通过抑制Efflux泵,提高造血干细胞和造血细胞中线粒体膜电位流细胞测定的准确性
Summary
异种生物排泄泵在造血干细胞和祖细胞(HSPCs)中高度活跃,并导致TMRM(线粒体膜潜在荧光染料)的挤出。在这里,我们提出了一个协议,以准确测量线粒体膜电位在HSPC与TMRM的存在,Verapamil,一个排泄泵抑制剂。
Abstract
由于细胞代谢是造血干细胞(HSC)自我更新的关键调节剂,因此HSC研究人员对线粒体在造血平衡中所起的各种作用进行了广泛的研究。线粒体活性水平反映在其膜电位 (μm) 中,可以通过细胞渗透阳离子染料(如 TMRM(四甲基二甲胺、甲基酯)进行测量。然而,排流泵从细胞中挤出这些染料的能力会限制其效用。在评估 HSC 时,由此产生的测量偏差尤为重要,因为异种生物转运剂在 HSC 中表现出比分化细胞更高的表达和活性水平。在这里,我们描述了一个利用Verapamil(一种排泄泵抑制剂)来精确测量多个骨髓群的μm的协议。由此产生的泵活性抑制表明,增加造血干细胞和祖细胞(HSPCs)的TMRM强度,同时在成熟馏分中保持相对不变。这突出表明,在使用_m依赖染料时,需要密切注意染料-排泄物活性,并且根据编写和可视化,此协议可用于准确比较骨髓内的不同种群或同一种群跨越不同的实验模型。
Introduction
造血干细胞(HSCs)是自我更新的,多能的,能够产生血液1,2的所有细胞。细胞代谢是HSC维持的关键调节器,与转录因子、内在信号和微环境3、4、5一起。因此,正确控制线粒体功能和质量对HSC维护至关重要。
线粒体膜电位(μm)是线粒体评估的关键参数,因为它直接反映了线粒体的功能,它来自电子传输链中的质子泵送活性平衡和通过F的质子流1/FO ATP 合成酶。这两者都是ADP至ATP8、9氧依赖磷酸化所必需的(取决于基因表达和基质可用性)。利用线粒体室的电子性,开发了各种电位染料来测量μm。其中之一是四甲胺甲基酯高氯酸盐(TMRM),已被广泛用于测量+m通过流动细胞测定在各种细胞10,包括造血干细胞和祖细胞11。
线粒体染料在HSC中必须谨慎使用,但是,因为这些细胞的异种生物排泄泵的高活性可能导致染料挤出12。事实上,线粒体染料(如罗达明123)的挤出已经使研究人员能够分离HSCs13,或者通过利用染料Hoechst Blue和Hoechst Red14的差分挤出来识别HSC”侧群”。15.还表明,Fumitremorgin C,ATP绑定盒式子家族G成员2(ABCG2)传输器的特定阻滞剂,不影响HSPCs16中MitoTracker的染色模式。这些结果公布后,在没有异种生物体排泄泵抑制剂的情况下,使用线粒体染料进行了多项研究,导致人们普遍的印象是,HSC只有少量线粒体,低μm16,17,18.
