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能和血管形成能力方面是有效的。然而，由于植入的矩阵塞的位置为其内的细胞产生相对孤立的环境，所以它不提供最佳的生理位置。最近，通过在血管上应用纤维蛋白贴片也开发了新的肺血管生成模型ce站点的小鼠肺13 。然而，由于这种技术在小鼠胸腔内侵入性地操作，所以操纵姿势又是另一个风险，因此使其难以在更广泛的测定设置中使用。 在这里描述的血管生成测定中，将小体积的细胞混合物移植到乳腺的脂肪垫内，其在青春期期间提供空间，基质和丰富的血管生成刺激。这允许所产生的血管符合并整合到重建主体脉管系统中，进行进一步的功能评估，并且表现出优于皮下栓塞测定的优点。此外，乳腺脂肪垫位于腹膜腔外，使其成为非常容易接近的部位。在乳腺脂肪垫上的操作对受体动物引入有限的痛苦，避免涉及视网膜和肺基测定的侵入性外科手术。 血液脉管系统机智通常在组织成分之间交织，以最大化血管覆盖，从而可以优化提供血氧和物质转移。为此，组织的常规切片经常拦截其血管的管状结构。血管特性的分析最好在血管保存并保持完好时进行。组织的整体制备可以在很大程度上保持其内的整个血管结构。该方法不仅可以观察器官内血管的真实的空间分布，还可以与其他组织成分的关系。 通过平衡细胞质量的产生和损失来维持含有干细胞的组织的体内平衡。特定居住器官内的干细胞通常被遮蔽并与周围的组织环境紧密接触，被称为干细胞生态位。因此，stu分析成体干细胞组织的死亡优选应在完整的生理环境中进行。移植测定可以提供评估细胞潜力的手段，而谱系跟踪技术可以探索本地栖息地内真正的细胞命运。近年来， 体内谱系追踪已成为干性能实验评估的有力技术。这个策略已被用于鉴定组织残余物干细胞和它们在多种组织的后代，包括肠10，14，毛囊15，胃16，和胰腺17。谱系追踪方法在很大程度上依赖于干细胞的遗传标记，并在确定的群体中启动。因此，难以排除在标记细胞内甚至更小的亚群体中发现实际干性的可能性。因此，必须精确地绘制跟踪克隆的原始细胞，并定量测量随时间的追踪效率。这样一来，可以抓住标签人口的自我更新能力。 所描述的血管生成测定可以针对特定的研究目的进行修改:它不仅可用于测试感兴趣的内皮细胞群的血管生成能力，还可用于评估血管生成因子对局部血管重塑的影响。然而，由于乳腺在不同的发育阶段（ 如青春期，发情周期和怀孕期间）发生动态形态学变化，所以在结果解释过程中，应考虑体内变化（如激素水平波动）的影响。该测定中的关键步骤包括主要内皮细胞分离。为了最佳地获得用于移植的活细胞，FACS分离的程序应该be轻轻地进行。应注意避免在细胞制备过程中严格的处理，如细胞沉淀松动和悬浮。细胞混合物注入脂肪的另一个关键步骤。在脂肪垫注射期间，重要的是确保细胞混合物精确地沉积在脂肪垫内。如果针插入太深/浅，或总体积超过推荐剂量，这可能很棘手。 该方案提供了一系列有助于鉴定内皮干细胞群体的实验方法。通过这些技术，可以通过移植来评估干细胞特性，以及跟踪和观察其在体内生理过程中的行为。这些方法是可以应用于多个学科的有效工具，包括肿瘤环境中内皮细胞群的研究及其细胞效能的改变。一世特别是，脂肪移植和整体准备是可重复和强大的方法，可能有助于今后努力调查血管生物学的不同方面。

