Name: combining intravital fluorescent microscopy (ivfm) with genetic models to study engraftment dynamics of hematopoietic cells to bone marrow niches
Uploaded: 2017-03-21T13:00:00.000+00:00
Duration: 11 min 6 s
Description: 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

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>

<ol>
	<li>Carlesso, N., Cardoso, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+regulatory+niches+and+their+role+in+normal+and+malignant+hematopoiesis.">Stem cell regulatory niches and their role in normal and malignant hematopoiesis.</a> Curr Opin Hematol. 17 (4), 281-286 (2010).</li><li>Lo Celso, C., Scadden, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+haematopoietic+stem+cell+niche+at+a+glance.">The haematopoietic stem cell niche at a glance.</a> J Cell Sci. 124 (PT 21), 3529-3535 (2011).</li><li>Calvi, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoblastic+cells+regulate+the+haematopoietic+stem+cell+niche.">Osteoblastic cells regulate the haematopoietic stem cell niche.</a> Nature. 425 (6960), 841-846 (2003).</li><li>Kiel, M. J., Yilmaz, O. H., Iwashita, T., Terhorst, C., Morrison, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SLAM+family+receptors+distinguish+hematopoietic+stem+and+progenitor+cells+and+reveal+endothelial+niches+for+stem+cells.">SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells.</a> Cell. 121 (7), 1109-1121 (2005).</li><li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+haematopoietic+stem+cell+niche+and+control+of+the+niche+size.">Identification of the haematopoietic stem cell niche and control of the niche size.</a> Nature. 425 (6960), 836-841 (2003).</li><li>Mendez-Ferrer, 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=Mesenchymal+and+haematopoietic+stem+cells+form+a+unique+bone+marrow+niche.">Mesenchymal and haematopoietic stem cells form a unique bone marrow niche.</a> Nature. 466 (7308), 829-834 (2010).</li><li>Naveiras, O., 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=Bone-marrow+adipocytes+as+negative+regulators+of+the+haematopoietic+microenvironment.">Bone-marrow adipocytes as negative regulators of the haematopoietic microenvironment.</a> Nature. 460 (7252), 259-263 (2009).</li><li>Scheiermann, 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=Adrenergic+nerves+govern+circadian+leukocyte+recruitment+to+tissues.">Adrenergic nerves govern circadian leukocyte recruitment to tissues.</a> Immunity. 37 (2), 290-301 (2012).</li><li>Faust, N., Varas, F., Kelly, L. M., Heck, S., Graf, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insertion+of+enhanced+green+fluorescent+protein+into+the+lysozyme+gene+creates+mice+with+fluorescent+granulocytes+and+macrophages.">Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with fluorescent granulocytes and macrophages.</a> Blood. 96 (2), 716-726 (2000).</li><li>Han, H., 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=Inducible+gene+knockout+of+transcription+factor+recombination+signal+binding+protein-J+reveals+its+essential+role+in+T+versus+B+lineage+decision.">Inducible gene knockout of transcription factor recombination signal binding protein-J reveals its essential role in T versus B lineage decision.</a> Int Immunol. 14 (6), 637-645 (2002).</li><li>Lo Celso, 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=Live-animal+tracking+of+individual+haematopoietic+stem/progenitor+cells+in+their+niche.">Live-animal tracking of individual haematopoietic stem/progenitor cells in their niche.</a> Nature. 457 (7225), 92-96 (2009).</li><li>Wang, L., 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=Notch-dependent+repression+of+miR-155+in+the+bone+marrow+niche+regulates+hematopoiesis+in+an+NF-kappaB-dependent+manner.">Notch-dependent repression of miR-155 in the bone marrow niche regulates hematopoiesis in an NF-kappaB-dependent manner.</a> Cell Stem Cell. 15 (1), 51-65 (2014).</li><li>Miyamoto, T., 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=Myeloid+or+lymphoid+promiscuity+as+a+critical+step+in+hematopoietic+lineage+commitment.">Myeloid or lymphoid promiscuity as a critical step in hematopoietic lineage commitment.</a> Dev Cell. 3 (1), 137-147 (2002).</li><li>Luc, 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=Down+Regulation+of+Mpl+marks+the+transition+to+lymphoid-primed+multipotent+progenitors+with+gradual+loss+of+granulocyte-monocyte+potential.">Down Regulation of Mpl marks the transition to lymphoid-primed multipotent progenitors with gradual loss of granulocyte-monocyte potential.</a> Blood. 111 (7), 3424-3434 (2008).</li><li>Suzuki, M., Moriguchi, T., Ohneda, K., Yamamoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+contribution+of+the+Gata1+gene+hematopoietic+enhancer+to+erythroid+differentiation.">Differential contribution of the Gata1 gene hematopoietic enhancer to erythroid differentiation.</a> Mol Cell Biol. 29 (5), 1163-1175 (2009).</li><li>Essers, M. A., 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=IFNalpha+activates+dormant+haematopoietic+stem+cells+in+vivo.">IFNalpha activates dormant haematopoietic stem cells in vivo.</a> Nature. 458 (7240), 904-908 (2009).</li><li>Pietras, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Re-entry+into+quiescence+protects+hematopoietic+stem+cells+from+the+killing+effect+of+chronic+exposure+to+type+I+interferons.">Re-entry into quiescence protects hematopoietic stem cells from the killing effect of chronic exposure to type I interferons.</a> J Exp Med. 211 (2), 245-262 (2014).</li><li>Scott, M. K., Akinduro, O., Lo Celso, C. <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+4-dimensional+tracking+of+hematopoietic+stem+and+progenitor+cells+in+adult+mouse+calvarial+bone+marrow.">In vivo 4-dimensional tracking of hematopoietic stem and progenitor cells in adult mouse calvarial bone marrow.</a> J Vis Exp. (91), e51683 (2014).</li></ol>

