在印第安纳大学的印第安纳生物显微镜中心进行了成像, 由 Dr. 的导演邓恩。立体定向装置是由 Soonpaa、水井中心的儿科研究的原型设计和制作的。这项工作得到了 NIH/R01DK097837-09 (nc)、NIH/R01HL068256-05 (nc)、NIH/NIDDK1U54DK106846-01 (nc)、MPN 研究基金会 (nc) 和 CTSI 合作项目 IUSM/圣母院 (nc) 的支持。

所有涉及使用动物的程序都是在印第安纳大学医学院的动物保育和使用委员会的授权下进行的。确保遵守该项工作所在国家的动物实验立法。
1. Mx1CreRBPJ--受体小鼠的制备
<ol><li>交叉 Mx1-Cre+小鼠与 RBPJ 液氧/液氧小鼠10获得 Mx1-Cre 阳性RBPJ液氧/液中小鼠12和 Mx1-Cre 阴性 RBPJ 液氧/液氧窝用作控件。通过 PCR10验证基因型。</li>
	<li>使用6-8 周老 Mx1Cre+/RBPJ 液氧/液氧和 Mx1Cre-/RBPJ 液氧/液氧小鼠执行 polyI: C 诱导。</li>
	<li>
注入 polyI: C 200 µg 的 ip 在综合考试中的+和-小鼠。给一个 polyI: 每隔一天注射一周3天。给一个 polyI: C 注射液的第二个星期, 7 天后, 上一次注射 (总共四注射)。
	<ol>
<li>使用 RBPJKO (诱导 Mx1Cre+/RBPJ 液氧/液氧) 和 RBPJWT (诱导 Mx1Cre-/RBPJ 液氧/液氧) 小鼠最后 polyI: C 注射后3周。 注: 推荐使用 pI 诱导的小鼠: 注射后3周。由 polyI 触发的 IFNα反应: C 诱导 BM 发生重大变化, 导致 HSC 的型扩张, 并将成熟祖细胞的输出降低到外周血16,17。造血亚群的表达在注射后3周正常化, 小鼠可以不用炎症的混杂作用而被利用。此感应协议已针对 RBPJ 进行了优化。如果删除不同的基因, 归纳协议可能因构造而异, 必须验证删除。在总共四 polyI: C 注射后, 我们通过 rt-pcr 验证了 100% loxP 位点之间 RBPJ 区域的缺失。</li>
	</ol>
</li>
</ol>2. 用于移植的赖氨酸-EGFP 供体骨髓细胞的制备
<ol><li>安乐一个赖氨酸-EGFP 小鼠 (二氧化碳后接颈椎脱位) 1 或2小时前移植。</li>
	<li>用70% 乙醇喷洒动物体表。</li>
	<li>使用外科剪刀, 使双腿周围的脚踝和手术钳的皮肤切口, 拉走皮肤和毛皮一起暴露干净的肌肉组织。</li>
	<li>使用外科剪刀, 以消除尽可能多的肌肉从腿部。使用剪刀, 削减骨骼 (在膝盖和踝关节) 和清洁任何剩余的肌肉组织从股骨和胫骨使用纱布海绵。将骨骼 (两个股骨和两个胫骨) 放入一个包含 DMEM 10% FBS 的6井板中。</li>
	<li>用10毫升冷的 2mM EDTA PBS 将骨骼粉碎, 吸管骨髓细胞使细胞进入单细胞悬浮。或者, 用1毫升注射器从每一侧用2毫米的 EDTA PBS 3 次冲洗骨头。</li>
	<li>使用 70-µm 过滤器进入15毫升离心管过滤骨髓细胞。冲洗过滤器2-3 毫升的 PBS。将细胞向下旋转10分钟, 在 460 x g, 重10毫升的新鲜 DMEM 10% FBS。</li>
	<li>计数骨髓细胞在一个例和调整浓度到 1.5 x 107细胞/毫升在 IMDM 没有血清。每只动物使用 3 x 106细胞, 体积为200µL。约1/3 细胞的总 BM 是髓系 GFP + 细胞。把细胞留在冰上, 直到注射完毕。使用 0.5 x 105单元格来确定由外地资产管制组织9的 GFP 表达式。</li>
</ol>3. 赖氨酸-GFP 细胞向 RBPJKO 小鼠骨髓移植
<ol><li>在一个馅饼笼中限制受赠老鼠。在 Cs 137 器上照射有致死剂量伽玛辐射 (1200 Rad) 的小鼠。使用分裂剂量协议: 900 拉德在晚上跟随了300拉德次日早晨 (16 h 分开)。</li>
	<li>移植致命照射的 RBPJWT 和 RBPJKO 受体小鼠5-6 小时后第二剂量的辐射。将从赖氨酸-EGFP 小鼠中获取的 BM 细胞注入 3 x 106细胞, 每个动物的浓度为via尾静脉注射 (详见2节中的收获细胞的详细信息)。</li>
	<li>不同时间点 IVFM 移植小鼠的影像独立组:24 小时, 在2、4和6周, 如下所述 (参见 4 & 5, 用于活体成像过程)。</li>
</ol>4. 活体成像的外科准备
<ol><li>消毒手术器械。两个优良的镊子 (一直, 一角), 一双罚款剪刀和一对针持有人。准备操作区域, 并提供所有需要的程序。</li>
