我们感谢弗朗西斯·克里克研究所（生物研究机构，体内成像和实验组织病理学）和Yolanda Saavedra-Torres和梅里德斯·桑切斯·加尔松的核心设施中的工作人员，分别是Crick和LRI的兽医，为他们提供了宝贵的帮助。我们非常感谢W. Gray博士批判性地阅读手稿。 DP得到EHA非临床研究奖学金的支持。这项工作得到了弗朗西斯·克里克研究所的支持，该研究所收到了英国癌症研究中心（FC001045），英国医学研究委员会（FC001045）和欢迎信托（FC001045）的核心资金。

所有动物实验均按照PPL 70/8904进行，经英国内政部批准，并符合"英国癌症研究"（Cancer Research UK）指南。人类脐带血（UCB）和原发性人类急性骨髓性白血病（AML）样品的使用在东伦敦伦理委员会收到同意后，经赫尔辛基宣言执行。 1.人造血和基质细胞的生物工程胶原基支架注意:整个方案应在无菌条件下和无菌材料下进行。细胞培养基1对应于hMSC培养基（MEM-α，P / S和10％hMSC-FBS）;细胞培养基2对应于EC培养基（M199,20％FBS，P / S，10mM HEPES，50μg/ mL肝素，2mM谷氨酰胺和50μg/ mL ECGS），细胞培养基3对应于造血细胞培养基（H5100和P / S）。 <ol><li>准备脐带血使用Ficoll-Paque密度梯度的单核细胞（CB-MNC）或原发性AML（骨髓或外周血MNCs），根据公认的方案53 。 <ol><li>对于AML，使用OKT.3抗体消耗T细胞。在室温洗涤细胞之前，在室温下孵育4μg的OKT.3 / 1×10 6个 AML MNC 30分钟。如果需要，用10％DMSO以2×10 8个细胞/ mL冷冻保存FBS中的MNC。 </li></ol></li><li>制备hMSC（培养基1）和EC（培养基2）细胞培养物。在hMSC培养基中的常规细胞培养瓶上培养板hMSC细胞（参见材料表 ）。 EC介质中的胶原1涂层表面的板EC（参见材料表 ）。 </li><li>在85-90％细胞汇合处去除培养基，用PBS洗涤两次，加入胰蛋白酶-EDTA溶液（20μL/ cm 2 ）。 <ol><li> 5分钟后，检查细胞是否分离。通过稀释1:3与细胞培养基。以300×g离心5分钟，并在每种细胞类型的相应培养基中重新悬浮，并使用Neubauer细胞计数细胞。 </li><li>使用2×10 6 - 10 7个细胞/ mL的细胞。如果两种细胞类型都需要一起使用，则以1:1的比例混合hMSC和EC悬浮液。 </li></ol></li><li>将细胞悬浮液转移到胰岛素注射器。 </li><li>使用解剖刀，将消毒的明胶海绵（ 例如，海洛因）初始支架（20mm×60mm×7mm）切割成24个相似尺寸（6.6mm×7.5mm×7mm; 图1A和B ）。 </li><li>通过浸泡在PBS（5分钟）中重建明胶支架。 </li><li>一个接一个地，将支架轻轻地放在无菌组织上以除去多余的PBS。将支架转移到24孔板（超低附着表面）的一个孔中，并使用注射器注射含有10 5 - 10 6个细胞（hMSC al一个或与hEC组合; 图1C ）。 </li><li>每个支架重复步骤1.7，直到所有需要的支架用基质细胞接种。 </li><li>在细胞培养箱（37℃和CO 2 5％）内孵育1小时。 </li><li>用3mL细胞培养基1（ 图1D ）填充各孔，并将支架返回到细胞培养箱（37℃和CO 2 5％）中孵育过夜。如果在脚手架中使用EC，则使用细胞培养基2。 </li><li>解冻CB-MNC，并按照适当的CD34 +部分试剂盒方案从其中分离CD34 +细胞。将选定的CD34 +细胞悬浮于培养基3中，并在Neubauer室中计数。 注意:这里，细胞以浓度为2×10 6个细胞/ mL使用。 <ol><li>如果使用AML患者来源的初级样本，解冻细胞，在FBS中稀释1:10，在300℃离心5分钟xg，将细胞重悬于PBS-2％FBS中，加入抗人CD3抗体（每10 6个细胞2-4μg），并在室温下孵育30分钟。以300×g离心5分钟，重悬于补充有细胞因子（20ng / mL粒细胞集落刺激因子（G-CSF），20ng / mL IL-3和20ng / mL血小板生成素（TPO））的造血细胞培养基中， 。 </li></ol></li><li>重复步骤1.7 - 1.9，但在这种情况下，种子1×10 5人造血细胞而不是基质成分，如上所述。 <ol><li>用3 mL细胞培养基3（ 图1D ）填充孔，并将支架返回到细胞培养箱（37℃和CO 2 5％）中孵育过夜。使用1:1混合细胞培养基2和3如果EC用于支架。 </li></ol></li><li>对于仅重组人骨形态发生蛋白-2（rhBMP-2）载体支架，轻轻将支架逐个放在无菌组织上以除去过量的PBS。将支架转移到u-底部96孔板（ 图1E ）的一个孔中，并加入5μLrhBMP-2，彻底覆盖支架。 <ol><li>加入20μL凝血酶，然后加入20μL纤维蛋白原，每次彻底覆盖支架（ 图1F ）。重复每个支架的步骤，直到所有必需的支架被处理，然后在37℃下孵育5-10分钟。检查凝血是否成功。 </li></ol></li></ol> 2.生物工程支架的手术植入注意:这里，使用雄性或雌性，6至12周龄的NSG小鼠。由于动物免疫缺陷，所有程序应在无菌条件下进行。步骤2.10 - 2.13与生存策略和手术后护理有关。 <ol><li>手术前60 - 120分钟皮下施用止痛药（卡洛芬，5 mg / kg体重/小鼠）。</li><li>在0.5％异氟烷和2 L / min O 2的室内诱导麻醉。应在手术过程中连续监测小鼠。将动物转移到俯卧位的手术区域，以便方便地进入背部。使用提供1.5％异氟烷和2升/分钟O 2的鼻锥将动物置于麻醉下。在手术过程中将鼠标放在麻醉下，经常检查动物状态。 </li><li>在麻醉下，使用眼睛凝胶在小鼠的眼睛，以防止干燥，并保持在37°C的小鼠。 </li><li>使用电动修剪器刮掉鼠标背面的手术区域。要对皮肤进行消毒，将棉絮浸于稀释的氯雷西西定（PBS中稀释1:10），并使用此提示清洁皮肤表面。重复此过程两次。 </li><li>使用无菌镊子和手术刀（或剪刀），进行皮肤的0.5至0.7厘米的前后全切口。使用插入的镊子在皮下组织做一个口袋。 </li><li>皮下插入支架，确保将其放置在口袋内（ 图1G和H ）。用手术胶合并切口（ 图1I和J ）。 </li><li>为了治疗手术后疼痛，皮下给予丁丙诺啡（0.1mg / kg体重）。 </li><li>在恢复期间，将动物放在预热笼中并监测恢复，直到观察到正常行为。 </li><li>在水中稀释疼痛药物（carprofen，0.1 mg / mL水），并在手术后4天将其作为饮用水提供给动物。 </li><li>在手术后48小时内经常检查动物和伤口，以获得不良反应。 </li></ol> 3.小鼠治疗，安乐死和样品检索成像注意:支架之间进行分析植入后8周和24周。 <ol><li>在成像前60分钟，将植入的小鼠保持在37℃的加热箱中，并静脉内施用100μL人免疫球蛋白以阻断非特异性位点。 </li><li>在成像前30分钟，静脉内施用10μg（每只小鼠）特异性抗体以标记感兴趣的细胞。 </li><li>在成像前5分钟，静脉内给予655纳米荧光团标记的血管聚集剂（655-VPA）15μL（在100μLPBS中稀释），以观察血管结构。 </li><li>通过颈椎脱位安乐死小鼠。 </li><li>使用锋利的剪刀，在原始植入位置附近在鼠标背面做一个纵向皮肤切口。 </li><li>在镊子和剪刀的帮助下，仔细地将皮肤从植入了脚手架的皮下袋中分离出来。 </li><li>用镊子握住脚手架，通过切割残留的膜a从皮肤轻轻地将其移出使用剪刀围绕支架的组织。参见图2中要回收的支架的实例。 </li><li>用快速胶粘剂将支架固定在成像板（35 mm x 10 mm培养皿）上，并在室温下注入盐水溶液（PBS）。 </li><li>对于BMP支架，在用PBS填充板之前，使用手术微型钻头在显微外科显微镜下稀释骨表面;这允许荧光团可视化和高分辨率图像捕获。使用1.2或1.6毫米毛刺，取决于脚手架的大小。 注意:根据骨骼的厚度，用户将意识到要钻多少。通常，当BMP支架血管化时，当接近正确的成像厚度时，骨稍微改变颜色并变得更红。 </li><li>将板插入共焦显微镜的台上。 </li></ol>使用双光子显微镜实时成像OPY 注意:使用非下行检测器（NDD）时，请始终使用NDD滑块进行成像以将荧光指向NDD。显微镜配置如图3所示 。 <ol><li>打开显微镜和电脑，点击"启动系统"启动软件，进入"采集"模式。 </li><li>勾选"显示手动工具"框。在"激光"菜单中，打开双光子激光器，使其能够预热和稳定。 </li><li>在"成像设置"菜单中，同时激活"通道模式"和"切换每帧的跟踪"。在"光路"菜单中，选择"未分解"和"主光束分配器MBS 760+"。勾选激活四个NDD并设置如图3所示的配置。 注意:通过这种配置，骨骼结构的胶原信号（秒）在380〜485nm，FITC-hCD31 +人内皮细胞和AF488-hCD45 +人造血细胞（500-550nm）和640-690nm的655-VPA收集SHG。 </li><li>在"通道"菜单中，将"激光波长"设置为890 nm，功率设置为50％。将"增益（主）"设置为500-600，"数字偏移"设置为0，每个通道的"数字增益"设置为15。采集开始后调整这些值。 </li><li>在"采集模式"中，设置所需的参数以获得高分辨率图像，而不会损坏组织并漂白荧光团。将"扫描模式"设置为"帧"，"帧大小"设置为"x512 y512"，"行步"为1，"速度"为9，"平均数"为8，"位深"为"8位"模式"到"线"，"方向"到"双向"，"方法"到"意思"。 <ol><li>设置"Zoom"为1，用于图像的初始扫描，如果需要，可以增加它以专注于特定区域。 </li></ol></li><li>将含有支架的平板放置在显微镜平台上的20X，1.0NA浸水透镜下，并降低镜片，直到其接触盐水溶液。使用显微镜目镜将镜头的焦点设置在支架上，使用灯作为光源。 </li><li>激活"Z-stack"菜单，选择"First / Last"功能，并设置两个子顺序片之间所需的间隔（ 例如 2-μmZ-stack）。将间隔保持在Z-stack内。 </li><li>选择"实时"以对样品进行实时扫描成像，并调整"数字增益"和"数字偏移"以获得最佳曝光。要同时显示多个通道，请选择"拆分"功能。 </li><li>在"舞台"菜单中，在"实时"模式下，扫描图像并"标记"区域est（ROI），例如人造血细胞和血管结构的位置。当样品的扫描完成后，移动到第一个ROI开始成像。 </li><li>在"实时"模式下，使用"首先设置"和"设置最后"功能设置围绕感兴趣区域的3D Z-stack的顶部和底部。一旦完成，设置中心，"C."使用"开始实验"按钮开始获取ROI。参见图4 ， 图5和图6中的图像示例。 </li><li>一旦获取完成，请将图像保存在指定的文件夹中。转到下一个投资回报率并重复步骤4.10。实验完成后，从显微镜上取出成像板，小心地从样品板上取出样品，并清洁残留的胶水。 </li><li>准备样品进行下一个分析技术。 </li></ol> 5.样品加工g用于组织学和免疫染色注意:样品根据描述样品固定，嵌入和切片过程的JoVE通用实验室技术54中描述的方案进行处理。骨形成样品应在固定和嵌入过程之间的EDTA脱钙剂中治疗7天。阻断/透化溶液是具有1％Triton X-100,1％牛血清白蛋白（BSA）和10％正常山羊血清（NGS）的10mM PBS pH 7.4缓冲液。 <ol><li>将切片置于二甲苯中10分钟），二甲苯5分钟，100％乙醇5分钟，70％乙醇5分钟，50％乙醇5分钟，H 2 O 5分钟。 </li><li>将切片转移到基于柠檬酸盐的抗原揭示工作溶液中。 </li><li>将切片煮沸15分钟，让它们冷却至室温。 </li><li>用10％PBS pH 7.4的溶液用1％Triton X-100（5分钟，3次）洗涤切片。 </li><li>转移sam封闭/渗透溶液并孵育30分钟。 </li><li>加入在封闭/透化溶液中稀释的一抗，并在4℃下孵育过夜。 </li><li>用10％PBS pH 7.4的溶液用1％Triton X-100（5分钟，3次）洗涤切片。 </li><li>在室温下在黑暗中加入在封闭/透化溶液中稀释1小时的二抗。 </li><li>用H 2 O洗涤（5分钟，3次）。 </li><li>为了减少背景荧光，将切片浸入苏丹黑色工作溶液中10分钟，在黑暗和室温下。 </li><li>在H 2 O（5分钟，3次）中洗涤切片。 </li><li>使用带有DAPI（0.5μg/ mL）的荧光安装介质安装切片。 </li><li>将切片储存在4°C，检查安装介质是否干燥，然后进行荧光成像。请参见图7中的示例图像。 </li></ol>