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><tbody><tr><td>Ketamine cocktail</td><td>IU School of Medicine</td><td></td><td>Ketamine 90-100 mg/kg, Xylazine 2.5-5.0 mg/kg, Acepromazine 1.0-2.5 mg/kg</td></tr><tr><td>TRITC dextran</td><td>Tdb Consultancy</td><td>TD150-100mg</td><td>Other color dextran may be used.</td></tr><tr><td>Andis hair trimmer</td><td>Braintree Scientific</td><td>CLP-323 75</td><td></td></tr><tr><td>Gauze sponge</td><td>Med Vet International</td><td>PK224</td><td>4-ply, 2 x 2</td></tr><tr><td>Nair depilatory cream</td><td>Commercial store</td><td></td><td></td></tr><tr><td>Saline</td><td>Med Vet International</td><td>RXSAL-POD1LT</td><td>0.9% Sodium Chloride poly bottle</td></tr><tr><td>Insulin syringe</td><td>Fisher Scientific</td><td>14-826-79</td><td>28 g, 1/2 cc</td></tr><tr><td>Fine Forceps</td><td>Fine Science Tools</td><td>00108-11, 00109-11</td><td>straight forcep, angled forcep</td></tr><tr><td>Scissor</td><td>Fine Science Tools</td><td>15018-10</td><td></td></tr><tr><td>Needle holder</td><td>Fine Science Tools</td><td>12002-14</td><td></td></tr><tr><td>5-0 silk suture</td><td>Fisher Scientific</td><td>MV-682</td><td>Other non-absorbable suture may be used</td></tr><tr><td>WillCo- glass bottom dish</td><td>WillCo</td><td>GWSt-5040</td><td></td></tr><tr><td>Optical microscope oil</td><td>Leica</td><td></td><td></td></tr><tr><td>Stereotaxic stage insert&amp;nbsp;</td><td>IU School of Medicine</td><td>Custom design</td><td></td></tr><tr><td>Olympus FV1000 confocal microscope system&amp;nbsp;</td><td>Olympus</td><td></td><td></td></tr><tr><td>Olympus XLUMPLFL 20XW, NA 0.95 objective&amp;nbsp;</td><td>Olympus</td><td></td><td></td></tr><tr><td>Small heating pad</td><td>Commercial store</td><td></td><td>Zoo Med reptile heating pad</td></tr><tr><td>Imaris 8.1 imaging software</td><td>Bitplane</td><td></td><td>3/4 D Image Visualization and Analysis software</td></tr></tbody></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="http://cloudflare.jove.com/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="http://cloudflare.jove.com/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="http://cloudflare.jove.com/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="http://cloudflare.jove.com/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="http://cloudflare.jove.com/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

