Name: real-time imaging of ccl5-induced migration of periosteal skeletal stem cells in mice
Uploaded: 2020-09-16T14:00:00.000+00:00
Duration: 6 min 10 s
Description: Watch this Scientific Journal Video about Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice at JoVE.com

这项工作得到了德克萨斯州骨病计划奖，Caroline Wiess法律基金奖和美国国立卫生研究院NIAMS的支持，奖项编号为R01 AR072018，R21 AG064345，R01 CA221946至D.P。 我们感谢M.E. Dickinson和T.J. Vadakkan在BCM Optical Imaging and Vital Microscopy Core 以及BCM Advanced Technology Cores的资助下，由NIH资助（AI036211，CA125123和DK056338）。
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所有小鼠都保持在无病原体的条件下，所有程序均由贝勒医学院的机构动物护理和使用委员会（IACUC）批准。
1. 鼠标准备
<ol><li>将Mx1-Cre17和Rosa26-loxP-stop-loxP-tdTomato（Tom）18只小鼠（从杰克逊实验室购买）与αSMA-GFP小鼠（由Ivo Kalajzic博士和Henry Kronenberg博士提供）杂交，生成Mx1-Cre + Rosa26-Tomato + α SMA-GFP+（Mx1 / Tomato / αSMA-GFP）报告小鼠（图2A）。</li>
	<li>对于 Mx1-Cre诱导，每隔一天向小鼠注射25mg / kg pIpC，持续10天。</li>
	<li>在静脉移植来自野生型C57BL / 6 小鼠（WT-BMT）的1 x 10 6全骨髓单核细胞之前1天用9.5 Gy照射小鼠19。</li>
	<li>在恢复6至8周后，对小鼠进行如下所述的骨膜细胞迁移方案的体内成像。 注意: Mx1-Cre小鼠的辐照和WT-BMT对于消除可能对损伤有反应的内源性 Mx1标记造血细胞是必要的。照射后六到八周，宿主的造血细胞计数为<5%，因此可以进行成像19。</li>
</ol>2. 鼠标约束和成像支架
<ol><li>使用50 mL锥形管创建鼠标约束（图1A）。<ol>
<li>使用尖头剪刀，在圆锥体的底部戳一个洞。从底部孔切割到锥形的顶部（切割侧将是约束的顶部）。</li>
		<li>切口与前一个切口平行约4-5厘米长，从约束装置顶部向下一半（约2厘米）。去除这一部分的圆锥形并平滑边缘是必要的。</li>
	</ol>
</li>
	<li>使用可调节角度板，0.5"光学柱和0.5"柱，0.1875"六角的直角夹具构建锥形管成像支架（图1B 和 材料表）。<ol>
<li>将角板调整为 35° 角。在角板的左侧，将光学柱从板的背面拧入第二个孔中。</li>
		<li>在可调节板的右侧，将弹簧加载的0.1875"六角锁定0.25"指旋螺钉从板上的前孔和最后一个孔插入第二个孔中。直角夹具将与光学柱一起使用，以将锥形鼠标约束固定到位。</li>
	</ol>
</li>
</ol>3. 术前外科手术
<ol><li>小鼠麻醉<ol>
<li>对于疼痛管理，在手术前30-60分钟皮下注射缓释丁丙诺啡（1mg / kg BW）。</li>
		<li>使用含有37.5mg / mL氯胺酮，1.9mg / mL木拉嗪和0.37mg / mL乙丙嗪的啮齿动物IIICCM组合（组合III）麻醉小鼠。小鼠的适当剂量为每1公斤体重0.75-1.50毫升。剂量将持续30-45分钟。</li>
