长期和短期造血干细胞的分离方法
Summary
我们提出了使用Hoxb5报告系统分离长期造血干细胞（LT-HSC）和短期造血干细胞（ST-HSC）的分步方案。
Abstract
自我更新能力和多谱系分化潜力通常被认为是造血干细胞（HSCs）的决定性特征。然而，大量研究表明，HSC隔室中存在功能异质性。最近的单细胞分析报告了HSC区室内具有不同细胞命运的HSC克隆，这被称为偏倚HSC克隆。异质性或可重复性差的结果背后的机制知之甚少，特别是关于通过常规免疫染色移植纯化的HSC级分时的自我更新时间。因此，建立一种可重复的长期HSC（LT-HSC）和短期HSC（ST-HSC）的分离方法，由其自我更新的长度定义，对于克服这个问题至关重要。使用无偏的多步骤筛选，我们鉴定了一种转录因子 Hoxb5，它可能是小鼠造血系统中LT-HSC的独家标志物。基于这一发现，我们建立了 Hoxb5 报告小鼠系，并成功分离出LT-HSC和ST-HSC。在这里，我们描述了使用 Hoxb5 报告系统分离LT-HSC和ST-HSC的详细协议。这种分离方法将有助于研究人员更好地了解自我更新的机制以及HSC隔室中这种异质性的生物学基础。
Introduction
造血干细胞（HSCs）具有自我更新能力和多能性，位于造血层次结构的顶点1，2。1988年，Weissman及其同事首次证明可以使用流式细胞术3实现小鼠HSC的分离。随后，据报道，由细胞表面标志物组合定义的部分Lineage-c-Kit+Sca-1 + CD150 + CD34 / loFlk2−含有小鼠4，5，6，7，8中的所有HSC。
免疫表型定义的（谱系−c-Kit+Sca-1+CD150+CD34−/loFlk2−）HSC（以下简称pHSC）以前被认为是功能同质的。然而，最近的单细胞分析显示，pHSCs在其自我更新能力9，10和多能性11，12方面仍表现出异质性。具体而言，pHSC分数中似乎存在两个自我更新能力的人群：具有持续自我更新能力的长期造血干细胞（LT-HSCs）和具有短暂自我更新能力的短期造血干细胞（ST-HSCs）9，10。
迄今为止，区分LT-HSC和ST-HSC的自我更新能力的分子机制仍然知之甚少。根据细胞的自我更新能力分离两个细胞群并发现潜在的分子机制至关重要。还引入了几种报告系统来纯化LT-HSC13，14，15;然而，每个报告系统定义的LT-HSC纯度是可变的，迄今为止尚未实现独家LT-HSC纯化。
因此，开发LT-HSC和ST-HSC的分离系统将加速pHSC部分自我更新能力的研究。在分离LT-HSC和ST-HSCs时，一项使用多步骤，无偏倚筛选的研究确定了单个基因Hoxb5，该基因在pHSC部分16中异质表达。此外，对Hoxb5报告小鼠的骨髓分析显示，大约20%-25%的pHSC部分由Hoxb5 pos细胞组成。使用Hoxb5 pos pHSC和Hoxb5阴性pHSC的竞争性移植测定显示，只有Hoxb5pos pHSC具有长期自我更新能力，而Hoxb5阴性pHSC在短时间内失去其自我更新能力，表明Hoxb5在pHSC部分16中识别LT-HSC。
在这里，我们演示了使用 Hoxb5 报告系统分离LT-HSC和ST-HSC的分步协议。此外，我们提出了一种竞争性移植测定法来评估Hoxb5pos / neg pHSC的自我更新能力（图1）。该 Hoxb5 报告系统使我们能够前瞻性地分离LT-HSC和ST-HSC，并有助于了解LT-HSC特异性特征。
Protocol
所有描述的动物实验都得到了RIKEN生物系统动力学研究中心的批准。 1.受体小鼠的预处理 准备8-10周龄的雄性C57BL / 6同源小鼠作为受体小鼠。受体小鼠的数量取决于实验方案。我们通常为每种情况准备10-20只小鼠。用补充有恩诺沙星（170mg / L）的灭菌水喂养小鼠。由于受辐照的受体小鼠极易感染，因此请保持笼子尽可能清洁。注意：在照射前24小时?…
Representative Results
以前，已经使用竞争性移植测定法测量了自我更新能力，其中供体HSC被认为只有在观察到受体外周血中的多谱系供体细胞时才保持其自我更新能力17。此外，一些报告将LT-HSC定义为在第二次骨髓移植几个月后继续产生外周血细胞的细胞10，18。因此，为了比较它们的自我更新能力，将从Hoxb5报告小鼠中分离出的10只Hoxb5pos或Hoxb5…
Discussion
传统上，已经制备了细胞表面标记定义的HSCs来研究HSC的功能，例如自我更新能力和多效性19，20，21。然而，免疫表型定义的（谱系−c-Kit+Sca-1+CD150+CD34−/loFlk2−）HSC部分包含两个离散的HSC群体：LT-HSC和ST-HSC9，10。因此，对真?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们非常感谢Hiroshi Kioninari在RIKEN BDR提供的动物护理和提供受体小鼠，以及神户大学实验室管理的小贺瞳，长坂加代子和宫桥正树。作者也非常感谢对这项工作的持续支持。Masanori Miyanishi得到了日本科学促进会（JSPS）KAKENHI资助号JP17K07407和JP20H03268，Mochida医学和药物研究纪念基金会，日本生命科学基金会，武田科学基金会，安斯泰来代谢紊乱研究基金会和AMED-PRIME，AMED的支持，资助号JP18gm6110020。 坂卷太郎由JSPS KAKENHI授权号JP21K20669和JP22K16334支持，并得到以下机构的支持JSPS核心到核心计划和RIKEN初级研究助理计划。Katsuyuki Nishi得到了JSPS授权号KAKENHI JP18J13408的支持。