然而,最近,它证明,Verapamil,一个宽谱抑制剂的排泄泵,显著改变了线粒体染料MitoTracker绿色19的染色模式。这种差异可能是由于Fumitremorgin C对Abcg2具有高度选择性,而HSCs也表示其他运输工具,如Abcb1a(它只对富米震金C敏感度弱)19。我们还报告说,其他线粒体染料,如TMRM、诺尼阿克里丁橙和米托斯特橙(MTO)表现出与Mitotracker Green相同的图案。更重要的是,我们观察到,除了线粒体质量11之外,HSPC的流细胞学模式也反映了它们的μm。
TMRM染料的摄入量严格取决于线粒体的负电荷,但染料的积累在摄入和排空泵20间隙之间保持恒定平衡。HSC和成熟细胞群之间异种生物排泄泵表达的差异会影响这种平衡,并可能导致有偏差的结果。在分析电位染料时,应考虑使用专用抑制剂,如Verapamil。在这里,我们描述了一个改进的协议,用于通过基于TMRM的流式细胞测定进行精确μm测量,该该协议通过使用专用抑制剂纠正异种生物运输者活性。
Protocol
Representative Results
Discussion
线粒体膜电位测量是线粒体分析和评估的基石,线粒体对细胞的代谢状态至关重要。在这里,我们描述了一个通过TMRM染色分析μm的协议。TMRM是一种细胞渗透荧光染料,由于μm在活性线粒体中积累,其各自的水平在细胞外、细胞质和线粒体室之间保持平衡。该协议适用于各种染料,包括四甲胺、乙酯(TMRE)和JC-1。适当的染色条件对于获得准确结果至关重要。这些措施包括:防止光线照射、?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢伊托实验室的所有成员,特别是伊都和H Sato,以及爱因斯坦干细胞研究所的评论和爱因斯坦流细胞测定和分析成像核心设施(由美国国家癌症研究所资助P30 CA013330)为帮助进行实验。K.I. 由美国国家卫生研究院(R01DK98263、R01DK115577和R01DK100689)和纽约州卫生局资助,担任爱因斯坦单细胞基因组学/基因组学(C029154)的核心主任。伊托是白血病和淋巴瘤学会的研究学者。
Materials
| ACK lysing buffer | Life Technologies | A1049201 | |
| B220-biotin | BD Bioscience | 553086 | |
| CD3e-biotin | Life Technologies | 13-0031-85 | |
| CD4-biotin | Fischer Scientific | BDB553782 | |
| CD8-biotin | Life Technologies | 13-0081-85 | |
| CD11b-biotin | BD Bioscience | 553309 | |
| CD19-biotin | BD Bioscience | 553784 | |
| CD34-FITC | eBioscience | 11-0341-85 | |
| CD48-APC | eBioscience | 17-0481-82 | |
| CD135-biotin | eBioscience | 13-1351-82 | |
| CD150-PerCP/Cy5.5 | Biolegend | 115922 | |
| c-kit-APC/Cy7 | Biolegend | 105826 | |
| Cyclosporin H | Millipore Sigma | SML1575-1MG | |
| DAPI solution (1mg/mL) | Life Technologies | 62248 | |
| Fetal Bovine Serum (FBS) | Denville | FB5001-H | |
| FCCP | Millipore Sigma | C2920-10MG | |
| Gr1-biotin | Biolegend | 108404 | |
| IgM-biotin | Life Technologies | 13-5790-85 | |
| Il7Rα-biotin | eBioscience | 13-1271-85 | |
| Nk1.1-biotin | Fischer Scientific | BDB553163 | |
| Phosphate buffered saline (PBS) | Life Technologies | 10010023 | |
| Sca-1-PE/Cy7 | eBioscience | 25-5981-81 | |
| SCF murine | PEPROTECH | 250-03-10UG | |
| StemSpan SFEM medium | STEMCELL technologies | 9605 | |
| Streptavidin-Pacific Blue | eBioscience | 48-4317-82 | |
| Ter119-biotin | Fischer Scientific | BDB553672 | |
| TMRM | Millipore Sigma | T5428-25MG | |
| TPO | PEPROTECH | 315-14-10UG | |
| Verapamil hydrochloride | Millipore Sigma | V4629-1G |
References
- Weissman, I. L., Anderson, D. J., Gage, F. Stem and progenitor cells: origins, phenotypes, lineage commitments, and transdifferentiations. Annual Review of Cell and Developmental Biology. 17, 387-403 (2001).
- Morrison, S. J., Shah, N. M., Anderson, D. J. Regulatory mechanisms in stem cell biology. Cell. 88 (3), 287-298 (1997).
- Wilson, A., et al. Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair. Cell. 135 (6), 1118-1129 (2008).