		<li>麻醉小鼠后，用无菌棉签将眼药膏涂抹在眼睛上，以防止眼睛脱水。</li>
		<li>对于长期影像学检查（>45分钟），再次进行麻醉以保持适当的麻醉深度，如下所示。<ol>
<li>准备一个1 mL注射器，其中含有足够量的组合III，以在计划的成像持续时间内保持麻醉（每30分钟额外镇静0.75 mL / kg BW）。将带有12英寸管的25 G蝴蝶针输液装置连接到注射器上，然后将麻醉剂推入管路和针头。</li>
			<li>将蝴蝶针插入腹腔内，然后将针头粘到位。 注意:这就是在成像期间施用附加组合III的方式，这样就不需要移动动物或破坏视平面。</li>
		</ol>
</li>
		<li>在整个过程中，通过保持在37°C的加热垫提供热支持。对于长时间的影像学检查（>1小时），需要额外的液体支持。 在影像学检查期间，可向 IP 患者提供补充液体和额外的 Combo III，或皮下注射 33 mL/kg 生理盐水 （0.9 NaCl）。</li>
	</ol>
</li>
	<li>小鼠约束和手术部位的准备<ol>
<li>使用剪刀尽可能多地去除头顶的头发。将脱毛膏涂抹在剃须区域，以去除手术部位的任何剩余毛发。</li>
		<li>确保鼠标因对脚趾捏合缺乏反应而完全麻醉。15分钟后，如果小鼠未完全麻醉，则给予第二小剂量的组合III（<0.75mL / kg BW）。</li>
		<li>首先将鼠标放在约束尾部，向后滑动足够远，使鼻子略微悬挂在圆锥形的边缘上。</li>
		<li>使用医用胶带将前爪粘在锥形管的底部。</li>
		<li>将带鼠标的锥形管放在设置为35°角的成像支架上，并使用直角夹具固定（图1C）。</li>
		<li>一旦小鼠固定在成像支架中，用β定磨砂膏和70%异丙醇交替擦洗3次手术部位。再次，检查动物是否完全镇静。</li>
		<li>将鼠标移动到干净的吸水垫上，并在鼠标上放置一个悬垂物以创建一个大的无菌区域。</li>
	</ol>
</li>
</ol>4. 外科手术
<ol><li>开口切口<ol>
<li>使用细尖镊子，立即将皮肤从内侧到右耳。用显微切割剪刀，切开被镊子固定的皮肤。 注意:使用上述方法启动切口会产生一个圆形切口，更适合在手术后缝合。</li>
		<li>从初始切口开始向左耳（<1厘米）做一个横向切口。 从初始切口开始，再切口朝向动物的鼻子（右眼后约2-3毫米）。</li>
		<li>使用镊子和剪刀将皮肤与骨膜分开，轻轻切开结缔组织，将皮肤与骨膜隔开。</li>
		<li>使用无菌棉签，将一些眼药膏涂抹在皮瓣的顶部。 轻轻拿起皮瓣，将其折叠在左眼上。矢状缝合线和冠状缝合线的交点应清晰地暴露（图1E）。 注意:眼科软膏用于保持皮瓣到位。</li>
		<li>用无菌PBS冲洗开放表面，以清洁任何血液和残留头发的区域。 注意:如有必要，请使用细尖镊子去除冲洗后残留的毛发，但注意不要损坏骨膜。</li>
	</ol>
</li>
	<li>微骨折<ol>
<li>为了在颅骨上产生微骨折，请取出29 G胰岛素注射器的柱塞，并切割注射器的后端，大约300-400 uL标记。 注意:此步骤使得在产生伤害时更容易在解剖镜下操作。</li>
		<li>将斜角的尖端（一到两个针宽）从冠状缝合线朝向鼻子，将一到两个针宽放在矢状缝合线的右侧（图1E）。</li>
		<li>顺时针轻轻旋转注射器，然后逆时针旋转几次。 注意:在年轻小鼠中，需要非常少量的向下压力来产生圆形微骨折。注意不要让针头穿透大脑，因为这会导致出血并干扰成像。</li>
		<li>使用27 G针将微骨折扩大至~0.4 mm。如果骨碎片仍留在微骨折中，请用PBS冲洗该区域。如果骨碎片仍然留在微骨折中，请使用29 G针轻轻地将其舀出。</li>
