从单个骨骼肌中分离静止干细胞群
Summary
该协议描述了从小鼠的单个骨骼肌中分离肌肉干细胞和纤维脂肪祖细胞。该方案涉及单肌肉解剖、通过荧光活化细胞分选分离干细胞、通过免疫荧光染色进行纯度评估以及通过 5-乙炔基-2′-脱氧尿苷掺入测定法定量测量 S 期进入。
Abstract
骨骼肌含有不同的成体干细胞群，有助于组织的稳态和修复。骨骼肌干细胞（MuSCs）具有制造新肌肉的能力，而纤维脂肪祖细胞（FAPs）有助于基质支持组织并具有制造成纤维细胞和脂肪细胞的能力。MuSC和FAP都处于延长的可逆细胞周期退出状态，称为静止。静止状态是其功能的关键。静止干细胞通常从单个样品中汇集在一起的多个肌肉组织中纯化。然而，最近的研究表明，从不同肌肉分离的MuSCs的分子谱和静止深度存在明显差异。本协议描述了从单个骨骼肌中分离和研究MuSC和FAP，并提出了进行干细胞活化分子分析的策略。它详细介绍了如何分离和消化不同发育起源、厚度和功能的肌肉，例如横膈膜、三头肌、腹股细肌、前胫骨 （TA）、腓肠肌 （GA）、比目鱼肌、指长伸肌 （EDL） 和咬肌。MuSC 和 FAP 通过荧光激活细胞分选 （FACS） 纯化，并通过免疫荧光染色和 5-乙炔基-2′-脱氧尿苷 （EdU） 掺入测定进行分析。
Introduction
由于肌肉干细胞（MuSCs）的存在，骨骼肌具有很高的再生能力。MuSC位于肌纤维上，基底层下方，并且处于延长的可逆细胞周期出口1，2，3，4的静止状态。损伤后，MuSCs激活并进入细胞周期，产生扩增的祖细胞，这些祖细胞可以分化和融合形成新的肌纤维2，5。先前的工作表明，MuSCs对于肌肉再生是绝对必要的6，7，8。此外，单个MuSC可以移植并产生新的干细胞和新的肌纤维9。骨骼肌还含有一组间充质基质细胞，称为纤维脂肪祖细胞（FAP），它们在肌肉再生期间支持MuSC功能中起着至关重要的作用6，10，11，12。
由于它们具有协调肌肉再生的潜力，人们对了解MuSC和FAPs如何工作产生了极大的兴趣。静态MuSC由转录因子Pax7和Sprouty1以及细胞表面蛋白降钙素受体的表达标记，而静止的FAP由细胞表面蛋白血小板衍生生长因子受体α（PDGFRa）10，12，13，14，15标记.先前的研究表明，可以使用细胞表面标志物和荧光激活细胞分选（FACS）从骨骼肌中纯化MuSCs和FAPs9，15，16，17，18，19，20，21。虽然这些协议极大地提高了研究MuSC和FAP的能力，但一个缺点是大多数这些协议需要从不同肌肉组织的池中分离MuSC。我们和其他人最近的工作揭示了从不同组织分离的MuSC之间的细胞表型和基因表达水平的差异22，23。来自横膈膜、三头肌和腹股细肌的 MuSC 比来自下后肢肌肉的 MuSC 活化更快22，而来自眼外肌的 MuSC 比来自横膈膜和下后肢肌肉的 MuSC 分化更快23。
该协议描述了从单个骨骼肌中分离MuSC和FAP（图1）。这包括横膈肌、三头肌、腹股细肌、胫骨前肌 （TA）、比目鱼肌、长指伸肌 （EDL）、腓肠肌 （GA） 和咬肌的解剖。随后使用胶原酶II（一种特异性靶向胶原蛋白中Pro-X-Gly-Pro氨基序列的蛋白酶，能够降解结缔组织和组织解离24）和分散酶（一种切割纤连蛋白和胶原蛋白IV的蛋白酶，能够进一步解离细胞25）通过酶消化解离解剖肌肉).MuSC和FAP通过FACS从单细胞悬浮液中分离出来。作为细胞分析下游测定的示例，通过测定5-乙炔基-2′-脱氧尿苷（EdU）掺入来确定干细胞活化，而细胞纯度通过细胞类型特异性标记物Pax7和PDGFRa的免疫荧光染色来确定。
Protocol
本协议是根据奥胡斯大学的动物护理指南和当地道德法规执行的。 注意：确保遵守当地动物实验和死后啮齿动物样本处理伦理委员会的规定。小鼠是过敏原的潜在来源;如果可用，请打开排气通风并将其放置在工作区上方，以避免过度接触过敏原。或者，如果定期进行实验，请戴上口罩。该协议涉及使用锐器，建议研究人员熟悉在割伤情况下进行急救的程序和后勤工作。 <p…
Representative Results
按照个体骨骼肌分离方案（图2），从三只已从当地育种计划中停止的瑞士雄性远交小鼠中分离出腹股细肌，TA，EDL，GA，比目鱼肌，三头肌，咬肌和横膈肌（图2）。在组织解离和抗体染色后，通过FACS纯化来自单个肌肉的MuSC和FAP（图3）。用未染色的样品获得初始设门，以鉴定细胞并将单体与双峰分离（图3A?…
Discussion
执行该协议的几个步骤是实现良好产量的关键。与散装隔离方案中使用的肌肉量相比，单个肌肉的体积较小。这会导致解剖过程中肌肉干燥的风险，从而降低产量。为了防止这种情况，重要的是在解剖后立即向肌肉添加介质。此外，如果解剖需要更长的时间，可以一次从一条肢体上去除皮肤，以减少肌肉暴露在空气中的时间。较小的体积也会导致过度消化的风险增加。为了解决这个问题，与块肌?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