	<li>给鼠标注射氯胺酮鸡尾酒麻醉剂 (嗪 2.5-5 毫克/千克 + 乙酰 1.0-2.5 毫克/千克 + 氯胺酮90-100 毫克/千克) 使用26-28 克针注射器。 动物将被监测每15分钟的过程中, 麻醉将补充必要的¼的原始剂量。</li>
	<li>将鼠标放在适当的热源上 (37 ° c 加热垫, 保护动物免受与加热垫的直接接触), 并直观地监控呼吸速率。</li>
	<li>使用脚趾夹紧响应检查反射。在开始任何手术前, 确保动物完全处于麻醉阶段。</li>
	<li>使用26-28 针注射器给小鼠尾静脉注射荧光血管标记物 (葡聚糖, 100 μ l 20 毫克/毫升溶液)。</li>
	<li>将兽医眼膏应用于双眼。用小电动剪刀夹住动物头部的背表面。涂抹5分钟的脱毛膏, 用纱布海绵除去奶油, 然后用生理盐水冲洗。用棉签准备70% 酒精的清洁头皮。</li>
	<li>使用细钳和剪刀, 使头皮上的一个小的中线皮肤切口 (10-20 毫米) 露出背后的背颅骨表面。使用5-0 手术丝在切口两侧的皮肤上放置两个停留缝合线, 创建一个皮瓣以暴露颅骨的影像。</li>
	<li>将老鼠放在背上, 将裸露的头皮浸入一个装满显微镜油的玻璃底盘中。将动物运送到 mutiphoton 成像室。</li>
	<li>将动物放在显微镜下的舞台上, 将颅骨放在目标上方的玻璃盘上, 然后用37° c 加热垫盖住 (动物必须避免与热直接接触).</li>
</ol>5. 活体小鼠颅骨的高分辨率成像
<ol><li>使用为多成像修改的倒置共焦系统 (请参阅材料表)。按照制造商的说明将2光子激光器调到 830 nm, 在显微镜鼻片中放置一个 20X W、NA 0.95 物镜, 并检查激光束对齐。 注意: 直立显微镜系统是最常用的这些研究, 但一个倒置的多系统也可以使用。本研究采用定制设计的创伤立体定向装置。虽然有几个商业上可用的立体定位设备的直立显微镜系统, 没有商业化的立体定向设备的倒置显微镜系统, 旨在确保鼠标头骨。作为一个自定义的立体定向装置的替代品, 头骨可以在目标上方的位置上使用各种胶带或胶水的稳定性方法来固定。</li>
	<li>打开一个图像采集软件。在 "购置设置" 面板中, 检查是否选择了一个定向扫描模式。将扫描速度设置为4μ s/像素, 帧速率为 512 x 512 像素, 缩放至1.5。从可用物镜的列表中选择 20X W Na 0.95 目标, 以匹配定位在鼻甲中的镜片。</li>
	<li>从 "图像采集控制" 面板中获取 "染料列表", 并选择 "两个光子"。打开 "光路 & 染料" 窗口, 选择 DM690-980 励磁 DM. 通过检查激光单元2中的复选框打开2P 激光快门。在 "显微镜控制器" 窗口中, 选择 RDM690 镜像。</li>
	<li>选择 "epi 灯", 选择 B/G epi 滤光片, 并将目标集中在标本上, 以可视化血管流和颅骨骨髓龛位, 以中心静脉 (a) 和冠状动脉缝合 (B) (图 2A) 的分岔为参考。</li>
	<li>
使用 non-descanned 模式收集图像。选择三外部探测器: PMT detector1 收集倍信号的胶原蛋白 (发射过滤器-430/100 nm), GaAsP detector2 收集 GFP 信号 (发射过滤器-525/50) 和 GaAsP detector3 收集信号 TRITC-葡聚糖 (排放过滤器-605/90 nm)。
	<ol>
<li>在 4μs/像素的扫描速率下执行成像, 无平均值以最小化光。在恒定激光功率下采集图像, 并对探测器增益进行调整, 以利用最小饱和度的探测器的全动态范围。</li>
		<li>通过组织的深度 (60 x 1 μ m Z 栈) 从颅骨骨髓的6区域收集一系列的切片。使用1微米的步长设置, 缩放1.5 和 512 x 512 像素帧大小 (423 µm x 423 µm)。 注: 图像一只鼠标所需的总时间为 1-1. 5 小时。</li>
	</ol>
</li>
</ol>6. 定量分析
<ol><li>按照制造商的说明执行图像定量和3D 重建, 使用专用的3维/4 维图像定量和可视化软件 (请参见材料表)。利用最大强度投影 (MIP)、alpha 混合或阴影投影体渲染算法, 在3D 中交互式地可视化 Z 栈。</li>
	<li>使用 "点对象分割模块"来分割 GFP 细胞。应用堆栈算术处理 (信道减法) 消除 GFP 细胞的假阳性计数 (这消除了在绿色和红色通道中显示强荧光的骨细胞信号)。</li>
	<li>利用表面分割模块对血管和骨表面进行分割。如果需要, 通过应用称为 "点对面距离" 的 X 拉伸算法, 计算出细胞与上述任何表面的距离。</li>
</ol>