		<li>重复步骤4.2.1-4.2.4，在矢状缝合线左侧造成损伤。 注意:此时，有两种选择:1）立即进行CCL5治疗（第4.3节）和成像（第5节）;或2）在第6节中的术后程序后关闭手术部位。24小时后，再次麻醉小鼠，重新打开手术视力，用CCL5治疗损伤（第4.3节），并进行活体成像（第5节）和术后护理（第6节）。</li>
	</ol>
</li>
	<li>CCL5 处理<ol>
<li>从兰特斯（100 ng / mL）的储备中，使用基底膜基质（材料表）制备10 ng / mL的溶液。将这种溶液保持在冰上，以保持基质为液体形式。</li>
		<li>使用2-20μL移液器用2μLCCL5（10ng / μL）治疗每个损伤（该移液器吸头具有更大的直径，使用粘性液体时更有效）。让基质凝固。</li>
		<li>一旦基质凝固，用皮瓣轻轻覆盖颅骨，以确保组织不会脱水。在成像前孵育1小时。</li>
	</ol>
</li>
</ol>5. 活体成像
<ol><li>显微镜设置<ol>
<li>打开多光子激光器（飞秒钛蓝宝石激光器）。将波长设置为 880 nm，并调整骨骼二次谐波发生 （SHG） 成像 （440 nm） 的功率。</li>
		<li>打开488 nm和561 nm固体激光器，分别激发GFP和tdTomato表达细胞。</li>
		<li>打开 PMT（光电倍增管）探测器，用于二次谐波生成（405–455 nm 检测）和 GFP（505–550 nm 检测）信号。打开 HyD 3 检测器以检测番茄（590–620 nm 检测）信号。</li>
	</ol>
</li>
	<li>安装<ol>
<li>在将鼠标移动到示波器之前，请轻轻按压鼠标后脑勺来检查颅骨的视觉平面。如果平面不水平，请通过向左或向右轻轻旋转鼠标约束来调整位置。 注:这是确保足够图像质量的关键步骤。</li>
		<li>将无菌的2%甲基纤维素在水中（w / v）中涂抹，以覆盖整个颅骨和皮瓣，并防止组织干燥。</li>
		<li>将鼠标放在XYZ轴电动显微镜载物台上（图1G）。使用落射荧光，对齐鼠标，使其1）面向显微镜，2）冠状和矢状缝合线的交点是载物台的中心参考点。</li>
		<li>将玻璃盖放在成像区域并仔细检查对齐情况（图1H）。</li>
	</ol>
</li>
	<li>延时成像<ol>
<li>使用低放大倍率镜头（25倍水浸物镜，NA = 0.95）扫描钙质。获取矢状肌和冠状缝合线交叉的参考图像。</li>
		<li>使用来自细胞的SHG和荧光信号，定位每个损伤视力并记录XYZ坐标以及与矢状肌和冠状缝合线的距离（图2B）。</li>
		<li>在冠状或矢状缝合线上选择一个位置进行长期成像。在成像过程中从参考中获取此区域的详细 Z 堆栈图像。 注意:由于缝合线代表已知的P-SSC生态位，这是细胞响应颅骨损伤而迁移的地方。</li>
		<li>使用延时软件设置每1分钟记录一张图像至少1小时。在快照之间的时间内，将当前视野与初始视野进行比较。如果它们不同，请使用 Z 轴图像来确定需要调整视野的方向。</li>
	</ol>
</li>
</ol>6. 术后动物护理
<ol><li>成像后，用无菌PBS冲洗暴露的钙质以除去甲基纤维素。</li>
	<li>要关闭切口，轻轻地将皮瓣放在颅骨上。使用无菌棉签去除残留的眼科软膏并干燥切口部位。</li>
	<li>采用单丝尼龙5-0尺寸不可吸收缝合线，附有C-1反向切割针，闭合手术部位。使用无菌棉签将三重抗生素软膏涂抹在封闭的切口上。 注意:通过高质量的缝合技术和最小的出血减少疤痕。</li>
	<li>将鼠标放在干净的笼子中，放在温暖的表面上，每15分钟检查一次，直到达到胸骨卧位。</li>
	<li>术后72小时给予第二剂缓释丁丙诺啡。</li>
	<li>每天监测蜷缩行为、褶皱的皮毛和/或缺乏行走，直到缝合线被移除（约 1 周）。</li>
</ol>

