小鼠巨核细胞祖细胞的分离
Summary
该方法描述了通过流式细胞术从小鼠股骨，胫骨和盆骨中纯化MEP和MKp。
Abstract
骨髓巨核细胞是确保血小板产生的大多倍体细胞。它们通过巨核生成从造血干细胞中产生。该过程的最后阶段很复杂，通常涉及双能巨核细胞 – 红细胞祖细胞（MEP）和单能巨核细胞祖细胞（MKp）。这些群体先于 真正的 巨核细胞的形成，因此，它们的分离和表征可以允许对巨核细胞的形成进行稳健和公正的分析。该协议详细介绍了从小鼠骨髓收集造血细胞的程序，通过磁消耗富集造血祖细胞，最后是产生高度纯化的MEP和MKp群体的细胞分选策略。首先，骨髓细胞从股骨，胫骨和髂嵴收集，髂嵴是含有大量造血祖细胞的骨骼。髂骨的使用大大增加了每只小鼠获得的总细胞数，从而有助于更合乎道德地使用动物。使用450 nm磁珠优化了磁谱系耗尽，允许通过流式细胞术进行非常有效的细胞分选。最后，该协议提出了两种高度纯化的巨核细胞祖细胞群体分类的标记和门控策略：MEP（Lin–Sca-1–c-Kit+CD16 / 32–CD150+CD9dim）和MKp（Lin– Sca-1–c-Kit+CD16 / 32–CD150+CD9明亮）).该技术易于实施，并提供足够的细胞材料来执行i）分子表征以更深入地了解其身份和生物学，ii） 体外 分化测定，这将更好地理解巨核细胞成熟的机制，或iii）与微环境相互作用的 体外 模型。
Introduction
血小板由巨核细胞产生。这些大的多倍体细胞位于骨髓中，对于所有血细胞，它们都来自造血干细胞（HSC）1。骨髓中巨核细胞产生的经典途径起源于HSC，涉及不同祖细胞的产生，这些祖细胞逐渐限制了它们的分化潜力2。签署对巨核细胞谱系承诺的第一个祖是巨核细胞 – 红细胞祖细胞（MEP），这是一种能够同时产生红细胞和巨核细胞3，4，5的双能祖。然后，MEP产生一个单能祖细胞/前体（MKp），它将分化成能够产生血小板的成熟巨核细胞。这些祖细胞的产生所涉及的机制，以及它们分化和成熟为巨核细胞是复杂的，只能部分理解。此外，MEP群体在分化潜力方面的异质性和这些细胞的内在承诺水平尚不清楚。为了破译这些过程，必须获得（或能够获得）纯化的MEP和MKp群体，以进行精细分子和单细胞分析。
一些研究表明，细胞表面标记物的特殊组合用于鉴定小鼠6，7，8中致力于巨核细胞谱系的祖细胞。从这些中设计出一种方法，允许从小鼠中纯化MEP和MKp。该方法经过优化，可获得足够数量和质量的细胞，以进行大量测定。考虑到伦理因素，为了尽量减少参与实验的动物数量，我们从股骨和胫骨以及髂嵴中收获骨髓。这种骨含有高频率和数量的造血祖细胞，并且在长骨收获过程中大部分时间都受损。这里介绍的是可靠收集该骨骼的详细方法。
优化的第二个标准是产生高度纯化的细胞群。荧光活化细胞分选（FACS）是获得纯化目标细胞群的首选方法。然而，当感兴趣的细胞群非常罕见时，就会达到低产量。因此，浓缩程序是必要的。在该协议中，使用磁珠选择负选择程序。
Protocol
Representative Results
Discussion
本文中描述的方法允许提取和纯化小鼠MEP和MKp。方案优化的一个重要参数是获得足够数量的细胞，这些细胞与大多数基于分子和细胞的测定相容。收集小鼠骨以进行造血细胞提取的一般做法通常包括收获每只小鼠的股骨和胫骨。因此，骨盆骨是造血材料的另一个来源，经常被忽视。不收集髂嵴的原因是对小鼠骨骼的内部解剖结构知之甚少，并且用户通常通过穿过或刚好在股骨头上方来收集后肢。?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者希望感谢Monique Freund，Catherine Ziessel和Ketty的技术援助。这项工作得到了ARMESA（医学和公共发展协会）和Grant ANR-17-CE14-0001-01的支持，以 Henri.de la。大厅。
Materials
| 21-gauge needles | BD Microlance | 301155 | |
| 7AAD | Sigma-Aldrich | A9400 | |
| Antibody Gr-1-biotin | eBioscience | 13-5931-85 | Magnetic depletion |
| Antibody B220-biotin | eBioscience | 13-0452-85 | Magnetic depletion |
| Antibody Mac-1-biotin | eBioscience | 13-0112-85 | Magnetic depletion |
| Antibody CD3e-biotin | eBioscience | 13-0031-85 | Magnetic depletion |
| Antibody CD4-biotin | eBioscience | 13-9766-82 | Magnetic depletion |