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 border="0" cellpadding="0" cellspacing="0" width="1265">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">0.05% Trypsin-Ethylene Diamine Tetraacetic Acid(EDTA) (1X)</td>
			<td style="width:263px;">Gibco (Life Technologies)</td>
			<td style="width:208px;">25300-062</td>
			<td style="width:387px;">0.05% Trypsin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.22 &#181;m Filter Unit</td>
			<td>Merck Millipore Ltd.</td>
			<td>SLGP033RB</td>
			<td>0.22 &#181;m Filter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-Methylbutanol-2, ReagentPlus</td>
			<td>Sigma-Aldrich</td>
			<td>24, 048-6</td>
			<td>Tert Amyl Alcohol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">222, Tribromethanol</td>
			<td>Sigma-Aldrich</td>
			<td>T48402-25g</td>
			<td>222, Tribromethanol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4&#39;,6-diamidino-2-phenylindole(DAPI)</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td>DAPI</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70 &#181;m Cell Strainer</td>
			<td>BD FAL</td>
			<td>352350</td>
			<td>70 &#181;m Cell Strainer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adhesion Microscope Slides</td>
			<td>CITOGLAS</td>
			<td>188105</td>
			<td>Glass slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-mouse CD105 APC</td>
			<td>eBioscience</td>
			<td>17-1051-82, clone MJ7/18</td>
			<td>for FACS analusis use at 1 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-mouse CD201 Biotin</td>
			<td>eBioscience</td>
			<td>13-2012-82</td>
			<td>for FACS analusis use at 2.5 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-mouse CD31 PE-Cyanine 7</td>
			<td>eBioscience</td>
			<td>25-0311-82, clone 390</td>
			<td>for FACS analusis use at 1 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD FACSJazz Cell Sorter</td>
			<td>BD Biosciences</td>
			<td>655489</td>
			<td>FACS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>100190332</td>
			<td>BSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td>Centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase type 3</td>
			<td>Worthington-Biochem</td>
			<td>#LS004183</td>
			<td>Collagenase III</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal microscopy</td>
			<td>Leica</td>
			<td>sp8</td>
			<td>Confocal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Deoxyribonuclease I from bovine pancreas</td>
			<td>Sigma</td>
			<td>D2463-5VL</td>
			<td>Dnase I</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Donkey anti-Rat Cy3</td>
			<td>Jackson ImmunResearch</td>
			<td>712-165-150</td>
			<td>Secondary antibody, use at 0.5 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont Forceps</td>
			<td>WPI</td>
			<td>500342</td>
			<td>Froceps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont Forceps - Micro-Blunted Tips</td>
			<td>FST</td>
			<td>11253-20</td>
			<td style="width:387px;">Forceps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td>Hyclone</td>
			<td>SH30396.03</td>
			<td>FBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 &#181;l per test</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Sigma</td>
			<td>G6279</td>
			<td>Glycerol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iscov&#39;s Modified Dulbecco&#39;s Medium</td>
			<td>Gibco (Life Technologies)</td>
			<td>12440-053</td>
			<td>IMDM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isolectin GS-IB4 from Griffonia Simplicifolia, Alexa Fluora&#160; 647</td>
			<td>Invitrogen</td>
			<td>Z32450</td>
			<td>Isolectin-647</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrigel&#160; Matrix (growth factor reduced)</td>
			<td>BD</td>
			<td>356231</td>
			<td>Matrigel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse strain (Actin-GFP)</td>
			<td>Jax Laboratories</td>
			<td>3773</td>
			<td>Actin-GFP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse strain (C57BL/6)</td>
			<td>SLAC</td>
			<td>C57BL/6</td>