作者没有什么可以披露的。

<html>对现有方法的意义: 在这个协议中，我们描述了一种在小鼠中产生不同人源化微环境的方法，并通过双光子显微镜和组织学观察其结构。提供的代表性数据显示了该方法的可行性，使用不同的基质细胞来设计人源化组织。该方案具有在正常和病理状态下研究人类造血细胞和骨髓小生境细胞的具体应用。这些应用包括克隆进化，药物筛选和人类HSCs与基质组分之间的串扰的研究。在新兴的组织工程领域，已经提出了几种替代方法。值得注意的途径包括体外 55，56，57 3D人性化的BM结构的发展，S = "外部参照"&gt; 58,59，60，61，62，63和人源化BM支架的小鼠64原位移植物。我们的方法具有将体内系统的复杂性与人源化组织移植物的易于解剖可及性相结合的优点。 修改和故障排除: 该方案的变异性来源可以在用于种植支架的细胞的选择中找到。在我们的工作中，我们使用BM衍生的hMSCs。然而，间充质细胞可以从几个组织获得，其可以根据来源显示出独特的性质。因此，可以考虑使用来自不同器官的hMSC。然而，它们在体内形成骨组织的能力应在本P中使用前进行测试该协议使用市售的人内皮细胞源（ 即 E4ORF1转导的HUVEC）。最近，使用器官特异性内皮细胞用于不同目的的已报道65，66。此外，使用从BM导出的原代hEC可能代表协议有意义的改进。因此，使用不同来源的内皮细胞可能产生不同的体内结果。 我们使用NSG免疫抑制受体小鼠有利于植入人源化支架并避免组织排斥。我们不排除使用本协议来设计其他小鼠品系异位骨髓组织的可能性。实际上，的rhBMP-2可以诱导不同哺乳动物模型47，48的骨形成，49，50 <sup&gt;， 52 。然而，使用不同的菌株/模型可能观察到细胞活力和长期移植的差异。 支架恢复的时间也可以灵活，这取决于实验的最终目的。在提出的方案中，我们在植入后8-12周恢复样品以评估长期造血移植。为了研究人类BM生物圈形成的早期步骤（ 例如骨软骨组织形成47或血管发育），可以选择不同的时间点。 我们在本协议中描述的活体成像技术用于外植体的短期成像。用于维持生理温度，氧张力，和CO 2浓度的使用的平衡室的应在长期成像的情况下可以考虑，如研究运动的行为。 临界街议定书内的eps 在与协议相关的挑战中，我们将强调一些步骤所需的技术技能。间质和内皮细胞应以低细胞通道数使用;否则，它们将不能够支持人造血细胞植入在体内或参加从头脉管系统和骨形成体内 。我们建议在第1至5代使用hMSC和hEC。支架制备和细胞播种步骤需要基本的细胞培养技术和对该过程中使用的特定细胞的性质的了解。手术方案很简单，但需要一些实践。维持无菌环境以避免免疫缺陷小鼠中植入的支架的污染对于确保实验成功至关重要。样本外植体和活体成像需要外科手术（特别是使用微量钻屑）和知识显微镜系统。最后，样本处理和组织学需要使用的技术的基本知识。 技术局限性: 我们描述的方法允许活人人造血细胞的可视化，种植人源化骨髓微环境，人内皮细胞形成血管结构和形成骨/骨髓空间的间充质细胞。当组织在体内形成时 ，最终的工程支架仍然是嵌合（人和鼠）。应该考虑这个问题，因为嵌合组织可能不能完全模仿人骨髓复杂性和环境。 植入的支架具有有限的尺寸（我们尝试最大为6.6×7.5×7mm），因此它们能够承载有限数量的用于异种移植的细胞。回收细胞的绝对数量也将受到限制;因此，植入支架的数量应该是作为实验所需细胞数的函数计算。 我们描述的成像应用对于从表面观察150-200μm深度的大面积活体组织特别有用，而不会破坏结构并损坏细胞。因此，它不允许整个脚手架的可视化。如果需要对组织进行完全扫描，则标准的免疫荧光方法将更为合适。 未来应用: 该生物工程模型的未来方向将是增加组织中人体成分的复杂性。人类BM小众的知识和特征近年来已有进展67 ，并且所描述的方案可能是研究这些新细胞组分和可溶性因子的功能的有趣的平台，以及它们在支持正常/恶性蚂蚁HSC。 此外，成像技术提供了在纵向研究中对支架进行活体成像的潜力，这将需要在手术后恢复中对活体麻醉的小鼠中支架进行成像的技术改进。这种方法将需要额外的步骤，目前正在实验室进行调查。

在干细胞室中观察到的细胞命运决定被内在因素和外在因素紧密地调节。特别是现在人们普遍认识到，BM微环境在控制HSC从静止状态到活跃状态以及自我更新或分化命运决定1方面发挥了重要作用。此外，最近的发现表明，血液恶性肿瘤影响BM微环境的功能，指向两个隔室2，3，4，5，6之间的串扰活性的存在。尽管最近有进展，但是关于如何使特定的BM-利基组件的活动有助于HSC行为和恶性转化的许多关键问题仍然存在。 BM微环境是一种高度杂种<html>许多不同细胞类型的复杂和复杂的混合物，每种具有专门的功能。丰富的内皮（EC）和血管组分调节营养物和代谢物周转，入口和不同小区的出口和从BM，和几个HSC功能7,8。间质基质细胞（MSC）是通过三种不同谱系（ 即成骨，软骨形成，脂肪形成）分化的未分化干细胞和祖细胞的异质群体，是BM生态位的另一个基本组成部分。这些骨髓间充质干细胞在BM的中心区域和邻近内膜区域都是局部化的。它们可以与血管结构相关联，并在调节牵涉HSC功能9，10，11，12，13，"&gt; 14，15。 许多报告表明，HSCs存在于骨髓内的各种定义的位点，并且它们的功能可能取决于它们的精确定位。关于HSC的大部分现有知识及其与BM微环境的相互作用源自鼠科研1 。利用异种移植物模型的已扩展此知识对人类正常和恶性造血干细胞，免疫缺陷小鼠16，17，18，19，20的鼠BM内移植。虽然这代表了一个有效的模型，但它仍然存在许多挑战，例如在大多数情况下需要调节受体小鼠以允许人类HSC归巢和植入或跨物种屏障及其对细胞 - 细胞相互作用的影响不明确，功能。 使用中和抗体和遗传修饰的小鼠以及异种移植一直在突出人类HSCs与其微环境建立的复杂对话。的引入和活体双光子共焦显微术的发展已经移动这些研究前进了一步，允许直接的，高分辨率，和骨髓19，20，21，22的动态成像和用于功能提供了一种强大的工具表征BM微环境及其在调节HSC功能中的作用。为了规避经典异种移植模型中出现的一些问题，将人性化骨架结构的工程概念引入了前提。合并生物材料和细胞移植概念，报告显示了模拟人类骨骼的可行性在异位区23，24，25，26，27箭头微环境。这将打开使用生物工程在小鼠模型中研究人正常和恶性造血28，29，30，31，32，33，34，35，36，37，38，39，肿瘤发生和转移40，41，42的可能性，43，44。 基于在骨组织工程和体内成像19，第 22，45，46，以前的经验47,48，49，50，51，52，我们描述了一个协议，以生物工程和现场图像器官人类BM组织。这些结构起源于将人BM衍生的基质细胞植入到在免疫缺陷小鼠中皮下移植的基于胶原的支架中。在以前的报告中，我们证明了人类干细胞确保人体微环境，足以满足人体的造血细胞45的植入的形成。此外，这里我们描述如何共同植入其他人类BM细胞成分S，如人内皮细胞（HEC），和/或细胞因子用于骨形成（ 例如，hBMP2转）重要的是，用的hMSCs协作以产生不同的人源微环境，其可以是实时成像原位 。