Materials
| 0.2 mL Strip of 8 Tubes, Dome Cap | SSIbio | 3230-00 | |
| 0.5M EDTA pH 8.0 | Iinvtrogen | AM9260G | |
| 100 µm Cell Strainer | Falcon | 352360 | |
| 30G insulin syringe | BD | 326668 | |
| 40 µm Cell Strainer | Falcon | 352340 | |
| 5 mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap | FALCON | 352235 | |
| 7-AAD Viability Staining Solution | BioLegend | 420404 | |
| 96 well U-Bottom | FALCON | 351177 | |
| Anti-APC-MicroBeads | Milteny biotec | 130-090-855 | |
| Aspirator with trap flask | Biosan | FTA-1 | |
| B220-Alexa Fluor 700 (RA3-6B2) | BioLegend | 103232 | |
| B220-Biotin (RA3-6B2) | BioLegend | 103204 | |
| B220-BV786 (RA3-6B2) | BD Biosciences | 563894 | |
| B6.CD45.1 congenic mice | Sankyo Labo Service | N/A | |
| Baytril 10% | BAYER | 341106546 | |
| BD FACS Aria II special order system | BD | N/A | |
| Brilliant stain buffer | BD | 566349 | |
| CD11b-Alexa Fluor 700 (M1/70) | BioLegend | 101222 | |
| CD11b-Biotin (M1/70) | BioLegend | 101204 | |
| CD11b-BUV395 (M1/70) | BD Biosciences | 563553 | |
| CD11b-BV711 (M1/70) | BD Biosciences | 563168 | |
| CD127-Alexa Fluor 700 (A7R34) | Invitrogen | 56-1271-82 | |
| CD150-BV421 (TC15-12F12.2) | BioLegend | 115943 | |
| CD16/CD32-Alexa Fluor 700 (93) | Invitrogen | 56-0161-82 | |
| CD34-Alexa Fluor 647 (RAM34) | BD Biosciences | 560230 | |
| CD34-FITC (RAM34) | Invitrogen | 11034185 | |
| CD3-Alexa Fluor 700 (17A2) | BioLegend | 100216 | |
| CD3ε -Biotin (145-2C11) | BioLegend | 100304 | |
| CD3ε -BV421 (145-2C11) | BioLegend | 100341 | |
| CD45.1/CD45.2 congenic mice | N/A | N/A | Bred in our Laboratory |
| CD45.1-FITC (A20) | BD Biosciences | 553775 | |
| CD45.2-PE (104) | BD Biosciences | 560695 | |
| CD4-Alexa Fluor 700 (GK1.5) | BioLegend | 100430 | |
| CD4-Biotin (GK1.5) | BioLegend | 100404 | |
| CD8a-Alexa Fluor 700 (53-6.7) | BioLegend | 100730 | |
| CD8a-Biotin (53-6.7) | BioLegend | 100704 | |
| Centrifuge Tube 15ml | NICHIRYO | 00-ETS-CT-15 | |
| Centrifuge Tube 50ml | NICHIRYO | 00-ETS-CT-50 | |