- Morrison, S. J., Scadden, D. T. The bone marrow niche for haematopoietic stem cells. Nature. 505 (7483), 327-334 (2014).
- Frenette, P. S., Pinho, S., Lucas, D., Scheiermann, C. Mesenchymal stem cell: keystone of the hematopoietic stem cell niche and a stepping-stone for regenerative medicine. Annual Review of Immunology. 31, 285-316 (2013).
- Ito, K., Bonora, M., Ito, K. Metabolism as master of hematopoietic stem cell fate. International Journal of Hematology. 109 (1), 18-27 (2019).
- Ito, K., et al. Self-renewal of a purified Tie2+ hematopoietic stem cell population relies on mitochondrial clearance. Science. 354 (6316), 1156-1160 (2016).
- Bonora, M., et al. ATP synthesis and storage. Purinergic Signal. 8 (3), 343-357 (2012).
- Walker, J. E. The regulation of catalysis in ATP synthase. Current Opinion in Structural Biology. 4 (6), 912-918 (1994).
- Scaduto, R. C., Grotyohann, L. W. Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives. Biophysical Journal. 76, 469-477 (1999).
- Bonora, M., Ito, K., Morganti, C., Pinton, P., Ito, K. Membrane-potential compensation reveals mitochondrial volume expansion during HSC commitment. Experimental Hematology. 68, 30-37 (2018).
- Goodell, M. A., et al. Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species. Nature Medicine. 3 (12), 1337-1345 (1997).
- Spangrude, G. J., Johnson, G. R. Resting and activated subsets of mouse multipotent hematopoietic stem cells. Proceedings of the National Academy of Sciences of the United States of America. 87 (19), 7433-7437 (1990).
- Scharenberg, C. W., Harkey, M. A., Torok-Storb, B. The ABCG2 transporter is an efficient Hoechst 33342 efflux pump and is preferentially expressed by immature human hematopoietic progenitors. Blood. 99 (2), 507-512 (2002).
- Zhou, S., et al. The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nature Medicine. 7 (9), 1028-1034 (2001).
- Simsek, T., et al. The distinct metabolic profile of hematopoietic stem cells reflects their location in a hypoxic niche. Cell Stem Cell. 7 (3), 380-390 (2010).
- Vannini, N., et al. Specification of haematopoietic stem cell fate via modulation of mitochondrial activity. Nature Communications. 7, 13125 (2016).
- Xiao, N., et al. Hematopoietic stem cells lacking Ott1 display aspects associated with aging and are unable to maintain quiescence during proliferative stress. Blood. 119 (21), 4898-4907 (2012).
- de Almeida, M. J., Luchsinger, L. L., Corrigan, D. J., Williams, L. J., Snoeck, H. W. Dye-Independent Methods Reveal Elevated Mitochondrial Mass in Hematopoietic Stem Cells. Cell Stem Cell. 21 (6), 725-729 (2017).
- Hall, A. M., Rhodes, G. J., Sandoval, R. M., Corridon, P. R., Molitoris, B. A. In vivo multiphoton imaging of mitochondrial structure and function during acute kidney injury. Kidney International. 83 (1), 72-83 (2013).
- Oguro, H., Ding, L., Morrison, S. J. SLAM family markers resolve functionally distinct subpopulations of hematopoietic stem cells and multipotent progenitors. Cell Stem Cell. 13 (1), 102-116 (2013).
- Nicolli, A., Basso, E., Petronilli, V., Wenger, R. M., Bernardi, P. Interactions of cyclophilin with the mitochondrial inner membrane and regulation of the permeability transition pore, and cyclosporin A-sensitive channel. The Journal of Biological Chemistry. 271 (4), 2185-2192 (1996).
- Vannini, N., et al. The NAD-Booster Nicotinamide Riboside Potently Stimulates Hematopoiesis through Increased Mitochondrial Clearance. Cell Stem Cell. 24 (3), 405-418 (2019).