<table border="0" cellpadding="0" cellspacing="0" width="1378">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Ficoll-Paque</td>
			<td style="width:277px;">Ge Healthcare</td>
			<td style="width:229px;">17-1440-03</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Cd34 positive selection Kit</td>
			<td style="width:277px;">Stemcell</td>
			<td style="width:229px;">18056</td>
			<td style="width:480px;">Store a 4&#176;C.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Magnet</td>
			<td style="width:277px;">Stemcell</td>
			<td style="width:229px;">18000</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Anti-human CD3 antibody, clone OKT-3</td>
			<td style="width:277px;">Bioxcell</td>
			<td style="width:229px;">BE0001-2</td>
			<td style="width:480px;">Store at 4&#176;C. Used for T cell depletion in primary AML samples.&#160;</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:392px;">Cytokines (IL3, G-CSF, and TPO)</td>
			<td style="width:277px;">PeproTech</td>
			<td style="width:229px;">200-03, 300-23 and 300-18</td>
			<td style="width:480px;">For doing the stock: Dilute each one of the cytokines in 100&#956;L of water and mix them. Add 200&#956;L, do alicuots of 45&#956;L and Store at -20&#176;C.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">DMSO</td>
			<td style="width:277px;">Sigma</td>
			<td style="width:229px;">D4540</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:392px;">FBS</td>
			<td style="width:277px;">Life technologies</td>
			<td style="width:229px;">10270106</td>
			<td style="width:480px;">Heat-inactivate at 56&#176;C during 30 minutes, do aliquots and freeze down. Warm in 37&#176;C water bath before use.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">MEM-&#945;</td>
			<td style="width:277px;">Invitrogen</td>
			<td style="width:229px;">32571-028</td>
			<td style="width:480px;">Store at 4&#176;C. Warm in 37&#176;C water bath before use.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Myelocult H5100</td>
			<td style="width:277px;">corning</td>
			<td style="width:229px;">5100</td>
			<td style="width:480px;">Store at 4&#176;C. Warm in 37&#176;C water bath before use.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">199</td>
			<td style="width:277px;">Gibco</td>
			<td style="width:229px;">41150-020</td>
			<td style="width:480px;">Store at 4&#176;C. Warm in 37&#176;C water bath before use.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:392px;">hMSC-FBS, Heat-inactivated</td>
			<td style="width:277px;">Gibco</td>
			<td style="width:229px;">12662-029</td>
			<td style="width:480px;">Store at -20&#176;C. Warm in 37&#176;C water bath before use.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">P/S</td>
			<td style="width:277px;">Sigma-Aldrich</td>
			<td style="width:229px;">P0781</td>
			<td style="width:480px;">Store at -20&#176;C. Warm in 37&#176;C water bath before use.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">ECGS</td>
			<td style="width:277px;">Millipore</td>
			<td style="width:229px;">02-102</td>
			<td style="width:480px;">Dilute in culture media and use 0.22 mm filter.</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:392px;">HEPES</td>
			<td style="width:277px;">Sigma-Aldrich</td>
			<td style="width:229px;">S1558-50ML</td>
			<td style="width:480px;">Store at 4&#176;C.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Heparin</td>
			<td style="width:277px;">Sigma-Aldrich</td>
			<td style="width:229px;">H3149</td>
			<td style="width:480px;">Store at 4&#176;C.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Glutamine</td>
			<td style="width:277px;">Gibco</td>
			<td style="width:229px;">25030</td>
			<td style="width:480px;">Store at -20&#176;C.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Collagen 1 coated cell culture plate</td>
			<td style="width:277px;">corning</td>
			<td style="width:229px;">354505</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Trypsin-EDTA solution</td>
			<td style="width:277px;">Thermo-Fisher</td>
			<td style="width:229px;">25200056</td>
			<td style="width:480px;">Store at -20&#176;C. Warm in 37&#176;C water bath before use.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:392px;">hMSC</td>
			<td style="width:277px;">Lonza</td>
			<td style="width:229px;">PT-2501</td>
			<td style="width:480px;">Alternatively, hMSCs we also kindly provided by Dr. Dosquet (University Paris Diderot, Paris) from human bone marrow obtained during orthopaedic surgery under ethical approval 10-038 from IRB00006477.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">VeraVec HUVEC endothelial cells</td>
			<td style="width:277px;">Angiocrine bioscience</td>
			<td style="width:229px;">hVera101</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:392px;">Human hematopoietic cells</td>
			<td style="width:277px;"></td>
			<td style="width:229px;"></td>
			<td style="width:480px;">Umbilical Cord blood or primary Acute Myeloid Leukemia (AML) samples were obtained from the Royal London Hospital (London, UK) after informed consent and protocol of use was approved by the East London Ethical Committee and carried out in accordance with the Declaration of Helsinki.&#160;</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:392px;">Gelfoam, Size 12-7mm</td>
			<td style="width:277px;">Pfizer</td>
			<td style="width:229px;">00009-0315-08</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">PBS</td>
			<td style="width:277px;">Thermo-Fisher</td>
			<td style="width:229px;">10010023</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Surgical material</td>
			<td style="width:277px;">Multiple</td>
			<td style="width:229px;"></td>
			<td style="width:480px;">Sterile forceps, tweezers and sharp scissors.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Sterile tissues</td>
			<td style="width:277px;"></td>
			<td style="width:229px;"></td>
			<td style="width:480px;">Heat-sterilize paper tissues.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">1 ml Syringe with needle of 25G</td>
			<td style="width:277px;">Terumo</td>
			<td style="width:229px;">SS+01H25161</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:392px;">Ultra Low Attachment Multiple Well Plates</td>
			<td style="width:277px;">Corning</td>
			<td style="width:229px;">3473 or 3471</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">BMP2</td>
			<td style="width:277px;">Noricum</td>
			<td style="width:229px;">rhBMP-2</td>
			<td style="width:480px;">Dilute at 5 mg/ml in acetic acid 50 mM and store at 4 &#176;C.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:392px;">Thrombin</td>
			<td style="width:277px;">Sigma</td>
			<td style="width:229px;">T9010</td>
			<td style="width:480px;">Dilute in CaCl2 2%, 500 mL per vial, and store at 4 &#176;C.</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:392px;">Fibrinogen</td>
			<td style="width:277px;">Sigma</td>
			<td style="width:229px;">F3879</td>
			<td style="width:480px;">Dilute at 4 mg/100 mL in PBS. Store at -20C. Warm before use.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:392px;">Tissue-culture dishes 35 mm x 10 mm</td>
			<td style="width:277px;">Falcon</td>
			<td style="width:229px;">353001</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:392px;">U-Botton 96 well plate</td>
			<td style="width:277px;">Falcon</td>
			<td style="width:229px;">353077</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:392px;">NSG mice</td>
			<td style="width:277px;">The Jackson Laboratory</td>
			<td style="width:229px;">5557</td>
			<td style="width:480px;">NSG mice were a kind gift from Dr Leonard Shultz (The Jackson Laboratory).</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Chlorhexidine</td>
			<td style="width:277px;">G9</td>