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作者没有什么可透露的。

该协议描述了一个实验设计的优化, 以研究活体荧光灯显微镜的造血细胞植入动力学。在这项研究中, 在骨髓细胞或在一个缺口信号有缺陷 bm 的扩张在骨颅骨被跟踪的赖氨酸-GFP 阳性髓细胞后, BMT 到 RBPJWT 或 RBPJKO 受体。这种方法被提出作为一个模型, 可用于解决类似的问题, 如: i), 以确定在 BM 的细胞的其他血统, 如淋巴, 红或巨细胞的扩展和本地化, 通过作为供体细胞造血细胞携带血统特异促进剂驱动 GFP 或番茄红;二) 利用其他特定的 KO 或转基因小鼠作为受体, 评估不同的微环境决定因素。
这种成像协议的强度在颅骨骨, 是用于选择 BM 区域的解剖标志, 中心静脉分岔和冠状动脉缝合, 是合理保存在所有小鼠, 允许个体之间的一致性实验, 同时放弃了自动化阶段的要求。此外, 使用 gfp 模型跟踪造血细胞证明是非常有效的, 因为 gfp 提供了一个稳定的信号在次优条件下, 如辐照后。最后, 成像设置描述, 使用倒置显微镜和定制的立体定位装置, 其中鼠标在仰卧位, 大大减少了呼吸文物。
这一实验设计的两个方面, 如果实施, 可能会导致更广泛和更有效地利用 IVFM 的颅骨。首先, 最重要的是要优化和标准化的纵向成像协议, 允许观察相同的鼠标在不同的时间点 (从2天到几个星期) 干预后 (即BMT 或治疗), 而不是使用小鼠的独立队列。由于这种方法需要适当的条件, 以后恢复和采取措施, 以抵消炎症, 感染和疤痕形成在现场的成像, 其应用目前是有限的。其次, 在 BM 小生境 (血管、内、血管和神经元) 的特定细胞中携带荧光蛋白的谱系追踪鼠模型的建立, 将是有价值的, 将造血细胞功能与特定BM 龛的特征。
与其他组织相比, 骨成像仍然存在一些挑战和局限性。虽然双光子显微镜可以穿透 100-1, 000 微米深的组织, 它仍然是挑战, 以图像通过整个厚度的颅骨骨。图像的质量随着深度的增加而丢失, 因此这里的协议描述了从60微米厚的栈中对 BM 区域的可靠分析。另一个可能导致不一致或次优成像的因素是颅骨骨的弯曲形状, 它可以使图像脱离焦点。这是至关重要的头骨定位完美的玻璃盘上方的目标, 可能与一个立体定向装置。事实上, 头骨的大小是重要的: 小鼠的头骨太年轻或太小可能不适合完美的在一个给定的立体定向设备。例如, 此处使用的设备不适合小于6周且小于20克的小鼠。
另外一个限制是, 本研究中使用的成像系统仅限于3通道。由于蓝色通道自动分配以收集 non-labeling 基倍信号的骨胶原, 只有2通道可用于特定荧光标记。此外, 该系统的速度限制为1帧/秒2μ s/像素停留时间和 512 x 512 像素帧大小, 这是不理想的快速动态过程 (如测量血流量和评价细胞动员在血液流)。
一个重要的挑战是, 为 BMT 进行的辐射损害了血管的完整性。辐照后的血管渗漏对个体结构的分割/定量有困难, 在定量分析中可能会造成困难。
最后, 重要的是要考虑一只老鼠的成像大约需要1小时, 并且在某一特定的日子里可以拍摄的老鼠数量是有限的 (~ 4 到6只老鼠)。因此, 增加样本大小以获得分析能力可能需要多个独立的实验, 这可以增加变异性。
根据本协议, IVFM 在 BM 中检测到的造血细胞的数量在24小时后由 BMT 30 细胞/区域, 它是一个相当小的数字相比, 初始输入的细胞 (3 x 10 6)。虽然, 这个问题的一部分是由于细胞诱捕在肺和肝脏, 然后再归巢到 BM 静脉注射后, 仍有待探讨是否有地区在颅骨以外的那些被选中的移植细胞的家在较高效率.
细胞归巢和植入后, BMT 后, 通常是流式细胞仪的测量 CD45.1/CD45.2 标记, GFP, 羧基琥珀酰亚胺酯 (CFSE) 或组合的血统和干细胞标记。使用 IVFM, 特别是在早期的时间点, 使可用的独特和额外的信息, 不能提供流式细胞仪。例如, 通常不容易区分为 "单元" 的事件的 "细胞" 事件的 "项目", 特别是当收集到的积极事件的数量很少 (即在24小时从 BMT)。IVFM 提供了有关细胞的形态学和定位的信息, 这有助于这种区分。同样地, 通过冲洗或撞击骨骼所收获的 BM 造血细胞的分析也将包括那些居住在小生境中的细胞和已经在血管系统中流通的细胞。事实上, IVFM 允许对在 BM 龛内的细胞和正在动员到血液中的细胞进行区分和评估。这种区分在研究给定模型中的归巢、定位、分化和动员动力学方面具有重要的价值。重要的是, IVFM 可以提供关于造血细胞相对于构成特定生态位的微环境细胞的位置的独特信息。
其他方法用于解决 BM 位的组成, 这种组织学分析已经被使用。活体显微镜与共聚焦显微镜的组织学分析进行了比较, 并对塞尔索·阿莫林et al.进行了全面、优雅的讨论。18。