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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Functionally+Distinct+Mx1+alphaSMA++Periosteal+Skeletal+Stem+Cells.">Identification of Functionally Distinct Mx1+alphaSMA+ Periosteal Skeletal Stem Cells.</a> Cell Stem Cell. 25 (6), 784-796 (2019).</li><li>Deveza, L., Ortinau, L., Lei, K., Park, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+gene+expression+identifies+distinct+molecular+signatures+of+bone+marrow-+and+periosteal-skeletal+stem/progenitor+cells.">Comparative analysis of gene expression identifies distinct molecular signatures of bone marrow- and periosteal-skeletal stem/progenitor cells.</a> PLoS One. 13 (1), 0190909 (2018).</li><li>Schindeler, A., McDonald, M. M., Bokko, P., Little, D. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leptin-receptor-expressing+mesenchymal+stromal+cells+represent+the+main+source+of+bone+formed+by+adult+bone+marrow.">Leptin-receptor-expressing mesenchymal stromal cells represent the main source of bone formed by adult bone marrow.</a> Cell Stem Cell. 15 (2), 154-168 (2014).</li><li>Grcevic, D., 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=In+vivo+fate+mapping+identifies+mesenchymal+progenitor+cells.">In vivo fate mapping identifies mesenchymal progenitor cells.</a> Stem Cells. 30 (2), 187-196 (2012).</li><li>Kuhn, R., Schwenk, F., Aguet, M., Rajewsky, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+gene+targeting+in+mice.">Inducible gene targeting in mice.</a> Science. 269 (5229), 1427-1429 (1995).</li><li>Srinivas, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cre+reporter+strains+produced+by+targeted+insertion+of+EYFP+and+ECFP+into+the+ROSA26+locus.">Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus.</a> BMC Developmental Biology. 1, 4 (2001).</li><li>Park, D., 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=Endogenous+bone+marrow+MSCs+are+dynamic,+fate-restricted+participants+in+bone+maintenance+and+regeneration.">Endogenous bone marrow MSCs are dynamic, fate-restricted participants in bone maintenance and regeneration.</a> Cell Stem Cell. 10 (3), 259-272 (2012).</li><li>Nitzsche, F., 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=Concise+Review:+MSC+Adhesion+Cascade-Insights+into+Homing+and+Transendothelial+Migration.">Concise Review: MSC Adhesion Cascade-Insights into Homing and Transendothelial Migration.</a> Stem Cells. 35 (6), 1446-1460 (2017).</li><li>Adler, J., Pagakis, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reducing+image+distortions+due+to+temperature-related+microscope+stage+drift.">Reducing image distortions due to temperature-related microscope stage drift.</a> Journal of Microscopy. 210, 131-137 (2003).</li><li>Roberts, S. J., Ke, H. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anabolic+Strategies+to+Augment+Bone+Fracture+Healing.">Anabolic Strategies to Augment Bone Fracture Healing.</a> Current Osteoporosis Reports. 16 (3), 289-298 (2018).</li></ol>