			<td style="width:229px;"></td>
			<td style="width:480px;">Dilute 1:10 before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Carprofen</td>
			<td style="width:277px;">Pfizer</td>
			<td style="width:229px;">Rimadyl</td>
			<td style="width:480px;">5 mg/g of mouse</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Buprenorphine</td>
			<td style="width:277px;">Alstoe</td>
			<td style="width:229px;">Vetergesic</td>
			<td style="width:480px;">0.1 mg/g of mouse body weight</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Isoflurane</td>
			<td style="width:277px;">Abbott</td>
			<td style="width:229px;">B506</td>
			<td style="width:480px;">Induction of anaesthesia 2%, maintenance 1%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Trimmer</td>
			<td style="width:277px;">Wella</td>
			<td style="width:229px;">Contura HS61</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Surgical glue</td>
			<td style="width:277px;">3M</td>
			<td style="width:229px;">Vetbond</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">carbomer (polyacrylic acid)&#160; as Ophthalmic gel</td>
			<td style="width:277px;">Novartis</td>
			<td style="width:229px;">Viscotears Liquid Gel</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:392px;">Human Normal Immunoglobulin</td>
			<td style="width:277px;">Gammaplex</td>
			<td style="width:229px;">10g vial</td>
			<td style="width:480px;">100 ml/mouse intravenously, 30 min before infusion of specific antibody.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:392px;">NT-QTracker</td>
			<td style="width:277px;">Invitrogen</td>
			<td style="width:229px;">Q21021MP</td>
			<td style="width:480px;">Vessel-pooling agent. Administrate 15 ml/mouse intravenously 5 min before imaging.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">AF488-hCD45</td>
			<td style="width:277px;">Biolegend</td>
			<td style="width:229px;">304017</td>
			<td style="width:480px;">100 ml/mouse intravenously, 30 min before imaging.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">FITC-hCD31</td>
			<td style="width:277px;">BD Pharmigen</td>
			<td style="width:229px;">555445</td>
			<td style="width:480px;">100 ml/mouse intravenously, 30 min before imaging.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Super glue</td>
			<td style="width:277px;">Loctite&#160;</td>
			<td style="width:229px;">Super Glue</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Micro-Drill Kit</td>
			<td style="width:277px;">IDEAL - Fisher Scientific</td>
			<td style="width:229px;">NC9010016</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microsurgical microscope</td>
			<td style="width:277px;"></td>
			<td style="width:229px;"></td>
			<td style="width:480px;">No specific brand/company is adviced.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:392px;">LSM 710 NLO</td>
			<td style="width:277px;">Zeiss</td>
			<td style="width:229px;"></td>
			<td style="width:480px;">Upright confocal microscope with motorized stage, two-photon laser and 20x 1.0 NA water immersion lens. Alternatively, a microscope with similar specifications could be used.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:392px;">MaiTai &#8220;High Performance&#8221; fully automated 1-box 517 mode-locked Ti:Sapphire laser with DeepSee dispersion compensation</td>
			<td style="width:277px;">&#160;Spectra-Physics</td>
			<td style="width:229px;"></td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Formalin solution, neutral buffered, 10%&#160;</td>
			<td style="width:277px;">Sigma-Aldrich</td>
			<td style="width:229px;">HT501128</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Ethanol</td>
			<td style="width:277px;">Sigma-Aldrich</td>
			<td style="width:229px;">32294</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Osteosoft</td>
			<td style="width:277px;">Millipore</td>
			<td style="width:229px;">1.01728.1000</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Polysine slices</td>
			<td style="width:277px;">Thermo scientific</td>
			<td style="width:229px;">J2800AMNZ</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:392px;">Antigen unmasking solution</td>
			<td style="width:277px;">Vector</td>
			<td style="width:229px;">H-3300</td>
			<td style="width:480px;">Store at 4 &#176;C. Dilute 1:100 in H2O for working solution.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Triton 100x</td>
			<td style="width:277px;">Sigma-Aldrich</td>
			<td style="width:229px;">T9284</td>
			<td style="width:480px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Bovine serum albumin (BSA)</td>
			<td style="width:277px;">Sigma-Aldrich</td>
			<td style="width:229px;">A96470</td>
			<td style="width:480px;">Store at 4 &#176;C.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Normal Goat serum (NGS)</td>
			<td style="width:277px;">Sigma-Aldrich</td>
			<td style="width:229px;">G9023</td>
			<td style="width:480px;">Store at 4 &#176;C.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Endomucin Antibody</td>
			<td style="width:277px;">Santa Cruz</td>
			<td style="width:229px;">sc-65495</td>
			<td style="width:480px;">Store at 4 &#176;C.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">hVimentin antibody</td>
			<td style="width:277px;">Santa Cruz</td>
			<td style="width:229px;">sc-6260</td>
			<td style="width:480px;">Store at 4 &#176;C.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">hCD31 antibody</td>
			<td style="width:277px;">DAKO</td>
			<td style="width:229px;">M0823</td>
			<td style="width:480px;">Store at 4 &#176;C.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">hCD45 antibody</td>
			<td style="width:277px;">DAKO</td>
			<td style="width:229px;">M0701</td>
			<td style="width:480px;">Store at 4 &#176;C.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">ADRP (Perilipin2)</td>
			<td style="width:277px;">antibodies-online</td>
			<td style="width:229px;">ABIN283918</td>
			<td style="width:480px;">Store at 4 &#176;C.</td>
		</tr>
		<tr height="59">
			<td height="59" style="height:59px;width:392px;">Goat anti mouse secondary antibodies</td>
			<td style="width:277px;">Invitrogen</td>
			<td style="width:229px;">A11029 or A11005</td>
			<td style="width:480px;">Store at 4 &#176;C.</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:392px;">Goat anti Rabbit secondary antibodies</td>
			<td style="width:277px;">Invitrogen</td>
			<td style="width:229px;">A11037 or A11008</td>
			<td style="width:480px;">Store at 4 &#176;C.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Goat anti Rat secondary antibodies</td>
			<td style="width:277px;">Invitrogen</td>
			<td style="width:229px;">A11007 or A21247</td>
			<td style="width:480px;">Store at 4 &#176;C.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:392px;">Sudan Black</td>
			<td style="width:277px;">Sigma</td>
			<td style="width:229px;">S2380</td>
			<td style="width:480px;">Prepare a stock of 1% sudan black in ethanol 70%. Store at RT. Prepare working solution of 0.1% sudan black in ethanol 70% and filter using filter-paper before use.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:392px;">DAPI</td>
			<td style="width:277px;">Sigma</td>
			<td style="width:229px;">D8417</td>
			<td style="width:480px;">Prepare stock in H20 at 100 mg/mg. Store at 4 &#176;C.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Fluorescent mounting media</td>
			<td style="width:277px;">Dako</td>
			<td style="width:229px;">S3023</td>
			<td style="width:480px;">Add DAPI before use (1:400 from DAPI stock).</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:392px;">Cover glass</td>
			<td style="width:277px;">VWR</td>
			<td style="width:229px;">631-0147</td>
			<td style="width:480px;"></td>
		</tr>
	</tbody>
</table>