活体多荧光显微镜 (IVFM) 是一种强大的成像技术, 允许高分辨率, real-time 成像的组织与深度高达 1mm, 根据组织。当应用于小鼠颅骨, 它允许观察 BM 内造血细胞的行为, 其非侵入性的方式可达60-100 μ m11。本方法用于测定 RBPJ 敲除小鼠缺乏标准缺口信号的植入的正常髓内祖细胞的动力学。
最近我们小组的工作表明, 在 BM 微环境中有缺陷的标准缺口信号会导致类似骨髓的疾病12。利用 Mx1-Cre 诱导重组10, 通过条件删除 RBPJ 的 DNA 结合域 (标准缺口信号下游的临界转录因子), 得到了缺口信令的损失。在本研究中, 使用了 Mx1-Cre/RBPJ 的液氧液/液氧小鼠模型。有条件地删除 RBPJ 的 DNA 结合母题会导致所有凹槽受体的信号丢失。在 Mx1-Cre 模型中, Mx1 启动后的表达是由激活后的 polyI 管理: C 导致诱导靶基因缺失的血细胞以及基质成分的多个器官, 包括 BM, 脾脏和肝脏。
Mx1-Cre+/RBPJ氧液/液氧和 Mx1-Cre-/RBPJ 液氧/液氧小鼠 polyI: C (在此表明 RBPJKO 和 RBPJWT 分别) 照射和移植正常, 野生型造血细胞。从4周开始移植后, RBPJKO 受体发育显著增多继发脾肿大。虽然 RBPJKO 小鼠在移植后8周和以后的时间点, 骨髓中髓祖细胞的比例增加, 但在4和6周, bm 的分析结果并没有显示出它们与对照组相比的髓内含量显著的差异 RBPJWT收件人.这一观察, 连同 Mx1-Cre 在不同的造血器官中表达的事实, 提出了 BM 微环境是否对骨髓表型的启动有直接影响的问题。
为了确定 bm 是否是疾病发展的一个重要的初始部位, IVFM 的小鼠颅骨与 bm 移植 (BMT)、RBPJ 敲除模型和血统跟踪系统结合使用。在特定溶菌酶启动子 (赖氨酸-GFP)9的控制下表达 EGFP 的转基因小鼠被用来获得在 BM 成像后 BMT 的供体细胞。溶菌酶表达是特定于髓细胞和赖氨酸-GFP 标记细胞从普通的髓系祖 (CMP) 到成熟粒细胞膜13。
IVFM 在不同时间点的 bm 表明, 赖氨酸-GFP 细胞与 RBPJWT 和 RBPJKO 受体的 bm 相似, 但在 RBPJKO 受体的 bm 中扩张和嫁接更快。这个区别是戏剧性的在早先时间点 (星期 2) 和减少随着时间 (星期4和 6)。然而, 在这些后来的时间点, 对同一受体的造血室的评价显示, 在 RBPJKO 小鼠体内的铅和脾内循环的髓细胞数量稳步增加, 表明细胞的产量增加从 BM 到循环。赖氨酸-GFP 细胞在移植小鼠骨髓中的定位分析6周后发现髓细胞在 RBPJKO 微环境中的血管中比对照组更远。
总的来说, IVFM 与这些特定动物模型的结合提供了 RBPJKO BM 微环境中髓细胞植入动力学的洞察。本文所描述的实验设计和定量方法是一种可以应用于解决类似问题的范例。例如, 使用其他特定于单元的沿袭跟踪模型 (如 RAG1-GFP14或 Gata1-GFP15小鼠) 可能允许在 BM 中分别遵循淋巴或红祖细胞的行为。