			<td>C57BL/6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse strain&#160; (BALB/c nude)</td>
			<td>SLAC</td>
			<td>BALB/c nude</td>
			<td>Nude mice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td>PFA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicilin Streptomycin</td>
			<td>Gibco (Life Technologies)</td>
			<td>5140-122</td>
			<td>Pen/Strep</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffered Saline (pH7.2) 1X</td>
			<td>Gibco (Life Technologies)</td>
			<td>c20012500BT</td>
			<td>PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PRIM1640</td>
			<td>Gibco (Life Technologies)</td>
			<td>c11875500CP</td>
			<td>PRIM1640</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ProLong Gold antifade reagent</td>
			<td>Life Technologies</td>
			<td>P36934</td>
			<td>Mounting Medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 ug/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Red Cell Lysis Buffer</td>
			<td>Sigma</td>
			<td>R7767-100ML</td>
			<td>Red blood cell lysing buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Straight/Sharp-Blunt/10cm</td>
			<td>FST</td>
			<td>14028-10</td>
			<td>Fine Scissors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptavidin eFluor 450</td>
			<td>eBioscience</td>
			<td>48-4317-82</td>
			<td>for FACS analysis use at 0.5 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tamoxifen</td>
			<td>Sigma</td>
			<td>101551374</td>
			<td>TAM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787-250ML</td>
			<td>TritonX</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile Disposable Scaplels</td>
			<td>Swann Morton</td>
			<td>#10</td>
			<td>Scalpel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Betadine</td>
			<td>Yifeng Medical</td>
			<td align="right">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）。 乳腺脂肪移植: 乳腺脂肪垫为血管生成研究提供了理想的依据。在基于FACS的分离后，将Procr + EC与0.5％基底膜基质和0.1％台盼蓝指示剂混合，然后进行转染到3周龄的SCID收件人的空脂肪垫。在青春期期间，乳腺脂肪垫产生血管生成环境，促进与乳腺上皮扩张一致的脉管系统的重塑。结果，移植的Procr + EC（GFP + ）并入宿主的大容器中（ 图2B ，箭头），并且还形成了第二和第三GFP +血管分支（ 图2B ，箭头）。虽然移植的Procr + ECs也可以在基质插塞测定（插入侧翼皮肤）中产生血管，但形成的血管仅出现毛细血管样（ 图2A ;经许可修改自Yu 等人 8 ）。 Procr + VESCs形成的船舶功能: 调查GFP + v乳腺脂肪垫中的精华素是功能性循环血管系统的一部分，在收获前静脉注射Isolectin。 GFP +血管是isolectin +，表明形成的血管被发光并与主动脉血管连接（ 图2B ;经许可修改自Yu 等人 8 ）。 乳腺VESC谱系追踪使用Procr CreERT2 ; Rosa26 mTmG鼠标型号: Procr CreERT2 ; Rosa26 mTmG小鼠用于体内 Procr + VESCs的命运追踪（ 图3A ）。为了研究Procr + VESCs如何促进乳腺发育期乳腺发育和血管重建， Procr CreERT2 ; Rosa26 mTmG是管理员d与他莫昔芬在Procr +内皮细胞群中诱导GFP表达。药物处理Procr CreERT2的乳腺脂肪垫;在短期（注射后2天; 图3B ）和长期（注射后至多10个月; 图3C -F;经许可改编自Yu 等人 8的 图 ）收获Rosa26mTmG小鼠。进行整体准备，以便可视化追踪结果。通过用内皮表面标记物VE-钙粘着蛋白（Cdh5; 图3B ，右图）染色可以验证血管内皮细胞同一性。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55795/55795fig1.jpg" /> 图1:小鼠乳腺中VESCs的分析。 8周龄C57BL / 6乳腺ECs中Procr表达的FACS分析。 8周岁将irgin C67BL / 6雌性小鼠用于乳腺收集。在制备细胞样品后，分析未染色的样品和每个单色对照以确定每种颜色的电压。计算补偿参数以避免自发荧光或背景染色。建立了适当门控模板并对所需细胞群进行分类。为了建立FACS分类的层次结构，消除了所有事件的碎片，然后丢弃了双层或粘合细胞簇。门控细胞门控，CD31和CD105用于定义内皮细胞群。与FMO对照相比，从CD31 + CD105 + ECs分离出Procr +细胞。这个数字已经从Yu 等人修改8 ，经许可。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55795/55795fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 图2:VESC乳腺脂肪移植测定。 （ A ）由Procr + ECs形成的容器，其在基底膜基质栓塞内皮下皮下插入。 （ B ）受体乳腺脂肪垫的共焦图像，表明移植的Procr + EC（GFP +）与宿主乳腺脉管系统的整合和贡献。 ECs用CD31复染。 （ C ）静脉注射含有Isolectin的受体小鼠，表明产生的成长形成了发光的血管。刻度棒，50μm。使用40X NA 1.3石油物镜获得图像。以1024×1024的光学分辨率获得Z-叠层的平铺扫描，并且每层的间隔为1.0-1.2μm。为了捕获正确的荧光信号，过滤器分束器设置为TD 488/553/638，其中g在PMT增益600-800下，使用以下激发/发射源:mGFP，激发/发射最大值为488/509nm; mTomato，激发/发射最大值为532/588 nm; Alexa-647，激发/发射最大值为594/665;和DAPI，激发/发射最大值为350/470。这个数字已经从Yu 等人修改8 ，经许可。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55795/55795fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" class="xfigimg" src="/files/ftp_upload/55795/55795fig3.jpg" /> 图3:Procr + EC的谱系跟踪演示了它们在开发过程中对EC克隆扩展的贡献。 （ A ）使用Procr CreERT2的血统追踪策略的图示 ; Rosa26 mTmG线（左图）。试验如（右图）所示，短期（2天）和长期（7天至10个月）使用的设置。 （ BF ）在不同长度的追踪期之后，乳腺脉管系统的整体共聚焦成像，指示标记的Procr + ECs的初始位置及其在各种追踪阶段对乳腺脉管系统的贡献。刻度棒=50μm。这个数字已经从Yu 等人修改8 ，经许可。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55795/55795fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

