从造血器官中分离基质细胞
Summary
在这里，我们提出了能够从小鼠骨骼、骨髓、胸腺和人胸腺组织中分离基质细胞的方案，这些组织与单细胞多组学兼容。
Abstract
单细胞测序使得造血器官基质中异质细胞群的定位成为可能。这些方法提供了一个视角，通过它来研究以前未解决的稳态异质性，以及由外在应激或衰老过程中诱导的细胞类型表示的变化。在这里，我们提出了从小鼠和人胸腺以及小鼠骨骼和骨髓中分离高质量基质细胞群的逐步方案。通过这些方案分离的细胞适用于生成高质量的单细胞多组学数据集。本文将讨论样本消化、造血谱系耗竭、FACS分析/分选的影响，以及这些因素如何影响与单细胞测序的兼容性。通过FACS谱的示例，表明测序后分析中成功和低效的解离以及下游基质细胞产量，为用户提供了可识别的指针。考虑基质细胞的特定要求对于获得高质量和可重复的结果至关重要，这些结果可以推动该领域的知识发展。
Introduction
在健康的成年人中，血细胞的从头产生发生在骨髓和胸腺中。这些部位的基质细胞对于维持造血至关重要，但基质占组织的不到 1%，2,3,4。因此，获得支持基质的造血干细胞的纯分离株是一个重大挑战，特别是对于单细胞多组学而言，需要权宜之计才能获得高质量的样本。不同消化混合物的成分可能会干扰多组学分析中的某些步骤5,6。本文介绍的方案详细介绍了从骨髓和胸腺组织中分离出多种基质细胞的方法。
骨髓和胸腺中基质成分的扰动会导致血细胞发育的严重破坏，并可能导致恶性肿瘤 7,8,9。在细胞毒性调理和骨髓移植后，支持基质的造血功能受到损害，导致维持造血干细胞和祖细胞 （HSPC） 的细胞因子和生长因子的分泌减少2,10,11。此外，衰老会影响骨髓和胸腺基质细胞，可能导致造血表型老化。胸腺是第一个经历广泛的年龄相关退化的器官。早在青春期开始时，脂肪和纤维化组织就开始取代 T 细胞支持基质12,13。在骨髓中，脂肪细胞含量随着年龄的增长而增加，血管和骨内膜壁龛显着重塑14,15,16。
为了能够在多种应激状态下以及人和小鼠组织的胸腺中研究造血支持基质，我们优化了先前发表的消化方案1、2、8、17、18。这些方案确保了细胞的高效和可重复的分离，并且它们与单细胞RNA测序（scRNAseq）和其他类型的多组学兼容。
Protocol
所有与人体组织有关的工作都是在马萨诸塞州总医院内部审查委员会 （IRB） 批准后进行的。所有动物程序均按照马萨诸塞州总医院机构动物护理和使用委员会 （IACUC） 指南进行。本研究采用C57Bl/6只小鼠，年龄8-10周龄，雄性和雌性均为小鼠。这些动物是从商业来源获得的（见 材料表）。 1.鼠胸腺组织的制备 准备以下缓冲液和溶液。培?…
Representative Results
这些方案从胸腺和骨髓中产生可重复的基质细胞品种，适用于流式细胞术分析，以及单细胞多组学，如scRNA测序。小鼠胸腺组织在应激源的作用下经历了显著的重塑，例如骨髓移植前的细胞毒性调节或自然衰老过程。因此，在这两种情况下，胸腺细胞性都急剧降低（图1A）。虽然来自8周大的野生型小鼠的胸腺含有大约1亿个细胞，但2岁小鼠的细胞密度可以预期是其中的一半，…
Discussion
造血器官中的基质细胞对于正常造血至关重要，而造血基质扰动可导致造血维持和对应激反应的严重损害 9,23,24。深入了解造血基质细胞对于理解血液疾病至关重要 7,9,10,24 以及开发对抗它们的治疗方法的能力 25,26</sup…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们得到了马萨诸塞州总医院的HSCI-CRM流式细胞术设施和哈佛大学Bauer Core设施的专家技术支持。T.K 和 K. G 得到了瑞典研究委员会的支持，C.M. 得到了德国研究基金会的支持。我们感谢 Sergey Isaev 和 I-Hsiu Lee 在分析单细胞 RNA 测序数据方面的帮助。
Materials
| 0.25% Trypsin-EDTA | Thermo Fisher Scientific | 25200-072 | |
| 7AAD (7-aminoactinomycin D) | BD Biosciences | 559925 | |
| Anti-Human Lineage Cocktail 3-FITC | BD Biosciences | 643510 | |
| Bovine Serum Albumin | Millipore Sigma | A9647 | |
| C57Bl/6 mice | Jackson | 664 | Males or females, 8-12 weeks old |
| Calcein | Fisher Scientific | 65-0853-78 | |
| Collagenase IV | Millipore Sigma | C5138 | |
| Corning Sterile Cell Strainers, White, Mesh Size: 70 µm | Fisher Scientific | 08-771-2 | |
| DAPI (4',6-Diamidino-2-Phenylindole, Dilactate) | Biolegend | 422801 | |