L.C.O.和D.P.公开了一项正在申请的专利，名为"骨骼修复中的骨膜骨骼干细胞"（BAYM.P0264US.P1）。其余作者声明没有竞争利益。

在骨愈合过程中，骨膜细胞是损伤愈伤组织内新分化的软骨细胞和成骨细胞的主要来源3。与骨髓类似，在骨膜中也发现了成骨/软骨SSC4，5，6。评估内源性P-SSC功能特征在技术上具有挑战性。通常，SSC的迁移动力学是在体外评估的，这使得对其相应体内系统的解释具有挑战性。由于活体动物活体活体显微镜检?...

骨折修复是一种动态的多细胞过程，与胚胎骨骼发育和重塑不同。在此过程中，从受损组织诱导损伤信号后，骨骼干/祖细胞的快速募集、增殖和随后分化，这些都对骨折的整体稳定性和固定至关重要1。特别是，骨折愈合的早期阶段需要软愈伤组织形成，这主要归因于骨膜常驻细胞2。当骨骼受伤时，骨膜细胞的一个亚群会迅速做出反应，并导致愈伤组织内新分化的软骨中间体和成骨细胞3，暗示骨膜内存在明显的骨骼干/祖细胞群。因此，骨膜驻留骨骼干细胞（SSC）的鉴定和功能表征是治疗退行性骨病和骨缺损的一种有前途的治疗方法4。
SSC被认为存在于多个组织位置，包括骨髓。与骨髓类似，在骨膜中也发现了成骨/软骨SSC4，5，6。 这些骨膜 SSC （P-SSC） 可在胎儿骨骼发育过程中使用早期间充质谱系标志物（即 Prx1-Cre5、Ctsk-Cre7 和 Axin2-CreER8）进行标记4，7，8，9，10。这些单一遗传谱系追踪模型的一个显着局限性是标记细胞群中存在实质性的异质性。此外，它们无法将标记的SSC与其体内的后代区分开来。为了解决这一限制，我们最近开发了一种双报告小鼠（Mx1-Cre+ Rosa26-Tomato+α SMA-GFP+），以清晰地可视化骨髓SSC（BM-SSC）中的P-SSC11。通过该模型，已经确定P-SSC以Mx1 + αSMA +双重标记标记标记，而更多分化的Mx1 +细胞驻留在骨髓，骨内和骨膜表面以及皮质和小梁骨11，12。
已知多种细胞因子和生长因子可调节骨重塑和修复，并且已经过测试，以增强关键节段缺损中的骨骼修复1，13。然而，由于通过破坏骨室的物理屏障产生的损伤模型的细胞复杂性，这些分子对内源性P-SSC迁移和愈合期间激活的直接影响尚不清楚。SSC的功能特征和迁移动力学通常在体外通过进行转孔或划痕测定来评估，并结合已知可诱导其他细胞群迁移的细胞因子或生长因子。因此，解释这些体外实验的结果以应用于其相应的体内系统是具有挑战性的。目前，骨骼干/祖细胞迁移的体内评估通常不是实时观察到的;相反，它是在骨折后的固定时间点测量的5，7，14，15，16。
这种方法的局限性是没有在单细胞水平上评估迁移;相反，它是通过细胞群的变化来测量的。由于活体动物活体显微镜检查和额外报告小鼠的产生的最新进展，现在可以对单个细胞进行体内跟踪。使用活体动物活体显微镜，我们观察到Mx1 / Tomato / αSMA-GFP小鼠在受伤后24-48小时内P-SSC从颅骨缝合龛位到骨损伤的不同迁移。
CCL5/CCR5最近被确定为一种调节机制，在早期损伤反应期间影响P-SSC的招募和激活。有趣的是，没有检测到大量的P-SSC迁移以响应实时损伤。然而，用CCL5治疗损伤会产生P-SSC的稳健，方向上不同的迁移，可以实时捕获。因此，该方案的目的是提供一种详细的方法，用于在用CCL5处理后实时记录P-SSC的体内迁移。