bioengineering of humanized bone marrow microenvironments in mouse and their visualization by live imaging

人类造血干细胞（HSCs）存在于骨髓（BM）小生境中，这是一个复杂的多因素网络，其产生细胞因子，生长因子和细胞外基质。 HSC保持静止，自我更新或分化，获得突变并变得恶性的能力取决于它们与不同基质成分的复杂相互作用。为了观察生理和病理状态下人类HSCs与人骨骼生物圈之间的串扰，我们设计了一个方案来对免疫缺陷小鼠的人源化BM细胞进行异位模拟和成像。我们表明使用不同的细胞成分可以形成人性化的结构，并有机会维持长期的人体造血移植。使用双光子显微镜，我们可以在单细胞分辨率下原位形成这些结构，为人类BM的功能表征提供了强大的新工具微环境及其在调节正常和恶性造血作用。

在图1中 ，示出了支架细胞接种和植入过程的代表性图像。在图1C中 ，注意到细胞被直接注入到支架中。在图1G中 ，注意到在小鼠背部进行切口，其中皮下袋被创建并且支架被植入。 图2显示了在NSG小鼠中植入的不同支架的总体形态，并在8周后检索。注意hMSC种子支架中的轻微血管形成（ 图2A ）。在支架中将人EC与hMSC共同接种可以在支架中形成更相关的脉管系统（ 图2B ）。最后，rhBMP-2的存在诱导骨形成。在这种情况下，检索到的脚手架较大，它们由...构成骨骼类似的硬组织。 图3显示了使用NDD实时成像的显微镜上的通道配置设置（图中的细节）。 图4和视频1显示hMSC包被的支架中的人类造血细胞。支架植入后8周，静脉注射AF488-hCD45抗体和655-VPA后移植。该过程允许通过双光子共焦显微镜观察植入的人造血细胞和血管结构。在这种情况下，图像显示支架中的血管（655-VPA）和人造血细胞（AF488-hCD45）在支架实质中的长期植入。 图5和视频2对应于用hECs和hMSC种植的人类支架。手术后8周，静脉内移植支架接种FITC-hCD31抗体和655-VPA，并且如前所述用双光子共聚焦显微镜获得图像。图像显示hEC在支架中的血管形成中的参与，导致鼠人类嵌合脉管系统。 图6A显示用于刺激MSC支架中的骨形成的方法的代表性数据。与以前的数据相似，植入后8周，进行了655-VPA的静脉注射，取出支架，用双光子共聚焦显微镜获取图像。 rhBMP-2刺激的支架诱导骨组织的形成，由于骨骼中的钙提供的SHG（图像中的青色）可以形象化。所提供的图像还显示空腔和血管化的内膜组织的形成，其与BM内膜组织非常相似。在图6B中 </strong&gt;和Video 3 ，hECs共同植入hMSC。在FITC-hCD31抗体和655-VPA的静脉内接种后检索支架，双光子共焦显微镜图像显示hEC参与骨形成支架的新生血管形成。 图7显示了组织学的代表性图像，该过程是为了证实先前描述的结果而进行的。免疫荧光图像显示支架结构中的小鼠血管系统，hECs，hMSC和长期植入人造血细胞。将从小鼠检索的支架固定并用于免疫荧光。在rhBMP-2载体骨形成支架（ 图7D- F ）中，注意组织的形态，类似成熟骨髓与脂肪组织。在这种骨形成支架中，我们显示hMSCs是成纤维细胞，这将表示t他们对新形成的组织作为基质细胞有贡献。我们还显示人类脂肪细胞标志物表达，这表明hMSCs也有助于脂肪组织形成。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55914/55914fig1.jpg" /> 图1.细胞播种和植入过程的代表性图像。 A）使用手术刀的初步支架及其切割方法。 B）从初始支架获得的24件。 C）使用注射器的脚手架细胞播种方法。 D）用培养基进行细胞种植的支架，准备植入。 EF）骨形成支架的具体步骤: E）将支架转移到96孔，u底板， F）用于将rhBMP2，凝血酶和纤维蛋白原添加到支架中的方法的代表性图像。 GJ） Surgi<html>全身麻醉下的植入手术: G）在皮肤和支架植入中创建的伤口， H）植入法和IJ）使用手术胶的伤口闭合程序。 <a href="http://ecsource.jove.com/files/ftp_upload/55914/55914fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/55914/55914fig2.jpg" /> 图2.从小鼠检索的不同支架。 MSC支架（左），MSC + EC支架（中）和MSC + EC + BMP支架（右）的代表性图像。 <a href="http://ecsource.jove.com/files/ftp_upload/55914/55914fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 4 / 55914fig3.jpg"/&gt; 图3.通道配置。显示显微镜过滤器设置。 A）有四个NDD检测器模块:在第一个模块中有两个过滤器立方体;第二和第三模块各有一个过滤器立方体;最后一个模块没有立方体（未使用）。第一个光电倍增管（PMT）用于远红外通道（640-690nm），反映较低的波长;以下是380-485nm，500-550nm和555-625nm（顺序总是从较低到较高的波长）。 B）用上述配置可检测的荧光团排放（颜色编码）。 <a href="http://ecsource.jove.com/files/ftp_upload/55914/55914fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图4" class="xfigimg" src="/files/ftp_upload/55914/55914fig4.jpg" /> 图4. MSC支架<html>允许形成人类造血细胞的利基。 A）和B）采用AF488-hCD45（绿色）静脉内接种后标记人类造血细胞和655-VPA（品红色）m标记脉管系统后，采取后移植后的Z-叠层的3D重建。刻度棒在A和B（上图）中为20μm，B为5μm（下图）。 <a href="http://ecsource.jove.com/files/ftp_upload/55914/55914fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="视频1" src="/files/ftp_upload/55914/55914video1.jpg" /> 视频1. MSC支架允许形成人类造血细胞的利基 。在MSC支架（胶原结构:SHG，青色）中与血管系统（655-VPA）相关的人类造血细胞（AF488-hCD45）的3D重建。每个堆叠尺寸为140 x 140μm。ef ="http://ecsource.jove.com/files/ftp_upload/55914/55914video1.mov"target ="_ blank"&gt;请点击此处查看此视频。 （右键单击下载。） <img alt="图5" class="xfigimg" src="/files/ftp_upload/55914/55914fig5.jpg" /> 图5.人类EC参与MSC支架中人造血管的形成。 MSC + EC支架中由人源EC（FITC-hCD31）排列的脉管系统（655-VPA）的3D重建。刻度尺表示20μm。 <a href="http://ecsource.jove.com/files/ftp_upload/55914/55914fig5large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="视频2" src="/files/ftp_upload/55914/55914video2.jpg" /> 视频2.人类EC参与MSC支架的人性化船只的形成。人类EC的三维重建（FITC-hCD31）衬里va<html>在MSC + EC支架中的血清（655-VPA）。每个堆叠尺寸为240 x 240μm。 <a href="http://ecsource.jove.com/files/ftp_upload/55914/55914video2.mov" target="_blank">请点击此处查看此视频。 （右键单击下载。）</a> <img alt="图6" class="xfigimg" src="/files/ftp_upload/55914/55914fig6.jpg" /> 图6. rhBMP-2载体支架具有类似于骨髓的骨表面和人源化血管。 A） MSC + BMP支架的3D重建，显示骨结构（SHG-青色）和脉管系统（655-VPA）的形成。刻度棒表示100μm（左），70μm（中）和50μm（右）。 B）显示人源化血管（655-VPA）排列人EC（FITC-hCD31）的MSC + EC + BMP支架的3D重建。刻度棒表示50μm（左）和30μm（中间和右侧）。14 / 55914fig6large.jpg"target ="_ blank"&gt;请点击此处查看此图的较大版本。 <img alt="视频3" src="/files/ftp_upload/55914/55914video3.jpg" /> 视频3. rhBMP-2载体支架具有类似于骨髓的骨表面和人源化血管。 MSC + VERA + BMP支架的三维重建（骨:SHG;血管:655-VPA;人ECs:FITC-hCD31）。每个堆叠尺寸为600 x 600μm。 <a href="http://ecsource.jove.com/files/ftp_upload/55914/55914video3.mov" target="_blank">请点击此处查看此视频。 （右键单击下载。）</a> <img alt="图7" class="xfigimg" src="/files/ftp_upload/55914/55914fig7.jpg" /> 图7.在固定支架上进行的免疫荧光的代表性图像。 AF）进行免疫荧光研究以定位植入的人类细胞n脚手架。 AC）植入hECs，hMSC和hHSC的支架。 DF）骨形成支架植入hECs，hMSC和hHSC。颜色通道如下: AD）人类EC（hCD31）和小鼠血管结构（endomucin，ENDOM）， BE） hMSC（hVimentin，hVIM）和小鼠血管细胞（endomucin，ENDOM）， C）人造血细胞（hCD45）和小鼠血管系统（endomucin，ENDOM）和F）人类脂肪分化相关蛋白（hADRP）和小鼠血管系统（endomucin，ENDOM）。刻度尺表示10μm（A和C），20μm（B和E）和40（D和F）μm。 <a href="http://ecsource.jove.com/files/ftp_upload/55914/55914fig7large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging at JoVE.com

