Name: analysis of hematopoietic stem progenitor cell metabolism
Uploaded: 2019-11-09T12:00:00
Description: Watch this Scientific Journal Video about Analysis of Hematopoietic Stem Progenitor Cell Metabolism at JoVE.com

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。

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
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水化传感器盒。
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<li>在非CO2 37°C培养箱中孵育5mL的校准（材料表）。</li>
		<li>打开助焊剂检测套件（材料表），</st...

<ol>
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C., Yamazaki, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hematopoietic+stem+cell+diet.">The hematopoietic stem cell diet.</a> International Journal of Hematology. 107, 634-641 (2018).</li><li>Kohli, L., Passegue, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surviving+change:+the+metabolic+journey+of+hematopoietic+stem+cells.">Surviving change: the metabolic journey of hematopoietic stem cells.</a> Trends in Cell Biology. 24, 479-487 (2014).</li><li>Abdel-Wahab, O., Levine, R. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+regulation+of+hematopoietic+stem+cells+in+the+hypoxic+niche.">Metabolic regulation of hematopoietic stem cells in the hypoxic niche.</a> Cell Stem Cell. 9, 298-310 (2011).</li><li>Zhang, C. C., Sadek, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+and+metabolic+properties+of+hematopoietic+stem+cells.">Hypoxia and metabolic properties of hematopoietic stem cells.</a> Antioxidants &amp; Redox Signaling. 20, 1891-1901 (2014).</li><li>Snoeck, H. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Niche-less+maintenance+of+HSCs+by+2i.">Niche-less maintenance of HSCs by 2i.</a> Cell Research. 23, 458-459 (2013).</li><li>Anderson, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+stem+cells+develop+in+the+absence+of+endothelial+cadherin+5+expression.">Hematopoietic stem cells develop in the absence of endothelial cadherin 5 expression.</a> Blood. 126, 2811-2820 (2015).</li><li>Lo Celso, C., Scadden, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+transplantation+of+hematopoietic+stem+cells+(HSCs).">Isolation and transplantation of hematopoietic stem cells (HSCs).</a> Journal of Visualized Experiment. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+monocarboxylate+transporter+family--role+and+regulation.">The monocarboxylate transporter family--role and regulation.</a> IUBMB Life. 64, 109-119 (2012).</li><li>Kim, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+in+Cancer+Energy+Metabolism:+Culprits+or+Bystanders.">Mitochondria in Cancer Energy Metabolism: Culprits or Bystanders.</a> Toxicological Research. 31, 323-330 (2015).</li><li>Fosslien, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+medicine--molecular+pathology+of+defective+oxidative+phosphorylation.">Mitochondrial medicine--molecular pathology of defective oxidative phosphorylation.</a> Annals of Clinical &amp; Laboratory Science. 31 (1), 25-67 (2001).</li><li>Wick, A. 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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...

