造血干细胞代谢分析
Summary
造血干细胞（HSPCs）由于血液形成过程中的代谢可塑性，从静止状态过渡到分化状态。在这里，我们提出了一个优化的方法，用于测量线粒体呼吸和HSPCs的糖解。
Abstract
造血干细胞（HSPCs）具有明显的代谢可塑性，允许它们从静止状态过渡到分化状态，以维持血液形成的需求。然而，由于HSPC数量有限，并且缺乏针对非粘附性、易碎HSPCs的优化方案，因此很难分析HSPC的代谢状态（线粒体呼吸和糖解）。在这里，我们提供一套清晰、分步的指示，以测量代谢呼吸（耗氧率;OCR）和糖解（细胞外酸化率;ECAR） 的鼠骨髓-血统内格Sca1+c-Kit= （LSK） HSPCs。该协议从小鼠骨髓中提供更高量的LSK HSPCs，提高孵育期间HSPCs的生存能力，促进非粘附性HSPCs的细胞外通量分析，并为针对氧化磷酸化和糖解途径的药物提供优化的注射方案（浓度和时间）。该方法能够预测血液发育和疾病期间的代谢状态和HSPCs的健康。
Introduction
由于大多数成熟血细胞的寿命较短，血液的平衡依赖于长寿但罕见的造血干细胞（HSPCs）1的自我更新和分化。HSPC是静止的，但它们在刺激下迅速增殖和分化，以维持血液系统的需求。由于每个HSPC细胞状态都需要独特的生物能量需求，代谢变化是HSPC命运决定的关键驱动因素。因此，代谢可塑性的损失，通过改变HSPCs的静止、自我更新和分化之间的平衡，往往导致骨髓或淋巴增殖性疾病。总之，对HSPC发育的代谢调节的理解对于发现血液恶性肿瘤2、3、4、5的深层机制至关重要。
线粒体呼吸和糖解产生ATP，以推动细胞内反应，并产生大分子合成所需的构建基块。由于HSPCs与分化细胞6相比线粒体质量较低，并且它们在低氧骨髓利基中维持静止，因此HSPCs主要依靠糖解。HSPCs的激活增强了其线粒体代谢，导致静止的丧失和随后进入细胞周期。HSPC的这种代谢可塑性允许在整个成人寿命6，7，8，9，10，11，12期间维持HSPC池。因此，分析HSPC活化和健康状况，必须研究其代谢活动，如耗氧率（OCR;氧化磷酸化指数）和细胞外酸化率（ECAR;糖解指数）。OCR 和 ECAR 都可以使用细胞外通量分析仪进行实时测量。然而，目前的方法需要大量的细胞，并针对附着细胞13进行了优化。由于HSPC不能从小鼠14中大量分离，需要分拣才能获得纯种群，是非粘附细胞15，并且不能在一夜之间培养，而不避免分化16，因此很难测量HSPC的OCR和ECAR。在这里，我们提供一组清晰的分步说明，以伴随视频教程如何测量代谢呼吸和糖解的几千个小鼠骨髓-血统negSca1+c-Kit+ （LSK） HSPCs。
Protocol
该协议得到了全国儿童医院动物护理和使用委员会（IACUC）的批准。 注：协议按时间顺序描述，时间顺序跨越两天。使用如下协议中所述的新鲜试剂。 1. 在测定前一天制备试剂 水化传感器盒。 在非CO2 37°C培养箱中孵育5mL的校准（材料表）。 打开助焊剂检测套件（材料表），</st…
Representative Results
我们的提取方法允许我们每只小鼠收获高达 80，000 个 LSK HSPC。LSK细胞的存活性和数量通过我们的方法得到了提高，因为我们：（1）将上肢和下肢、髋骨、胸骨、肋骨、肋骨和脊柱的骨髓组合在一起，（2）避免使用红细胞裂化缓冲液，这将增加细胞死亡和结块，（3）使用单核细胞的密度梯度介质分离，避免使用预冷却缓冲液，这会导致团块中兴趣细胞的丧失。 虽然细胞外通?…
Discussion
在这里，我们演示了最大数量的纯和活的鼠LSK HSPCs种群的分离，以及使用细胞外通量分析仪测量其糖解和线粒体呼吸。具体来说，该协议克服了使用LSK HSPCs的下列技术问题：i） LSK HSPCs在小鼠骨髓14中的低频率，ii） LSK HSPCs26的低基础代谢活性，iii） LSK HSPCs27的脆弱;iv） LSK HSPCs 不粘附于培养容器15。此外，我们还优化了药物浓…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作部分得到了国家卫生研究院（HL131645、CA016058）、圣巴尔德里克基金会和佩洛多尼亚基金会的资助支持。
Materials
| 0.01% (w/v) poly-L-lysine solution | Sigma | P8920 | Used for LSK attachment |
| 40 µm cell strainer | Fisher Scientific | 22-363-547 | Used for cell filtration after bone crushing |
| Anti-Biotin MicroBeads | Miltenyi | 130-090-485 | Used for Lin- separation |
| Biotin Rat Anti-Mouse CD45R/B220 Clone RA3-6B2 | BD Biosciences | 553086 | Used for Lin- separation |
| Biotin Rat Anti-Mouse CD5 Clone 53-7.3 | BD Biosciences | 553019 | Used for Lin- separation |
| Biotin Rat Anti-Mouse CD8a Clone 53-6.7 | BD Biosciences | 553029 | Used for Lin- separation |