<table><tbody><tr><td>&amp;frac12;&amp;rdquo; optical post</td><td>ThorLabs</td><td>TR2</td><td>For imaging mount</td></tr><tr><td>1 mL syringe</td><td>BD</td><td>309659</td><td></td></tr><tr><td>27G needle</td><td>BD PercisionGlide</td><td>305111</td><td></td></tr><tr><td>29G insulin syringe</td><td>McKesson</td><td>102-SN05C905P</td><td></td></tr><tr><td>50 mL conicol tube</td><td>Falcon</td><td>352098</td><td>For mouse restraint</td></tr><tr><td>Adjustable angle plate</td><td>Renishaw</td><td>R-PCA-5023-50-20</td><td>For imaging mount</td></tr><tr><td>Alcohol wipes</td><td>Coviden</td><td>6818</td><td></td></tr><tr><td>betadine surgical scrub</td><td>Henry Schen</td><td>67618-151-16</td><td></td></tr><tr><td>Buprenorphine SR-LAB</td><td>ZooPharm</td><td></td><td>1mg/mL Sustained Release</td></tr><tr><td>Combo III</td><td>Obtained from staff veterinarian</td><td>N/A</td><td>37.6 mg/mL Ketamine; 1.9 mg/mL Xylazine; 0.37 mg/mL Acepronazine</td></tr><tr><td>Coverslip</td><td>Fisher</td><td>12-545-87</td><td>24 x 40 premium superslip</td></tr><tr><td>Fine tip forcepts</td><td>FST</td><td>11254-20</td><td></td></tr><tr><td>Ketamine</td><td>KetaVed</td><td>50989-161-06</td><td>100 mg/mL</td></tr><tr><td>Leica TCS SP8MP with DM6000CFS</td><td>Leica Microsystems</td><td>N/A</td><td></td></tr><tr><td>Matrigel</td><td>R &amp;amp; D Systems</td><td>344500101</td><td></td></tr><tr><td>Medical tape</td><td>McKesson</td><td>100199</td><td>3&quot; x 10 yds (7.6 cm x 9.1 m)</td></tr><tr><td>Methocellulose</td><td>Electron Microscopy Sciences</td><td>19560</td><td></td></tr><tr><td>Microdissection scissors</td><td>FST</td><td>1456-12</td><td></td></tr><tr><td>Motorized stage</td><td>Anaheim automation</td><td>N/A</td><td></td></tr><tr><td>Needle holder</td><td>FST</td><td>12500-12</td><td></td></tr><tr><td>Nonabsorbable sutures</td><td>McKesson</td><td>S913BX</td><td>monofilament nylon 5-0 nonabsorbable sutures with attached C-1 reverse cutting needle</td></tr><tr><td>Opthalmic ointment</td><td>Rugby</td><td>0536-1086-91</td><td></td></tr><tr><td>RANTES</td><td>Biolegend</td><td>594202</td><td>10 &amp;micro;g/50 &amp;micro;L</td></tr><tr><td>Right-angle clamp for &amp;frac12;&amp;rdquo; post, 3/16&amp;rdquo; Hex</td><td>ThorLabs</td><td>RA90</td><td>For imaging mount</td></tr><tr><td>Spring-loaded 3/16&amp;rdquo; Hex-locking &amp;frac14;&amp;rdquo; thumbscrew</td><td>ThorLabs</td><td>TS25H</td><td>For imaging mount</td></tr><tr><td>Sterile cotton swabs</td><td>Henry Schen</td><td>100-9249</td><td></td></tr><tr><td>Sterile DPBS (1x)</td><td>Corning</td><td>21-030-CV</td><td></td></tr><tr><td>Sterile drapes</td><td>McKessen</td><td>25-517</td><td></td></tr><tr><td>Surgical gloves</td><td>McKessen</td><td>3158VA</td><td></td></tr><tr><td>Triple antibiotic ointment</td><td>Taro Pharmaceuticals U.s.a., Inc.</td><td>51672-2120-2</td><td></td></tr><tr><td>Vacutainer blood collection set</td><td>BD</td><td>REF 367298</td><td>25G butterfly needle infusion set with 12&quot; tubing</td></tr></tbody></table>

real-time imaging of ccl5-induced migration of periosteal skeletal stem cells in mice

骨膜骨骼干细胞（P-SSC）对于终身骨骼维护和修复至关重要，使其成为开发增强骨折愈合的疗法的理想焦点。 骨膜细胞迅速迁移到损伤处，为骨折愈合提供新的软骨细胞和成骨细胞。传统上，细胞因子诱导细胞迁移的功效仅在体外通过进行转孔或划痕测定进行。随着使用多光子激发的活体显微镜检查的进步，最近发现1）P-SSC表达迁移基因CCR5和2）使用称为CCL5的CCR5配体处理改善了骨折愈合和P-SSC响应CCL5的迁移。这些结果是实时捕获的。这里描述的是一种方案，用于可视化P-SSC从颅骨架骨骼干细胞（SSC）生态位迁移到CCL5治疗后的损伤。该协议详细介绍了小鼠约束装置和成像支架的构造，小鼠颅骨的手术准备，颅骨缺陷的诱导以及延时成像的采集。

骨骼祖细胞被认为具有迁移或循环潜力20。最近，生成了Mx1-Cre+ Rosa26-Tomato+α SMA-GFP+（Mx1/Tomato/αSMA-GFP）报告小鼠，其中P-SSC用Mx1 + α SMA +双重标记标记（图2A，B）。Mx1+ α SMA+ P-SSC在损伤后24-48小时内从缝合间充质中大量迁移<sup cl...

Watch this Scientific Journal Video about Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice at JoVE.com

Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice

该协议描述了使用活体动物活体显微镜实时检测CCL5介导的骨膜骨骼干细胞迁移。

CCL5诱导小鼠骨膜骨骼干细胞迁移的实时成像

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of ...

real-time-imaging-ccl5-induced-migration-periosteal-skeletal-stem

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

2.2K 次观看.  Baylor College of Medicine. 该协议描述了使用活体动物活体显微镜实时检测CCL5介导的骨膜骨骼干细胞迁移。

视频: Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice

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

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

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 ...

