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
Summary
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.
Abstract
共聚焦显微镜是用于复杂的组织中的多个细胞类型中的定位的分析选择的方法，如骨髓。然而，细胞定位的分析和定量是困难的，因为在许多情况下，它依赖于人工计数，从而轴承引入评价者依赖性偏差和减少间信的风险。此外，它往往是难以判断是否之间从随机定位两个小区的结果的共定位，特别是当细胞类型在它们发生的频率强烈不同。这里，用于蜂窝共定位于骨髓的无偏量化的方法被引入。该协议描述了用于获得全鼠长骨的组织切片，包括骨髓样品制备，以及在染色方案和获得高清晰度图像。分析工作流程从识别造血和非HEMAT的跨越在2维（2D）的骨髓的图像的那些细胞之间的直接接触的量化opoietic细胞类型呈现。这还包括一个邻域分析，以获得关于周围某一细胞类型的细胞微环境的信息。为了评价共定位两种细胞类型是否是随机的细胞定位的光效或反映了细胞间优先关联，一个模拟的工具，它适用于测试这个假设在造血以及基质细胞的情况下，是使用。这种方法不限定于骨髓，并可以扩展到其他组织中，以允许组织学数据的重现性好，定量分析。
Introduction
由于在显微镜最近快速的技术发展，包括光学成像，整个组织的范围内的细胞的分析已成为免疫学家越来越容易获得。单细胞的悬浮液的表征代表有价值的，不可缺少的方法来了解细胞和分子的功能。然而，该细胞的内它们的（微）-anatomical环境分析对于理解在复杂过程互相协作，如免疫反应的发展各种细胞类型之间的相互作用是必不可少的。
虽然这是相对容易的显微镜，以获得从图像质量信息，但它仍然量化这些数据是一个挑战，部分原因是由于在这个领域的分析方法进行滞后相比，现在有可能在图像采集的事实。许多研究人员仍然依靠耗时在他们的组织学图像手动细胞计数，从而带来了一种偏见之间不同的评价者和其他团体阻碍复制。通常情况下，一个代表图像被选择为强调对细胞的位置或共定位的声明在一个出版物，难以为读者来判断这样的事件的统计相关性。
再加上事实，即图像数据的全部信息内容很少利用，这强调了需要一个更公正，更快速和全面的方法来分析组织的图像。
骨髓是一个复杂的组织，这需要对重要生命功能作为造血在成年脊椎动物的器官。除了 是发源地为造血细胞1,2和打在B淋巴细胞发育3中起重要作用，它也可以作为一个网站，免疫反应引发4和支持成熟，再循环的B细胞5。此外，其在维持我的作用mmunological存储器已经越来越意识到，在过去的十年中，作为多种类型构成免疫记忆细胞已被发现位于那里6-9。
骨髓复杂的组织结构和功能之间的关系仍然难以捉摸。不同于次级淋巴器官，这些组织在宏观室如T和B细胞区，骨髓缺乏明确宏观条块。到目前为止鲜明隔间骨髓由其邻近骨皮质或脉管限定。在骨髓中的多个进程的各个驻地基质细胞群如支持干细胞，B细胞或维护免疫记忆细胞群（如长寿命的浆细胞（PC机），CD4 +和发育的重要性CD8 +记忆T细胞）清楚地表明，存在一定程度的微区室中的骨髓。
<p类=“jove_content”>这些观察导致了不同的显微解剖龛，这是专门在某些功能（干细胞维持，B细胞发育的各个阶段，和维护的免疫记忆）在骨髓中的概念。尽管似乎有一定程度的异质性的服务于不同功能的壁龛中，一些由基质细胞，如CXC趋化因子配体12（CXCL12）或白介素-7（IL-7），所产生的因素是非常重要的组件这几条龛10。在骨髓中的可视化和基质细胞的表征是困难的，因为它们与长而细的树突延伸形成整个骨髓的网络的形态特征，并且缺乏适当的标记，以辨别基质亚群。目前尚不清楚在何种程度上这些利基相对于它们的细胞和分子构成因素的共同特点n和哪些元素呈现一定的利基独特。除了基质细胞，造血细胞类型已经显示通过提供某些信号的至少一段的壁龛中发挥关键作用。显然，小生组合物的复杂性，需要他们的原位分析，它已成为日益重要的免疫学家和血液科放大对骨髓微， 例如，通过分析其细胞成分之间的空间关系。
这里，一个策略来量化在骨髓中的自动化和无偏方式蜂窝共定位和邻里关系呈现。详细的工作流程，包括嵌合小鼠的产生，窝藏荧光基质细胞和非荧光的造血细胞，制备从脱钙骨，采集覆盖整个骨的共焦图象的组织切片，以及蜂窝C的自动图像分析邻定位及验证/从随机定位由一个模拟工具判别被设置（ 图8）。
Protocol
Representative Results
Discussion
尽管在现代光学成像方法的进步，组织学数据的分析仍经常因缺乏适当的量化工具和方法，或偏颇的分析，重点关注小面积的阻碍。这里介绍的协同方法结合图像分析覆盖整个骨髓区域，自动分割和识别物体的各种造血和间质细胞类型，共定位分析，并通过了新的定做提供的非随机发生的接触最终验证工具设计仿真软件。
整个骨骼包括骨髓组织学一直难以进行，由于各种组?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们感谢安德烈亚斯·拉德布鲁赫的有价值的讨论。我们感谢萨宾Gruczek，帕特里克Thiemann和曼努埃拉Ohde援助与动物护理和罗伯特·冈瑟优秀的技术援助。我们感谢我们训练有素的评价者劳拉OEHME，Jannike巴亚特（Bayat） – Sarmadi，Karolin模特肖像权，卡特琳·罗斯，佛罗伦萨帕什和卡塔琳娜喇叭的组织学样品的评估和兰迪·林德奎斯特的手稿校对。我们感谢J.和N·李，梅奥诊所，斯科茨代尔，亚利桑那州，美国的MBP特异性抗体。
这项工作是支持的DFG HA5354 / 4-1，以JIMI – 一个用于活体显微镜DFG核设施网络补助和TRR130 / TP17和DFG FOR 2165（HA5354 / 6-1），以AEHSZ由国际马克斯·普朗克支持学校传染病和免疫（次展示-IDI），柏林。
Materials
| Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
| Neomycin | Sigma | N6386 SIGMA | Neomycin trisulfate salt hydrate, EU hazard code: GHS08; www.sigmaaldrich.com |
| Ursovit AD3EC | Serumwerke Bernburg | 1 ml contains: 50.000 I.E retinyl palmitate, 5.000 I.E. cholecalciferol, 30 mg tocopheryl acetate, 100 mg ascorbic acid, 1 mg sorbic acid, 200 mg polyoxyl 35 castor oil, 0,5 mg propyl gallate | |
| Transfer buffer (100ml PBS, 1ml 1M HEPES, 50U/ml Penicillin/Streptomycin) | Sigma | P4333 Sigma H3375 Sigma | www.sigmaaldrich.com |
| 4-Hydroxy-3-nitrophenylacetyl hapten conjugated to chicken gamma globulin) | |||