<table border="0" cellpadding="0" cellspacing="0" width="1033">
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			<td height="21" style="height:21px;width:216px;">Ketamine cocktail</td>
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			<td>straight forcep, angled forcep</td>
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			<td style="width:167px;">Bitplane</td>
			<td style="width:139px;"></td>
			<td>3/4 D Image Visualization and Analysis software</td>
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</table>

combining intravital fluorescent microscopy (ivfm) with genetic models to study engraftment dynamics of hematopoietic cells to bone marrow niches

越来越多的证据表明, 正常的造血是由不同的微提示在 BM, 其中包括专门的细胞龛调节关键造血干细胞 (HSC) 功能1,2。事实上, 更详细的造血微环境的图片现在正在出现, 其中内和内皮龛形成正常 HSC 及其子代的调节功能单元3,4,5.新的研究揭示了血管细胞、脂肪细胞和神经细胞在维持和调节 HSC 功能方面的重要性6,7,8。此外, 有证据表明, 来自不同血统的细胞,即髓细胞和淋巴细胞, 位于 BM 微环境中的特定龛位。但是, BM 微环境及其占用者的完整映射仍在进行中。
表达血统特异荧光标记或转基因小鼠的转基因小鼠菌株, 在 BM 龛的特定细胞中缺乏选定的分子, 现已问世。淘汰赛和血统跟踪模型, 结合移植的方法, 提供了机会, 以完善有关特定的 "利基" 细胞的作用, 明确的造血种群, 如 HSC, B 细胞, T 细胞, 髓细胞和红细胞。这一战略可以进一步强化通过合并使用双光子显微镜的颅骨。通过提供体内高分辨率成像和 bm 颅骨的3维渲染, 我们现在可以精确地确定 bm 中特定的造血亚群的位置, 并评估它们随时间扩展的动力学。在这里, 赖氨酸-GFP 的转基因小鼠 (标记髓细胞)9和 RBPJ 敲除小鼠 (缺乏标准缺口信号)10与 IVFM 结合使用, 以确定髓细胞植入的缺口缺陷 BM 微环境。