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。

<table>
	<tbody>
		<tr>
			<td>0.01% (w/v) poly-L-lysine solution</td>
			<td>Sigma</td>
			<td>P8920</td>
			<td>Used for LSK attachment</td>
		</tr>
		<tr>
			<td>40 &#181;m cell strainer</td>
			<td>Fisher Scientific</td>
			<td>22-363-547</td>
			<td>Used for cell filtration after bone crushing</td>
		</tr>
		<tr>
			<td>Anti-Biotin MicroBeads</td>
			<td>Miltenyi</td>
			<td>130-090-485</td>
			<td>Used for Lin- separation</td>
		</tr>
		<tr>
			<td>Biotin Rat Anti-Mouse CD45R/B220 Clone RA3-6B2</td>
			<td>BD Biosciences</td>
			<td>553086</td>
			<td>Used for Lin- separation</td>
		</tr>
		<tr>
			<td>Biotin Rat Anti-Mouse CD5 Clone 53-7.3</td>
			<td>BD Biosciences</td>
			<td>553019</td>
			<td>Used for Lin- separation</td>
		</tr>
		<tr>
			<td>Biotin Rat Anti-Mouse CD8a Clone 53-6.7</td>
			<td>BD Biosciences</td>
			<td>553029</td>
			<td>Used for Lin- separation</td>
		</tr>
		<tr>
			<td>Biotin Rat Anti-Mouse Ly-6G and Ly-6C Clone RB6-8C5</td>
			<td>BD Biosciences</td>
			<td>553125</td>
			<td>Used for Lin- separation</td>
		</tr>
		<tr>
			<td>Biotin Rat Anti-Mouse TER-119/Erythroid Cells Clone TER-119</td>
			<td>BD Biosciences</td>
			<td>553672</td>
			<td>Used for Lin- separation</td>
		</tr>
		<tr>
			<td>CD117 (c-Kit) Monoclonal Antibody (2B8), APC</td>
			<td>eBioscience</td>
			<td>17-1171-83</td>
			<td>Used for LSK sorting</td>
		</tr>
		<tr>
			<td>Falcon 15 ml Conical Centrifuge Tubes</td>
			<td>Falcon-Fischer Scientific</td>
			<td>14-959-53A</td>
			<td>Used in cell isolation</td>
		</tr>
		<tr>
			<td>Falcon 50 ml Conical Centrifuge Tubes</td>
			<td>Falcon-Fischer Scientific</td>
			<td>14-432-22</td>
			<td>Used in cell isolation</td>
		</tr>
		<tr>
			<td>Falcon Round-Bottom Polypropylene Tubes</td>
			<td>Falcon-Fischer Scientific</td>
			<td>14-959-11A</td>
			<td>Used for LSK sorting</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Neuromics</td>
			<td>FBS001-HI</td>
			<td>Used in FACS buffer</td>
		</tr>
		<tr>
			<td>Histopaque-1083</td>
			<td>Sigma</td>
			<td>10831</td>
			<td>Used for ficoll gradient separation</td>
		</tr>
		<tr>
			<td>L-glutamine 100x</td>
			<td>Fisher Scientific</td>
			<td>25-030-081</td>
			<td>Used for the assay media</td>
		</tr>
		<tr>
			<td>LS Column</td>
			<td>Miltenyi</td>
			<td>130-042-401</td>
			<td>Used for Lin- separation</td>
		</tr>
		<tr>
			<td>Ly-6A/E (Sca-1) Monoclonal Antibody (D7), PE-Cyanine7</td>
			<td>eBioscience</td>
			<td>25-5981-82</td>
			<td>Used for LSK sorting</td>
		</tr>
		<tr>
			<td>Murine Stem Cell Factor (SCF)</td>
			<td>PeproTech</td>
			<td>250-03-100UG</td>
			<td>Used for the assay media</td>
		</tr>
		<tr>
			<td>Murine Thrombopoietin (TPO)</td>
			<td>PeproTech</td>
			<td>315-14-100UG</td>
			<td>Used for the assay media</td>
		</tr>
		<tr>
			<td>PBS 1%</td>
			<td>Fisher Scientific</td>
			<td>SH3002802</td>
			<td>Used for FACS buffer</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Fisher Scientific</td>
			<td>15140122</td>
			<td>Used for the assay media</td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Fisher Scientific</td>
			<td>P1304MP</td>
			<td>Used for LSK sorting</td>
		</tr>
		<tr>
			<td>Seahorse XFp Cell Culture Miniplate</td>
			<td>Agilent Technologies</td>
			<td>103025-100</td>
			<td>Used for LSK seeding</td>
		</tr>
		<tr>
			<td>Sodium Pyruvate (100 mM)</td>
			<td>ThermoFisher</td>
			<td>11360070</td>
			<td>Used for the assay media</td>
		</tr>
		<tr>
			<td>Streptavidin eFluor 450 Conjugate</td>
			<td>eBioscience</td>
			<td>48-4317-82</td>
			<td>Used for LSK sorting</td>
		</tr>
		<tr>
			<td>XF Calibrant</td>
			<td>Agilent Technologies</td>
			<td>100840-000</td>
			<td>Used for cartridge equilibration</td>
		</tr>
		<tr>
			<td>XF media</td>
			<td>Agilent Technologies</td>
			<td>103575-100</td>
			<td>Used for the assay media</td>
		</tr>
		<tr>
			<td>XFp Glycolysis Stress Test Kit</td>
			<td>Agilent Technologies</td>
			<td>103017100</td>
			<td>Drugs for glycolysis stress test</td>
		</tr>
		<tr>
			<td>XFp Mitochondrial Stress Test Kit</td>
			<td>Agilent Technologies</td>
			<td>103010100</td>
			<td>Drugs for mitochondrial stress test</td>
		</tr>
		<tr>
			<td>XFp Sensor Cartridge</td>
			<td>Agilent Technologies</td>
			<td>103022-100</td>
			<td>Used for glycolysis and mitochondrial stress test</td>
		</tr>
	</tbody>
</table>