| c-Kit-APC-eFluor780 (2B8) | Invitrogen | 47117182 | |
| D-PBS (-) without Ca and Mg, liquid | Nacalai | 14249-24 | |
| Fetal Bovine Serum | Thermo Fisher | 10270106 | |
| Flk2-PerCP-eFluor710 (A2F10) | eBioscience | 46135182 | |
| FlowJo version 10 | BD Biosciences | https://www.flowjo.com/solutions/flowjo | |
| Gmmacell 40 Exactor | Best theratronics | N/A | |
| Gr-1-Alexa Fluor 700 (RB6-8C5) | BioLegend | 108422 | |
| Gr-1-Biotin (RB6-8C5) | BioLegend | 108404 | |
| Hoxb5-tri-mCherry mice (C57BL/6J background) | N/A | N/A | Bred in our Laboratory |
| IgG from rat serum, technical grade, >=80% (SDS-PAGE), buffered aqueous solution | Sigma-Aldrich | I8015-100MG | |
| isoflurane | Pfizer | 4987-114-13340-3 | |
| Kimwipes S200 | NIPPON PAPER CRECIA | 6-6689-01 | |
| LS Columns | Milteny biotec | 130-042-401 | |
| Lysis buffer | BD | 555899 | |
| MACS MultiStand | Milteny biotec | 130-042-303 | |
| Microplate for Tissue Culture (For Adhesion Cell) 6Well | IWAKI | 3810-006 | |
| MidiMACS Separator | Milteny biotec | 130-042-302 | |
| Mouse Pie Cages | Natsume Seisakusho | KN-331 | |
| Multipurpose refrigerated Centrifuge | TOMY | EX-125 | |
| NARCOBIT-E (II) | Natsume Seisakusho | KN-1071-I | |
| NK-1.1-PerCP-Cy5.5 (PK136) | BioLegend | 108728 | |
| Penicillin-Streptomycin Mixed Solution | nacalai | 26253-84 | |
| Porcelain Mortar φ120mm with Pestle | Asone | 6-549-03 | |
| Protein LoBind Tube 1.5 mL | Eppendorf | 22431081 | |
| Sca-I-BUV395 (D7) | BD Biosciences | 563990 | |
| Stainless steel scalpel blade | FastGene | FG-B2010 | |
| Streptavidin-BUV737 | BD Biosciences | 612775 | |
| SYTOX-red | Invitrogen | S34859 | |
| Tailveiner Restrainer for Mice standard | Braintree | TV-150 STD | |
| TCRb-BV421 (H57-597) | BioLegend | 109230 | |
| Ter-119-Alexa Fluor 700 (TER-119) | BioLegend | 116220 | |
| Ter-119-Biotin (TER-119) | BioLegend | 116204 | |
| Terumo 5ml Concentric Luer-Slip Syringe | TERUMO | SS-05LZ | |
| Terumo Hypodermic Needle 23G x 1 | TERUMO | NN-2325-R |
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Cite This Article
Nishi, K., Nagasaka, A., Sakamaki, T., Sadaoka, K., Miyanishi, M. Isolation Method for Long-Term and Short-Term Hematopoietic Stem Cells. J. Vis. Exp. (195), e64488, doi:10.3791/64488 (2023).