| Chicken gamma globulin (CGG) 100 mg | Rockland | D602-0100 | www.rockland-inc.com |
| 20 % Paraformaldehyde solution (EM-grade) | Science Services | 15713 | EU hazard codes: GHS02, GHS05, GHS07, GHS08 |
| D(+)-Sucrose | Carl Roth | 4621.1 | www.carlroth.com |
| Dry ice | |||
| Acetone | Sigma.Aldrich | 179124 SIGMA-ALDRICH | EU hazard codes: GHS02, GHS07; www.sigmaaldrich.com |
| Hexane | Sigma-Aldrich | 208752 SIGMA-ALDRICH | EU hazard codes: GHS02, GHS07, GHS08, GHS09; www.sigmaaldrich.com |
| Tissue-Tek cryomolds (standard) | Sakura | 4557 | 25 x 20 x 5 mm; www.sakuraus.com |
| Tissue-Tek O.C.T. | Sakura | 4583 | www.sakuraus.com |
| Kawamoto's SCEM embedding medium | Section-Lab, JP | http://section-lab.jp/index.html | |
| Kawamoto's cryosection preparation kit | Section-Lab, JP | http://section-lab.jp/index.html | |
| Kawamoto's cryofilm type 2C(9) | Section-Lab, JP | http://section-lab.jp/index.html | |
| Microtome blade MX35 Premier Plus, Low Profile | Thermo Scientific | 3052835 | L X W: 80 x 8 mm (31.5 x 3.13"), thickness: 0.25 mm (0.01");www.thermoscientific.com |
| polyclonal rabbit anti-RFP antibody, biotinylated | Rockland | 600-406-379 | www.rockland-inc.com |
| Alexa Fluor 555 streptavidin | Life Technologies | S-32355 | www.lifetechnologies.com |
| rat anti-MBP | J. and N. Lee | available from: J. and N. Lee, Mayo Clinic, Scottsdale, AZ , U.S.A., clone MT-14.7 | |
| goat anti-rat-Alexa Fluor 647 | Life Technologies | A-21247 | www.lifetechnologies.com |
| rat anti-B220 – Alexa Fluor 594 | produced and coupled in-house (DRFZ), clone RA3.6B2, Alexa Fluor 594 from Life Technologies | ||
| mouse anti-l1 light chain -FITC | produced and coupled in-house (DRFZ), clone LS136 | ||
| rat anti-k light chain – FITC | produced and coupled in-house (DRFZ), clone 187.1 | ||
| DAPI (4′,6-diamidino-2-phenylindole dihydrochloride) | Sigma | D9542 SIGMA | www.sigmaaldrich.com |
| Fluorescent mounting medium | DAKO | S3023 | www.dako.com |
| Cover slips (24mm x 24mm x 0.13-0.16 mm) | Carl Roth | H875.2 | www.carlroth.com |
| Superfrost slides glasses (75 mm x 25 mm) | VWR | 48311-703 | www.vwr.com |
| Laser scanning confocal microscope | equipped with laser lines of 405, 488, 561, 594, 633 nm and a 20x/0.8 NA air objective lens; We used a Zeiss LSM710 and Zen 2010 Version 6.0 software. | ||
| Automated image analysis tools for bone marrow | Wimasis, Munich | The cell contact tool and cell vicinity tool will be made available by Wimasis upon request: www.wimasis.de | |
| VC2012 Runtime | Microsoft | free download: http://www.microsoft.com/en-us/download/details.aspx?id=30679 | |
| Simulation tool for random cell positioning | available from us, upon request | ||
| Image analysis software with image segmentation functions | We used Volocity (Perkin Elmer) for measuring cell size distributions of hematopoietic cell types in bone marrow (Figure 5). Alternatives are Definiens Image Analysis (Definiens) or Cell Profiler (free download: http://www.cellprofiler.org) | ||
| Fiji image analysis software | free download: http://fiji.sc/Fiji. Fiji was used by trained raters for manual cell count (Figure 3). |
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