analysis of hematopoietic stem progenitor cell metabolism

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。

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?...

Watch this Scientific Journal Video about Analysis of Hematopoietic Stem Progenitor Cell Metabolism at JoVE.com

Analysis of Hematopoietic Stem Progenitor Cell Metabolism

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。

造血干细胞代谢分析

Hematopoietic stem progenitor cells (HSPCs) have distinct metabolic plasticity, which allows them to transition from their quiescent state to a ...

造血干细胞和祖细胞的代谢变化使它们能够从静止状态过渡到分化状态，以维持血液形成的需求。造血干细胞和祖细胞代谢可塑性的变化导致血液紊乱。我们的协议将有助于测量血液发育和疾病期间造血干细胞和祖细胞的代谢调节和健康。分析线粒体呼吸和造血干细胞和祖细胞的糖解是困难的，因为它们是脆弱和罕见的种群。我们优化的协议允许从骨髓中分离出更高数量的造血干细胞和祖细胞，提高孵育过程中的可行性，促进非粘附性HSPCs的细胞外通量分析，并为药物靶向氧化磷酸化和糖解通路提供优化的注射参数。要开始此过程，请用无菌水填充公用板和井周围的水室的井。将传感器盒淹没到公用板中，确保传感器完全被水覆盖。然后将墨盒浸入公用板中，在37摄氏度的非CO2培养箱中孵化过夜。在 II 类生物安全柜中，在检测板的每个井中加入 40 微升市售 0.1%PLL 溶液。用套件中提供的盖子盖住检测板，并在罩下室温下孵化封闭板一小时。在此之后，使用无菌真空吸气器去除多余的溶液，并在发动机罩下空气干燥板。要收获单核骨髓细胞，在15毫升锥形管中加入5毫升密度梯度介质。慢慢加入五毫升的骨髓细胞悬浮液，确保细胞保持为密度梯度介质上方的层。在 500 倍 g 下离心，在室温下 30 分钟，确保离心机的加速度最低。将单核细胞的中间界面收获成一个新鲜的15毫升管。然后用含有2%FBS的5毫升1X PBS洗涤细胞。在500倍g和4摄氏度下离心5分钟。取出上一除以，在含有2%FBS的1X PBS的300微升中重新暂停细胞。接下来，在FACS管中为无污染或单色控制单元的10微升的等分。要从单核骨髓细胞中收获LSK HSPCs，在300微升单核骨髓细胞中加入15微升生物素抗体鸡尾酒。要从单核骨髓细胞中收获LSK HSPCs，在300微升单核骨髓细胞中加入15微升生物素抗体鸡尾酒。将管子放在冰箱摇床上的冰桶中，在4摄氏度下孵育，搅拌30分钟。然后向细胞中加入10毫升含有2%FBS的预冷却1XPBS。在500倍g和4摄氏度下离心5分钟。丢弃上一提液，将细胞颗粒重新在含有 2%FBS 的 400 微升 1X PBS 中。使用前，请短暂地旋涡抗生物素微珠。在每个样品中加入80微升微升微珠，并混合好。在4摄氏度下再孵育20分钟，搅拌。在此之后，向细胞中加入含有2%FBS的10毫升预冷却PBS。在500倍g和4摄氏度下将管子离心5分钟。丢弃上一提液，将细胞颗粒重新在含有2%FBS的PBS中。