2 RBPJKO 和 2 RBPJWT 接受者在不同时间点的单个成像会话中被成像:24 小时和2、4和6周, 移植后的 BM 赖氨酸-GFP 细胞 (工作流在图 1A中说明)。
在每只老鼠中, 图像是从 BM 颅骨的6标准区域获得的, 它们的位置与中心静脉的分岔有关 (图 2A, a) 和冠状动脉缝合 (图 2A, b)。注射葡聚糖得克萨斯-红色在成像之前, 可以识别这些地标和选择6区域 (图 2A): 左上、中和右 (UL、UM、UR) 和左下、中和右 (LL、LM、LR)。从每个区域收集 60 x 1 μ m z 栈, 并呈现为3维最大强度投影 (MIP) (图 2B);3维阴影投影, 其中包括第二次谐波生成 (倍) 显微镜探测胶原组织 (图 2C) 产生的骨成分 (灰色);最后, 一个3维分割的图像, 它允许定量的细胞和测量他们的距离从骨骼和血管 (图 2D)。
由于骨髓颅骨移植后不同区域造血细胞的归巢偏好存在着潜在的差异, 因此在每个小鼠的颅骨中取样多个区域是很重要的。图 3显示了在一个 RBPJWT 中分析的6区域中的单元分布的示例, 在 BMT (绿髓细胞、灰骨) 使用阴影投影3D 渲染算法后的2周内, 一个 RBPJKO 受体。每个区域的单元格数从 RBPJWT 鼠标的64到258不等, 在 RBPJKO 鼠标中从265到573。最后的结果, 然后表示为6地区的平均数量分析: 每个地区/每只老鼠的细胞数 (平均135在 RBPJWT 与469在 RBPJKO)。这个代表性的实验表明, 在 RBPJWT 中, 每个区域的细胞分布比 RBPJKO 受体有较大的变化。此外, 除了在一只老鼠的颅骨区域内发现的变异, 这是常见的, 在同一组内的单个小鼠之间也有差异。因此, 重要的是, 每一个时间点至少有4只老鼠被评估每一个实验, 以获得一个最小的总24至30地区的每一个条件分析。
图 4显示了髓系祖细胞在移植后6周内与对照组的动态变化。在这个代表性的实验中, 8 RBPJWT 和 8 RBPJKO 小鼠被移植和图像在指定的时间点 (2 RBPJWT 小鼠和 2 RBPJKO 小鼠在每个时间点)。图 4A显示了具有代表性的3维重建, 其中6个区域中的1是获得的。每个6区域的细胞都被计算在内, 每个时间点 (总共12区域) 的两只小鼠的细胞/区域的平均数量 (图 4C) 都是为 RBPJWT 和 RBPJKO 的。分析表明: (i) RBPJWT 和 RBPJKO 受体中的供者赖氨酸-GFP 细胞具有类似的归巢效率;二) RBPJKO 受体细胞的扩张速度较快, 在 BMT 后2周 RBPJWT 受体细胞数量的两倍;iii) RBPJWT 受体的细胞在以后扩大, 2 倍高于 RBPJKO 受体的细胞, 在4周后 BMT;和 iv) RBPJWT 和 RBPJKO 受体的细胞数在 BMT 后6周变得相似。对 PB 中的供体髓细胞的分析表明, RBPJKO 接受者 bm 的髓细胞的输出量高于4周 RBPJWT 受体的 bm, 当时 RBPJKO 小鼠的 bm 显示的是赖氨酸-GFP 细胞的低含量。骨髓细胞和髓细胞在 PB 中循环的联合分析表明, 在 RBPJKO bm 微环境中, 赖氨酸-GFP 细胞扩展, 并准备更迅速地进行动员。对长骨中的赖氨酸-GFP 细胞进行平行的分析, 从同一小鼠的骨髓中发现了类似于 IVFM 的一种趋势;但是, 条件之间的差异较不明显 (图 4B)。
在图 5中显示了来自骨骼或血管的单个赖氨酸-GFP 细胞距离的测量。在这个代表性的研究中, 使用了 RBPJWT 和 RBPJKO 颅骨的 LM 区域的3维分割分析, 从 BMT 的6周 (图 5A)。该地区每个细胞的距离从骨和血管计算: 200 细胞在 RBPJWT 和255细胞在 RBPJKO, 从而表示为平均。分析表明, 赖氨酸-GFP 细胞在 RBPJWT 和 RBPJKO 小鼠 (11 μ m) 中与骨的相似距离定位, 但它们在 RBPJKO 小鼠的血管中比 RBPJWT 小鼠 (分别为15μ m vs 5 μ m) 更遥远。这一结果是耐人寻味的, 因为在 RBPJKO 小鼠的赖氨酸-GFP 细胞动员到循环比在 RBJWT 小鼠, 因此预计将本地化更接近血管。但是, 本地化的差异可能反映了这两种群体 (RBPJWT 和 RBPJKO) 不同的分化阶段, 这一点不能通过分析来解决。这一结果的意义将在所有6区域内的一个更大的细胞池中进行随访, 并结合了外地资产管制分析。
总的来说, 当每只老鼠分析少量的区域时, 就得到了次优结果。成像是特别具有挑战性的早期时间点后, BMT, 由于致命的辐射损害的血管和漏葡聚糖从毛细血管减少图像的定义。因此, 重要的是要有大量的重复, 特别是在最早的时间点。
倍是研究3D 胶原纤维组织的有用工具。它是一种二阶非线性光学过程, 它来源于具有 non-centrosymmetry 的胶原纤维和高二阶非线性系数的结构。当强入射光与此类结构相互作用时, 它会以入射频率的两倍或入射波长的一半产生光。因此, 在2光子显微镜中, 不需要标记来捕获倍信号。与 830 nm 的励磁波长, 我们捕获倍信号在蓝色通道与发射过滤器 430/100 nm。