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

Nanyang Technological University

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

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

小鼠胚胎Hemogenic内皮细胞的分离

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

科研

发育生物学

An Efficient Method to Obtain Dedifferentiated Fat Cells

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used therapeutically, in the near future, to restore function to damaged organs. Nevertheless, several technical issues, including the highly invasive procedure of isolating MSCs and the inefficiency surrounding their amplification, currently hamper the potential clinical use of these therapeutic modalities. Herein, we introduce a highly efficient method for the generation of dedifferentiated fat cells (DFAT), MSC-like cells. Interestingly, DFAT cells can be differentiated into several cell types including adipogenic, osteogenic, and chondrogenic cells. Although other groups have previously presented various methods for generating DFAT cells from mature adipose tissue, our method allows us to produce DFAT cells more efficiently. In this regard, we demonstrate that DFAT culture medium (DCM), supplemented with 20% FBS, is more effective in generating DFAT cells than DMEM, supplemented with 20% FBS. Additionally, the DFAT cells produced by our cell culture method can be redifferentiated into several tissue types. As such, a very interesting and useful model for the study of tissue dedifferentiation is presented.

We have modified the conditions for DFAT cell generation and provide herein information regarding the use of an improved growth medium for the production of these cells.

一个有效的方法获取反分化脂肪细胞

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

水平整体安装:厚三维组织横截面皮肤的新型加工和成像方案

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells (hiPSC-ECs)

Endothelial cells (ECs) are essential for the regulation of inflammatory responses by either limiting or facilitating leukocyte recruitment into affected tissues via a well-characterized cascade of pro-adhesive receptors which are upregulated on the leukocyte cell surface upon the inflammatory trigger. Inflammatory responses differ between individuals in the population and the genetic background can contribute to these differences. Human induced pluripotent stem cells (hiPSCs) have been shown to be a reliable source of ECs (hiPSC-ECs), thus representing an unlimited source of cells that capture the genetic identity and any genetic variants or mutations of the donor. hiPSC-ECs can therefore be used for modeling inflammatory responses in donor-specific cells. Inflammatory responses can be modeled by determining leukocyte adhesion to the hiPSC-ECs under physiological flow. This step-by-step protocol provides a detailed description of the experimental setup and data analysis for the assessment of inflammatory responses in hiPSC-ECs and the analysis of leukocyte adhesion under physiological flow.

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells hiPSC-ECs

This step-by-step protocol provides a detailed description of the experimental setup and data analysis for the assessment of inflammatory responses in hiPSC-ECs and the analysis of leukocyte adhesion under physiological flow.

微流控法评价人诱导的多能干细胞内皮细胞 (hipsc-ec) 对白细胞粘附的评价

Endothelial cells (ECs) are essential for the regulation of inflammatory responses by either limiting or facilitating leukocyte recruitment into affected ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

从脐带血源性多能干细胞中提取三维皮肤有机细胞

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C） 损伤的反应

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

通过微分试穿素生成马赛克乳腺有机体

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

Isolation of Endocardial and Coronary Endothelial Cells from the Ventricular Free Wall of the Rat Heart

It has been shown that endocardial endothelial cells (EECs) and coronary endothelial cells (CECs) differ in origin, development, markers, and functions. Consequently, these two cell populations play unique roles in cardiac diseases. Current studies involving isolated endothelial cells investigate cell populations consisting of both EECs and CECs. This protocol outlines a method to independently isolate these two cell populations for cell-specific characterization. Following the collection of the left and right ventricular free wall, endothelial cells from the outer surface and inner surface are separately liberated using a digestion buffer solution. The sequential digestion of the outer surface and the inner endocardial layer retained separation of the two endothelial cell populations. The separate isolation of EECs and CECs is further verified through the identification of markers specific to each population. Based on previously published single cell RNA profiling in the mouse heart, the Npr3, Hapln1, and Cdh11 gene expression is unique to EECs; while Fabp4, Mgll, and Cd36 gene expression is unique to CECs. qPCR data revealed enriched expression of these characteristic markers in their respective samples, indicating successful EEC and CEC isolation, as well as maintenance of cell phenotype, enabling further cell-specific functional analysis.

We present a protocol for the isolation of endocardial and coronary endothelial cells from rat hearts through sequential tissue digestion in a digestion buffer, cell collection from recurrent centrifuge cycles, and cell purification using anti-rat CD31 microbeads.

从鼠心心室自由壁分离内膜细胞和冠状内皮细胞

It has been shown that endocardial endothelial cells (EECs) and coronary endothelial cells (CECs) differ in origin, development, markers, and functions. ...