| Antibody CD5-biotin | eBioscience | 13-0051-85 | Magnetic depletion |
| Antibody CD8a-biotin | eBioscience | 13-0081-85 | Magnetic depletion |
| Antibody TER119-biotin | eBioscience | 13-5921-85 | Magnetic depletion |
| Antibody CD127-biotin | eBioscience | 13-1271-85 | Magnetic depletion |
| Antibody CD45-FITC | eBioscience | 11-0451-85 | Cell sorting |
| Antibody CD45-PE | eBioscience | 12-0451-83 | Cell sorting |
| Antibody TER119-APC | eBioscience | 17-5921-83 | Cell sorting |
| Antibody CD45-PECy7 | eBioscience | 25-0451-82 | Cell sorting |
| Antibody CD45-biotin | eBioscience | 13-0451-85 | Cell sorting |
| Antibody CD9-FITC | eBioscience | 11-0091-82 | Cell sorting |
| Antibody c-kit-APC | eBioscience | 17-1171-83 | Cell sorting |
| Antibody Sca-1-PE | eBioscience | 12-5981-83 | Cell sorting |
| Antibody CD16/32-PE | eBioscience | 12-0161-83 | Cell sorting |
| Antibody CD150-PECy7 | eBioscience | 25-1502-82 | Cell sorting |
| Culture medium StemSpan-SFEM | Stemcell technologies | #09650 | |
| Dissection pad | Fisher Scientific | 10452395 | |
| DPBS | Life Technologies | 14190-094 | |
| Ethanol | vWR Chemicals | 83813.360 | |
| Forceps | Euronexia | P-120-AS | |
| Glass pasteur pipette | Dutscher | 42011 | |
| Magnet : DynaMag-5 | Thermo Fisher Scientific | 12303D | |
| Magnetic beads: Dynabeads Sheep Anti-Rat IgG | Thermo Fisher Scientific | 11035 | |
| Megacult | Stemcell technologies | #04970 | |
| MethoCult SF M3436 | Stemcell technologies | #03436 | |
| Newborn Calf Serum | Dutscher | 50750-500 | |
| Red Cell Lysis solution | BD Bioscience | 555899 | |
| Scalpels | Fisher Scientific | 12308009 | |
| Scissors | Euronexia | C-165-ASB | |
| Sterile 1 mL syringes | BD Bioscience | 303172 | |
| Sterile 15mL tubes | Sarstedt | 62.554.502 | |
| Sterile 5mL polypropylene tubes | Falcon | 352063 | |
| Sterile 5mL polystyrene tubes | Falcon | 352054 | |
| Sterile tubes with 70µm cell strainer cap | Falcon | 352235 | |
| Sterile petri dish | Falcon | 353003 | |
| Streptavidin-APC-Cy7 | BD Biosciences | 554063 | Cell sorting |
| Tube roller | Benchmark Scientific | R3005 |
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