| Dispase II | Thermo Fisher Scientific | 17105041 | |
| Dnase I Solution | Thermo Fisher Scientific | 90083 | 2500 U/mL |
| Easysep mouse streptavidin RapidSpheres Isolation kit | StemCell Technologies | 19860 | |
| Fetal Bovine Serum | Gibco | A31605-01 | Qualified One Shot |
| Human Fc Block | BD Biosciences | 564220 | |
| Liberase TM | Millipore Sigma | 5401127001 | Research Grade |
| Medium 199 | Gibco | 12350 | |
| Mouse anti-human CD235a-BV77 | BD Biosciences | 740785 | |
| Mouse anti-human CD31-PE/Dazzle594 | Biolegend | 303130 | |
| Mouse anti-human CD45-BV77 | Biolegend | 304050 | |
| Mouse anti-human CD4-BV605 | BD Biosciences | 562658 | |
| Mouse anti-human CD66b-FITC | BD Biosciences | 555724 | |
| Mouse anti-human CD8-APC/Cy7 | BD Biosciences | 557760 | |
| Mouse anti-human EpCam-BV421 | Biolegend | 324220 | |
| Protector RNase Inhibitor | Millipore Sigma | 3335402001 | |
| Rat anti-mouse CD105-PE /dazzle594 | Biolegend | 120424 | |
| Rat anti-mouse CD11b-Biotin | Biolegend | 101204 | |
| Rat anti-mouse CD140a-APC | Fisher Scientific | 17-1401-81 | |
| Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block) | BD Biosciences | 553142 | |
| Rat anti-mouse CD31-BUV737 | BD Biosciences | 612802 | |
| Rat anti-mouse CD31-BV421 | Biolegend | 102424 | |
| Rat anti-mouse CD3-Biotin | Biolegend | 100244 | |
| Rat anti-mouse CD45.2-Biotin | Biolegend | 109804 | |
| Rat anti-mouse CD45-PE/Cy7 | Biolegend | 103114 | |
| Rat anti-mouse CD45-PE/Cy7 | Biolegend | 103114 | |
| Rat anti-mouse CD45R/B220-Biotin | Biolegend | 103204 | |
| Rat anti-mouse CD51-PE | Biolegend | 104106 | |
| Rat anti-mouse EpCam-BV711 | BD Biosciences | 563134 | |
| Rat anti-mouse Ly-6A/E(Sca-1)-AF700 | Biolegend | 108142 | |
| Rat anti-mouse Ly-6G/Ly-6C(Gr1)-Biotin | Biolegend | 108404 | |
| Rat anti-mouse Ter119-Biotin | Biolegend | 116204 | |
| Rat anti-mouse Ter119-PE | Biolegend | 116208 | |
| Rat anti-mouse Ter119-PE/Cy7 | Biolegend | 116222 | |
| Stemxyme | Worthington Biochemical | LS004107 |
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Cite This Article
Kristiansen, T., Mayerhofer, C., Gustafsson, K., Scadden, D. T. Stromal Cell Isolation From Hematopoietic Organs. J. Vis. Exp. (203), e66231, doi:10.3791/66231 (2024).