<img alt="Figure 1" src="/files/ftp_upload/54253/54253fig1.jpg"> 图 1:实验工作流(A) 诱导 Mx1-Cre/RBPJ 液氧/液氧小鼠生成 RBPJKO 和 RBPJWT 小鼠 ~ 4 周前成像;(B) 从赖氨酸-GFP 转基因小鼠中制备 BM 细胞;(C) 将 BM 赖氨酸-GFP 细胞移植到致命辐照 RBPJKO 或 RBPJWT 受体;(D) IVFM 鼠颅骨在 BMT 后24小时、2、4和6周, 其次为无痛苦的死亡和 BM 分析。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54253/54253fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" src="/files/ftp_upload/54253/54253fig2.jpg"> 图 2:BM 血管龛的 IVFM.(A) 光子 EGFP 鼠标颅骨显示6标准成像区域的马赛克图像 (UL: 左上角, 嗯: 上中部, 右下角: 左下, 右下角: 下半角, LR: 下部, (绿色, 髓细胞; 红色, 血管; 灰色, 骨骼)10X 放大倍数;(B-D)BM 细分细节渲染为: (B) MIP, (C) 3 维阴影投影, (D) 3 维分割图像。图像处理-使用专用的3维/4 维图像定量和可视化软件 (表 1)。缩放条形图 = 50 μ m 在 B, C, D.<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54253/54253fig2large.jpg" target="_blank">请点击这里查看这个数字的更大版本.</a>
<img alt="Figure 3" src="/files/ftp_upload/54253/54253fig3.jpg"> 图 3:2 周的髓细胞植入.赖氨酸-GFP 细胞 (绿色) 和骨表面 (灰色) 的图像在 6 BM 地区从一个 RBPJWT 和一个 RBPJKO 鼠的颅骨骨 (U: 上部, L: 左, M: 中间, R: 右)。缩放条形图 = 50 μ m。条形图 (右) 表示每个区域中计数的单元格数 (RBPJWT 范围 64-258;RBPJKO 范围 265-573) 和他们的平均数字 135 STD+/-73, 和469性病 +/-121。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54253/54253fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" src="/files/ftp_upload/54253/54253fig4.jpg"> 图 4:髓细胞的动力学植入和输出后 BMT.(A) 赖氨酸-GFP 细胞 (绿色) 和骨骼表面 (灰色) 的图像, 从一个 RBPJWT 和 RBPJKO 鼠标的颅骨在每个时间点表示。刻度条 = 50 微米;(B) 斑点杂交显示了赖氨酸-GFP 阳性细胞的百分比的流式细胞仪内的 long-bones 从相同的鼠标成像在体内 (以上) 收获的 BM。(C) 条形图表示每个时间点的2只小鼠 (总共12区域) 的细胞/区域的平均数量, +/-性病. (D) 折线图显示在同一小鼠的 PB 中出现的中性粒细胞总数的折叠增加 (细胞计数)比在 (A) 在指定的时间点。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54253/54253fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" src="/files/ftp_upload/54253/54253fig5.jpg"> 图 5:髓细胞在 BM 中的定位与骨和血管的关系.(A) 代表3维图像的赖氨酸-gfp 细胞和血管, 和赖氨酸-gfp 细胞和骨在同一地区的颅骨骨在 RBPJWT 和 RBPJKO 受体在6周从 BMT。缩放条形图 = 50 μ m。(B) 条形图汇总了从骨骼表面或血管中的赖氨酸-GFP 细胞的平均距离 (以微米为单位) 的 SEM。测量的单元格数: RBPJWT n = 200 单元格和 RBPJKO n = 255 单元格。在 RBPJKO 中, RBPJWT 0.001 (学生的 t 检验), 从血管中的赖氨酸-GFP 细胞的距离更大。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54253/54253fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Combining Intravital Fluorescent Microscopy IVFM with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches at JoVE.com