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

提出了一种在小鼠中创建和实时映射不同人源化骨髓小生境的方法。基于由人间充质细胞产生的支持性利基，人内皮细胞的添加诱导人血管的形成，而添加rhBMP-2诱导人 - 小鼠嵌合成熟骨组织的形成。

人类骨髓微环境在小鼠中的生物工程及其通过实时成像的可视化

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

bioengineering-humanized-bone-marrow-microenvironments-mouse-their

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

11.6K 次观看.  The Francis Crick Institute. 提出了一种在小鼠中创建和实时映射不同人源化骨髓小生境的方法。基于由人间充质细胞产生的支持性利基，人内皮细胞的添加诱导人血管的形成，而添加rhBMP-2诱导人 - 小鼠嵌合成熟骨组织的形成。 

视频: Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Isolation of Human Mesenchymal Stem Cells and their Cultivation on the Porous Bone Matrix

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs bovine bone matrix Nukbone (NKB) as a scaffold. Thus, the application of MSCs in repair and tissue regeneration processes depends principally on the efficient implementation of the techniques for placing these cells in a host tissue. For this reason, the design of biomaterials and cellular scaffolds has gained importance in recent years because the topographical characteristics of the selected scaffold must ensure adhesion, proliferation and differentiation into the desired cell lineage in the microenvironment of the injured tissue. This option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) employs bovine bone matrix as a cellular scaffold and is an efficient culture technique because the cells respond to the topographic characteristics of the bovine bone matrix Nukbone (NKB), i.e., spreading on the surface, macroporous covering and colonizing the depth of the biomaterial, after the cell isolation process. We present the procedure for isolating and culturing MSCs on a bovine matrix.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs the bovine bone matrix Nukbone (NKB) as a scaffold.

人类骨髓间充质干细胞的分离及其培养的多​​孔骨基质

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific ...

科研

发育生物学

An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade clots in the bone marrow and release MSCs for further use. This protocol provides a rapid and inexpensive alternative to bone marrow resampling. Bone marrow is a major source of MSCs, which are interesting for tissue engineering and autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system. The resulting clots contain also MSCs that are lost for expansion culture or direct stem cell therapy. We experienced that 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Thus, we developed a protocol for enzymatic digestion of those clots to avoid labor-intense and costly bone marrow resampling. Urokinase - a clinically approved and readily available thrombolytic drug &#8211; clears away the bone marrow clots almost completely. As a consequence, treated bone marrow aspirates yield similar numbers of MSCs as unclotted samples. Also, after urokinase treatment the cells kept their metabolic activity and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. Our protocol salvages clotted blood and bone marrow samples without affecting the quality of the cells. This obsoletes resampling, considerably reduces sampling costs and enables the use of clotted samples for research or therapy.

Mesenchymal stem cells are usually obtained from bone marrow and require expansion culture. When samples clot before processing, a protocol using the (enzymatic) thrombolytic drug urokinase can be applied to degrade the clot. Thus, cells are released and available for expansion culture. This protocol provides a rapid and inexpensive alternative to resampling.

酶法从凝块骨髓样本拯救间充质干细胞

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade ...

Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel cell population specifically selected for growth in human serum supplemented medium. These cells are characterized by morphological, phenotypic, and molecular features distinct from MSCs and we named them Mesodermal Progenitor Cells (MPCs). MPCs are round, with a thick highly refringent core region; they show strong, trypsin resistant adherence to plastic. Failure to expand MPCs directly revealed that they are slow in cycling. This is as also suggested by Ki-67 negativity. On the other hand, culturing MPCs in standard medium designed for MSC expansion, gave rise to a population of exponentially growing MSC-like cells. Besides showing mesenchymal differentiation capacity MPCs retained angiogenic potential, confirming their multiple lineage progenitor nature. Here we describe an optimized highly reproducible protocol to isolate and characterize hBM-MPCs by flow cytometry (CD73, CD90, CD31, and CD45), nestin expression, and F-actin organization. Protocols for mesengenic and angiogenic differentiation of MPCs are also provided. Here we also suggest a more appropriate nomenclature for these cells, which has been re-named as "Mesangiogenic Progenitor Cells".

Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow

Here we describe an optimized, highly reproducible protocol to isolate Mesodermal Progenitor Cells (MPCs) from human bone marrow (hBM). MPCs were characterized by flow cytometry and nestin expression. They showed the ability to give rise to exponentially growing MSC-like cell cultures while retaining their angiogenic potential.

从人骨髓分离Mesangiogenic祖细胞（多用途储值卡）

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel ...

Live Confocal Imaging of Developing Arabidopsis Flowers

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. Recent advances in confocal microscopy, combined with the development of numerous fluorophores, have overcome these issues and opened the possibility to study the expression of several genes simultaneously, with a good cellular resolution, in live samples. Live confocal imaging provides plant biologists with a powerful tool to study development, and has been extensively used to study root growth and the formation of lateral organs on the flanks of the shoot apical meristem. However, it has not been widely applied to the study of flower development, in part due to challenges that are specific to imaging flowers, such as the sepals that grow over the flower meristem, and filter out the fluorescence from underlying tissues. Here, we present a detailed protocol to perform live confocal imaging on live, developing Arabidopsis flower buds, using either an upright or an inverted microscope.

Live confocal imaging provides biologists with a powerful tool to study development. Here, we present a detailed protocol for the live confocal imaging of developing Arabidopsis flowers.

住发展拟南芥花的共焦成像

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. ...

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where they act as front line sentinels of infection and tissue damage. While other immune cells are continuously renewed from hematopoietic stem and progenitor cells (HSPC) located in the bone marrow, a lineage of macrophages, known as resident macrophages, have been shown to be self-maintained in tissues without input from bone marrow HSPCs. This lineage is exemplified by microglia in the brain, Kupffer cells in the liver and Langerhans cells in the epidermis among others. The intestinal and colon lamina propria are the only adult tissues devoid of HSPC-independent resident macrophages. Recent investigations have identified that resident macrophages originate from the extra-embryonic yolk sac hematopoiesis from progenitor(s) distinct from fetal hematopoietic stem cells (HSC). Among yolk sac definitive hematopoiesis, erythromyeloid progenitors (EMP) give rise both to erythroid and myeloid cells, in particular resident macrophages. EMP are only generated within the yolk sac between E8.5 and E10.5 days of development and they migrate to the fetal liver as early as circulation is connected, where they expand and differentiate until at least E16.5. Their progeny includes erythrocytes, macrophages, neutrophils and mast cells but only EMP-derived macrophages persist until adulthood in tissues. The transient nature of EMP emergence and the temporal overlap with HSC generation renders the analysis of these progenitors difficult. We have established a tamoxifen-inducible fate mapping protocol based on expression of the macrophage cytokine receptor Csf1r promoter to characterize EMP and EMP-derived cells in vivo by flow cytometry.

While infiltrating macrophages are continuously recruited to adult tissues from circulating precursors, resident macrophages seed their tissue during development, where they are maintained without further input from progenitors. The progenitors for resident macrophages were recently identified. Here, we present methods for the genetic fate mapping of the resident macrophage progenitors.

通过流式细胞术鉴定小鼠胚胎中的红细胞生成素及其后代

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos.

体外在拟南芥中受精卵分化和胚模式的活体细胞成像的胚珠培养

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

小鼠第二腭融合的实时成像

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result in a series of bone metabolic diseases, including osteoporosis. To develop pharmaceutical targets for the prevention of pathological bone mass loss, the mechanisms by which osteoclasts differentiate from precursors must be understood. The ability to isolate and culture a large number of osteoclasts in vitro is critical in order to determine the role of specific genes in osteoclast differentiation. Inactivation of the mammalian/mechanistic target of rapamycin complex 1 (TORC1) in osteoclasts can decrease osteoclast number and increase bone mass; however, the underlying mechanisms require further study. In the present study, a RANKL-based protocol to isolate and culture osteoclasts from mouse bone marrow and to study the influence of mTORC1 inactivation on osteoclast formation is described. This protocol successfully resulted in a large number of giant osteoclasts, typically within one week. Deletion of Raptor impaired osteoclast formation and decreased the activity of secretory tartrate-resistant acid phosphatase, indicating that mTORC1 is critical for osteoclast formation.

This manuscript describes a protocol to isolate and culture osteoclasts in vitro from mouse bone marrow, and to study the role of the mammalian/mechanistic target of rapamycin complex 1 in osteoclast formation.

RANKL 小鼠骨髓破骨细胞培养法研究 mTORC1 在破骨细胞形成中的作用

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result ...

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

In this article, we present methods to isolate and differentiate bone marrow stromal cells and hematopoietic stem cells from mouse long bones. Two different protocols are presented yielding different cell populations suitable for expansion and differentiation into osteoblasts, adipocytes, and osteoclasts.

小鼠骨髓基质细胞与骨破骨祖的分离、培养及分化

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

有丝分裂过程中染色体分离的活体细胞成像

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

人类骨髓微环境在小鼠中的生物工程及其通过实时成像的可视化

摘要

探索更多视频

此视频中的章节

人类骨髓间充质干细胞的分离及其培养的多孔骨基质

酶法从凝块骨髓样本拯救间充质干细胞

从人骨髓分离Mesangiogenic祖细胞（多用途储值卡）

住发展拟南芥花的共焦成像

通过流式细胞术鉴定小鼠胚胎中的红细胞生成素及其后代

体外在拟南芥中受精卵分化和胚模式的活体细胞成像的胚珠培养

小鼠第二腭融合的实时成像

RANKL 小鼠骨髓破骨细胞培养法研究 mTORC1 在破骨细胞形成中的作用

小鼠骨髓基质细胞与骨破骨祖的分离、培养及分化

有丝分裂过程中染色体分离的活体细胞成像

人类骨髓微环境在小鼠中的生物工程及其通过实时成像的可视化

摘要

探索更多视频

此视频中的章节

人类骨髓间充质干细胞的分离及其培养的多​​孔骨基质

酶法从凝块骨髓样本拯救间充质干细胞

从人骨髓分离Mesangiogenic祖细胞（多用途储值卡）

住发展拟南芥花的共焦成像

通过流式细胞术鉴定小鼠胚胎中的红细胞生成素及其后代

体外在拟南芥中受精卵分化和胚模式的活体细胞成像的胚珠培养

小鼠第二腭融合的实时成像

RANKL 小鼠骨髓破骨细胞培养法研究 mTORC1 在破骨细胞形成中的作用

小鼠骨髓基质细胞与骨破骨祖的分离、培养及分化

有丝分裂过程中染色体分离的活体细胞成像

人类骨髓间充质干细胞的分离及其培养的多孔骨基质