| Biotin Rat Anti-Mouse Ly-6G and Ly-6C Clone RB6-8C5 | BD Biosciences | 553125 | Used for Lin- separation |
| Biotin Rat Anti-Mouse TER-119/Erythroid Cells Clone TER-119 | BD Biosciences | 553672 | Used for Lin- separation |
| CD117 (c-Kit) Monoclonal Antibody (2B8), APC | eBioscience | 17-1171-83 | Used for LSK sorting |
| Falcon 15 ml Conical Centrifuge Tubes | Falcon-Fischer Scientific | 14-959-53A | Used in cell isolation |
| Falcon 50 ml Conical Centrifuge Tubes | Falcon-Fischer Scientific | 14-432-22 | Used in cell isolation |
| Falcon Round-Bottom Polypropylene Tubes | Falcon-Fischer Scientific | 14-959-11A | Used for LSK sorting |
| Fetal Bovine Serum | Neuromics | FBS001-HI | Used in FACS buffer |
| Histopaque-1083 | Sigma | 10831 | Used for ficoll gradient separation |
| L-glutamine 100x | Fisher Scientific | 25-030-081 | Used for the assay media |
| LS Column | Miltenyi | 130-042-401 | Used for Lin- separation |
| Ly-6A/E (Sca-1) Monoclonal Antibody (D7), PE-Cyanine7 | eBioscience | 25-5981-82 | Used for LSK sorting |
| Murine Stem Cell Factor (SCF) | PeproTech | 250-03-100UG | Used for the assay media |
| Murine Thrombopoietin (TPO) | PeproTech | 315-14-100UG | Used for the assay media |
| PBS 1% | Fisher Scientific | SH3002802 | Used for FACS buffer |
| Penicillin-Streptomycin (10,000 U/mL) | Fisher Scientific | 15140122 | Used for the assay media |
| Propidium Iodide | Fisher Scientific | P1304MP | Used for LSK sorting |
| Seahorse XFp Cell Culture Miniplate | Agilent Technologies | 103025-100 | Used for LSK seeding |
| Sodium Pyruvate (100 mM) | ThermoFisher | 11360070 | Used for the assay media |
| Streptavidin eFluor 450 Conjugate | eBioscience | 48-4317-82 | Used for LSK sorting |
| XF Calibrant | Agilent Technologies | 100840-000 | Used for cartridge equilibration |
| XF media | Agilent Technologies | 103575-100 | Used for the assay media |
| XFp Glycolysis Stress Test Kit | Agilent Technologies | 103017100 | Drugs for glycolysis stress test |
| XFp Mitochondrial Stress Test Kit | Agilent Technologies | 103010100 | Drugs for mitochondrial stress test |
| XFp Sensor Cartridge | Agilent Technologies | 103022-100 | Used for glycolysis and mitochondrial stress test |
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Citer Cet Article
Scapin, G., Goulard, M. C., Dharampuriya, P. R., Cillis, J. L., Shah, D. I. Analysis of Hematopoietic Stem Progenitor Cell Metabolism. J. Vis. Exp. (153), e60234, doi:10.3791/60234 (2019).