科研

发育生物学

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

在中央小鼠中枢神经系统的干细胞移植的多模态成像

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

神经科学

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the efficacy of cell persistence and biodistribution of haMSCs in animal models. We demonstrated a method to label the cell membrane of haMSCs with lipophilic fluorescent dye. Subsequently, intra-articular injection of the labeled cells in rats with surgically induced KOA was monitored dynamically by an in vivo imaging system. We employed the lipophilic carbocyanines DiD (DilC18 (5)), a far-red fluorescent Dil (dialkylcarbocyanines) analog, which utilized a red laser to avoid excitation of the natural green autofluorescence from surrounding tissues. Furthermore, the red-shifted emission spectra of DiD allowed deep-tissue imaging in live animals and the labeling procedure caused no cytotoxic effects or functional damage to haMSCs. This approach has been shown to be an efficient tracking method for haMSCs in a rat KOA model. The application of this method could also be used to determine the optimal administration route and dosage of MSCs from other sources in pre-clinical studies.

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

This protocol describes an efficient way to monitor the cell persistence and biodistribution of human adipose-derived mesenchymal stem cells (haMSCs) by far-red fluorescence labeling in a rat knee osteoarthritis (KOA) model via intra-articular (IA) injection.

在体内荧光亲脂膜染料对大鼠膝骨关节炎模型中人脂肪间充质干细胞的追踪

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the ...

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

顺序<em&gt;在体内</em&gt;成骨干/祖细胞在骨折修复的影像学

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

医学

Tracking Mouse Bone Marrow Monocytes In Vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

跟踪小鼠骨髓单核细胞<em&gt;在体内</em

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

免疫与感染

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, including hematopoiesis, immune regulation and tumor regulation. Commonly used methods for understanding cellular actions in the bone marrow, such as histology and blood counts, provide static information rather than capturing the dynamic action of multiple cellular components in vivo. To complement the standard methods, a window chamber (WC)-based model was developed to enable serial in vivo imaging of cells and structures in the murine bone marrow. This protocol describes a surgical procedure for installing the WC in the femur, in order to facilitate long-term optical access to the femoral bone marrow. In particular, to demonstrate its experimental utility, this WC approach was used to image and track neutrophils within the vascular network of the femur, thereby providing a novel method to visualize and quantify immune cell trafficking and regulation in the bone marrow. This method can be applied to study various biological processes in the murine bone marrow, such as hematopoiesis, stem cell transplantation, and immune responses in pathological conditions, including cancer.

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

股骨窗口商会模型<em&gt;在体内</em在小鼠骨髓&gt;手机跟踪

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

在鼠标提睾肌为外周干细胞迁移分析的微循环活体显微镜

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

生物学

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

在造血干细胞和祖细胞利用共聚焦和多光子显微镜五荧光蛋白标记的活体克隆跟踪

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

In vitro Time-lapse Live-Cell Imaging to Explore Cell Migration toward the Organ of Corti

To study the effects of mesenchymal stem cells (MSCs) on cell regeneration and treatment, this method tracks MSC migration and morphological changes after co-culture with cochlear epithelium. The organ of Corti was immobilized on a plastic coverslip by pressing a portion of the Reissner's membrane generated during the dissection. MSCs confined by a glass cylinder migrated toward cochlear epithelium when the cylinder was removed. Their predominant localization was observed in the modiolus of the organ of Corti, aligned in a direction similarly to that of the nerve fibers. However, some MSCs were localized in the limbus area and showed a horizontally elongated shape. In addition, migration into the hair cell area was increased, and the morphology of the MSCs changed to various forms after kanamycin treatment. In conclusion, the results of this study indicate that the coculture of MSCs with cochlear epithelium will be useful for the development of therapeutics via cell transplantation and for studies of cell regeneration that can examine various conditions and factors.

In this study, we present a real-time imaging method using confocal microscopy to observe cells moving toward damaged tissue by ex vivo incubation with the cochlear epithelium containing the organ of Corti.

体外延时活细胞成像探索细胞向科尔蒂器官迁移

To study the effects of mesenchymal stem cells (MSCs) on cell regeneration and treatment, this method tracks MSC migration and morphological changes after ...

CCL5诱导小鼠骨膜骨骼干细胞迁移的实时成像

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