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

开发，扩展，<em&gt;在体内</em&gt;从人类胚胎干细胞（hESCs）监测人类NK细胞和诱导多能干细胞（iPS细胞）

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

生物工程

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 ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

采用实时定量PCR检测供体细胞植入一个具有竞争力的小鼠骨髓移植模型

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

医学

Quantitative Multispectral Analysis Following Fluorescent Tissue Transplant for Visualization of Cell Origins, Types, and Interactions

With the desire to understand the contributions of multiple cellular elements to the development of a complex tissue; such as the numerous cell types that participate in regenerating tissue, tumor formation, or vasculogenesis, we devised a multi-colored cellular transplant model of tumor development in which cell populations originate from different fluorescently colored reporter gene mice and are transplanted, engrafted or injected in and around a developing tumor. These colored cells are then recruited and incorporated into the tumor stroma. In order to quantitatively assess bone marrow derived tumor stromal cells, we transplanted GFP expressing transgenic whole bone marrow into lethally irradiated RFP expressing mice as approved by IACUC. 0ovarian tumors that were orthotopically injected into the transplanted mice were excised 6-8 weeks post engraftment and analyzed for bone marrow marker of origin (GFP) as well as antibody markers to detect tumor associated stroma using multispectral imaging techniques. We then adapted a methodology we call MIMicc- Multispectral Interrogation of Multiplexed cellular compositions, using multispectral unmixing of fluoroprobes to quantitatively assess which labeled cell came from which starting populations (based on original reporter gene labels), and as our ability to unmix 4, 5, 6 or more spectra per slide increases, we&#39;ve added additional immunohistochemistry associated with cell lineages or differentiation to increase precision. Utilizing software to detect co-localized multiplexed-fluorescent signals, tumor stromal populations can be traced, enumerated and characterized based on marker staining.1

Complex tissue masses, from organs to tumors, are composed of various cellular elements. We elucidated the contribution of cellular phenotypes within a tissue utilizing multi-labeled fluorescent transgenic mice in combination with multiparameter immunofluorescent staining followed by spectral unmixing to decipher cell origin as well as cell characteristics based on protein expression.

多光谱定量分析继荧光组织移植的细胞起源，类型和相互作用的可视化

With the desire to understand the contributions of multiple cellular elements to the development of a complex tissue; such as the numerous cell types that ...

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, ...

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 ...

生物学

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) ...

<meta charset="utf8"></head><body>股骨内注射用于活小鼠骨髓研究

Simplified Intrafemoral Injections Using Live Mice Allow for Continuous Bone Marrow Analysis

Despite the complexity of hematopoietic cell transplantation in humans, researchers commonly perform intravenous or intrafemoral (IF) injections in mice. In murine models, this technique has been adapted to enhance the seeding efficiency of transplanted hematopoietic stem and progenitor cells (HSPCs). This paper describes a detailed step-by-step technical procedure of IF injection and the following bone marrow (BM) aspiration in mice that allows for serial characterization of cells present in the BM. This method enables the transplantation of valuable samples with low cell numbers that are particularly difficult to engraft by intravenous injection. This procedure facilitates the creation of xenografts that are critical for pathological analysis. While it is easier to access peripheral blood (PB), the cellular composition of PB does not reflect the BM, which is the niche for HSPCs. Therefore, procedures providing access to the BM compartment are essential for studying hematopoiesis. IF injection and serial BM aspiration, as described here, allow for the prospective retrieval and characterization of cells enriched in the BM, such as HSPCs, without sacrificing the mice.

<meta charset="utf8"></head><body>使用活小鼠进行简化的股骨内注射允许连续骨髓分析

This protocol describes the intrafemoral injection of a few hematopoietic or leukemic stem cells, including gene-edited cells, in murine xenograft models, which will not only enable the quick and safe transplantation of cells but also serial analyses of the bone marrow.

使用活小鼠进行简化的股骨内注射允许连续骨髓分析

Despite the complexity of hematopoietic cell transplantation in humans, researchers commonly perform intravenous or intrafemoral (IF) injections in mice. ...

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

摘要

探索更多视频

此视频中的章节