠卵巢脂肪垫

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic ...

新型乳腺脂肪移植技术可视化血管内皮干细胞的血管生成

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此视频中的章节

通过细胞外囊泡/外来体转移乳腺基质上皮细胞和乳腺腔细胞之间的乳腺形成能力

大鼠乳腺内皮细胞移植到间膜白脂肪垫

组织工程血管在小鼠模型的生成和嫁接

在组织工程由内在血管化

建立用于癌症研究的生理性人血管化微肿瘤模型

建立用于癌症研究的生理性人血管化微肿瘤模型

建立用于癌症研究的生理性人血管化微肿瘤模型

乳腺上皮移植手术

小鼠脂肪组织来源的微血管片段作为血管化单元的分离组织工程

移植到小鼠卵巢脂肪垫