Combining Intravital Fluorescent Microscopy IVFM with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

活体荧光显微镜 (IVFM) 的颅骨是应用与遗传动物模型, 研究造血细胞归巢和植入的骨髓 (BM) 龛。

结合活体荧光显微镜 (IVFM) 和遗传模型研究造血细胞对骨髓生态位的植入动力学

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular ...

combining-intravital-fluorescent-microscopy-ivfm-with-genetic-models

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

Combining Intravital Fluorescent Microscopy (IVFM) with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

7.9K 次观看.  Indiana University School of Medicine. 活体荧光显微镜 (IVFM) 的颅骨是应用与遗传动物模型, 研究造血细胞归巢和植入的骨髓 (BM) 龛。

视频: Combining Intravital Fluorescent Microscopy IVFM with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Automated Quantification of Hematopoietic Cell &#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.

A strategy to quantitatively analyze histological data in the bone marrow is presented. Confocal microscopy of fluorescently labeled cells in tissue sections results in 2-dimensional images, which are automatically analyzed. Co-localization analyses of different cell types are compared to data from simulated images, giving quantitative information about cellular interactions.

造血干细胞的自动定量 - 在脱钙骨的组织学图像间质细胞的相互作用

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. ...

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 ...

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema and diminished visual acuity. However, human CECs are prone to compromised proliferative potential. Furthermore, stimulation of cell growth is often complicated by gradual endothelial-mesenchymal transition (EnMT). Therefore, understanding the mechanism of EnMT is necessary for facilitating the regeneration of CECs with competent function. In this study, we prepared a primary culture of bovine CECs by peeling the CECs with Descemet's membrane from the corneal button and demonstrated that bovine CECs exhibited the EnMT process, including phenotypic change, nuclear translocation of &#946;-catenin, and EMT regulators snail and slug, in the in vitro culture. Furthermore, we used a rat corneal endothelium cryoinjury model to demonstrate the EnMT process in vivo. Collectively, the in vitro primary culture of bovine CECs and in vivo rat corneal endothelium cryoinjury models offers useful platforms for investigating the mechanism of EnMT.

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

A primary culture of bovine corneal endothelial cells was used to investigate the mechanism of corneal endothelial-mesenchymal transition. Furthermore, a rat corneal endothelium cryoinjury model was used to demonstrate corneal endothelial-mesenchymal transition in vivo.

在体外和体内模型来研究角膜内皮-间充质转换

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema ...

Preparation of Plasma Membrane Vesicles from Bone Marrow Mesenchymal Stem Cells for Potential Cytoplasm Replacement Therapy

We have previously reported on the generation of plasma membrane vesicles (PMVs) through the mechanical extrusion of mammalian cells. The fusion of PMVs with mitochondrial deficient Rho0 cells restored mitotic activity under normal culture conditions. Atherosclerosis, type 2 diabetes, Alzheimer&#39;s disease, and cancer are age-related diseases that have been reported to be associated with multiple mechanical and functional defects in the cytosol and organelles of a variety of cell types. Bone marrow mesenchymal stem cells (BMSCs) represent a unique cell population from the bone marrow that possess self-renewal capabilities while maintaining their multipotency. The supplementation of senescence cells with young cytoplasm from autologous BMSCs via the fusion of PMVs provides a promising approach to ameliorate or even reverse age-associated phenotypes. This protocol describes how to prepare PMVs from BMSCs via extrusion through a polycarbonate membrane with 3 &#181;m pores, determine the existence of mitochondria and examine the maintenance of membrane potential within PMVs using a confocal microscope, concentrate PMVs by centrifugation, and carry out the in vivo injection of PMVs into the gastrocnemius muscle of mice.

Age-related diseases are associated with multiple defects in components of the cytoplasm. Here, we present a protocol to prepare plasma membrane vesicles from bone marrow mesenchymal stem cells. This technique could potentially be used as a means of cytoplasm replacement therapy to ameliorate or even reverse age-associated phenotypes.

从骨髓间充质干细胞制备血浆膜囊泡用于潜在的细胞质替代治疗

We have previously reported on the generation of plasma membrane vesicles (PMVs) through the mechanical extrusion of mammalian cells. The fusion of PMVs ...

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 ...

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 ...

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows within roughly two weeks after injury. Furthermore, zebrafish are able to heal bone rapidly after trepanation of the skull, and repair fractures that can be easily introduced into zebrafish bony fin rays. These injury assays represent feasible experimental paradigms to test the effect of administered drugs on rapidly forming bone. Here, we describe the use of these 3 injury models and their combined use with systemic glucocorticoid treatment, which exerts bone inhibitory and immunosuppressive effects. We provide a workflow on how to prepare for immunosuppressive treatment in adult zebrafish, illustrate how to perform fin amputation, trepanation of calvarial bones, and fin fractures, and describe how the use of glucocorticoids affects both bone forming osteoblasts and cells of the monocyte/macrophage lineage as part of innate immunity in bone tissue.

Here, we describe 3 adult zebrafish injury models and their combined use with immunosuppressive drug treatment. We provide guidance on imaging of regenerating tissues and on detecting bone mineralization therein.

成人斑马鱼损伤模型研究泼尼松对再生骨组织的影响

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...

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 ...