在设置磁性分离装置时，将电池悬浮液存放在四摄氏度。将柱放在磁场中，使磁辅助细胞分拣分离器在四摄氏度下。在4摄氏度的重力流下，用含有2%FBS的三毫升1X PBS冲洗，为磁分离准备柱。在四摄氏度下将细胞悬浮添加到预湿柱中。让所有细胞在摄氏四度时通过柱子，并在15毫升的圆锥管中收集污水。接下来，用三毫升 1X PBS 和 2%FBS 在 4 摄氏度下清洗柱。重复此洗涤三次。收集流经，并保持在四摄氏度。将含有流量的圆锥管在500倍g和4摄氏度下离心5分钟。丢弃上一液，用2%FBS将细胞用1X PBS的0.5毫升重新悬浮，然后将悬浮液转移到FACS管中。然后向每1000万个细胞添加24微升的LSK抗体鸡尾酒。用铝箔盖住管子，在黑暗中孵育四摄氏度，搅拌一小时。在此之后，在 FACS 管中加入三毫升 1X PBS 和 2%FBS。在500倍g和4摄氏度下离心5分钟。丢弃上流剂，将抗体标记的细胞重新在含有2%FBS的1X PBS的毫升中。在分拣之前，在细胞悬浮液中加入每毫升一毫克的微升，并使用40微米滤株过滤FACS管的内容。首先，在500倍g和室温下将收集的LSK细胞离心5分钟。丢弃上能，将完整介质中的细胞重新暂停至每40微升至少7万个细胞的最终浓度。种子40微升这个细胞悬浮到PLL涂层八孔板的井，确保留下所有的角井空。在 450 倍 g 和室温下将板离心一分钟。使用倒置显微镜确保细胞附着在井底。在此之后，将135微升的完整介质添加到每井的细胞中，使每井的最终体积达到175微升。将 175 微升完整介质添加到板的两角作为空白。在37摄氏度的非CO2培养箱中孵育细胞两小时。然后为分析器设置一个程序，将药物添加到板的每个井中，如文本协议表二和表三中所述。对于线粒体应激测试，从培养箱中检索公用板中的传感器盒，以便每井的最终浓度为两个微摩尔寡白霉素、1.5 微摩尔 FCCP 和 0.5 微摩尔或罗泰诺和抗霉素 A。从传感器墨盒上取下盖子并将其放在仪器托盘上。开始校准过程，需要 20 分钟。校准完成后，拆下实用板并将其替换为包含 LSK 细胞的检测板。按继续启动以前设置的程序。程序完成后，检索数据并对其进行分析。在这项研究中，被分离出最大数量的可行乳激素LSK HSPCs，用细胞外通量分析仪测量其糖解和线粒体代谢。虽然细胞外通量分析传统上用于粘附细胞，但这种方法使LSK HSPCs粘附在PLL涂层孔中，从而能够测量细胞外通量，从而测量LSK HSPCs的代谢健康。为了通过耗氧率和糖解通过基底和应激条件下的细胞外酸化率测量线粒体呼吸，按顺序注射干扰糖解和线粒体呼吸的药物。正如预期的那样，在葡萄糖和丙酮酸盐阳性介质中培养的LSK HSPCs的细胞外酸化率的基础水平高于在负介质中培养的LSK HSPCs。葡萄糖注射并没有改变正介质中培养的细胞的细胞外酸化率，但它促进了负介质中细胞的糖解。然而，这些细胞的基础水平仍然较低，在正组。使用寡霉素注射在阳性组的最高水平上激活了糖解，但不影响负组。在2-DG时，葡萄糖模拟注射将细胞外酸化率恢复到非糖解水平。对于线粒体应激测试，注射寡青霉素超极化线粒体膜，防止更多的质子泵送通过 ETC 复合物，从而降低线粒体呼吸率。当细胞试图恢复线粒体膜电位时，FCCP 的注射将耗氧率推至最大。与另外两个电子传输链抑制剂的注射导致线粒体呼吸完全停止，因此耗氧率恢复到其最低水平。请避免使用红细胞解液缓冲液进行单核细胞分离。我们建议使用菲科尔梯度分离，以防止团形成，减少LSK的损失。利用我们优化的方法，我们可以测量线粒体呼吸和糖解罕见和脆弱的造血干细胞和祖细胞，并研究代谢线索调节正常和恶性造血。

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