细胞分选在丹麦奥胡斯大学的FACS核心设施进行。图形是使用 Biorender.com 创建的。我们感谢J. Farup博士分享兔抗PDGFRa抗体。这项工作得到了对E.P.的AUFF Start赠款和NovoNordiskFonden向E.P.（0071113）和ADM（0071116）提供的Start Package赠款的支持。
Materials
| 1.5 mL tube( PCR performance tested, PP, 30,000 xg, DNA/DNase-/RNase-free, Low DNA binding, Sterile ) | Sarstedt AG & Co. KG, Hounisen Laboratorieudstyr A/S | 72.706.700 | 1.5 mL tube |
| 15 mL tube (PP/HD-PE, 20,000 xg, IVD/CE, IATA, DNA/DNase-/RNase-free, Non-cytotoxic, pyrogen free, Sterile) | Sarstedt AG & Co. KG, Hounisen Laboratorieudstyr A/S | 62.554.502 | 15 mL tube |
| 5 mL polystyrene round-bottom tube | Falcon, Fisher Scientific | 352054 | FACS tube without strainer cap |
| 5 mL polystyrene Round-bottom tube with cell-strainer cap | Falcon, Fisher Scientific | 352235 | FACS tube with strainer cap |
| 5 mL tube (PP, non sterile autoclavable) | VWR collection | 525.0946 | 5 mL tube |
| 50 mL tube( PP/HD-PE, 20,000 xg, IVD/CE, ADR, DNA/DNase-/RNase-free, non-cytotoxic, pyrogen free, Sterile) | Sarstedt AG & Co. KG, Hounisen Laboratorieudstyr A/S | 62.547.254 | 50 mL tube |
| Alexa Fluor 555 Donkey anti-rabbit IgG (H+L) | Invitrogen, Thermo Fisher | Lot: 2387458 (Cat # A31572) | |
| Alexa Fluor 647 donkey-anti mouse IgG (H+L) | Invitrogen, Thermo Fisher | Lot: 2420713 (Cat#A31571) | |
| ARIA 3 | BD | FACS, Core facility Aarhus University | |
| Centrifuge 5810 | eppendorf | EP022628188 | Centrifuge |
| Click-iT EdU Cell Proliferation Kit for Imaging, Alexa Fluor 488 dye | Invitrogen, Thermo Fisher | Lot: 2387287 (Cat# C10337) | Cell Proliferation Kit |
| Collagen from calf-skin | Bioreagent, Sigma Aldrich | Source: SLCK6209 (Cat# C8919) | |
| Collagenase type II | Worthington, Fisher Scientific | Lot: 40H20248 (cat# L5004177 ) | Collagenase |
| Dispase | Gibco, Fisher Scientific | Lot: 2309415 (cat# 17105-041 ) | Dispase |
| Donkey serum (non-sterile) | Sigma Aldrich, Merck | Lot: 2826455 (Cat# S30-100mL) | |
| Dumont nr. 5, 110 mm | Dumont, Hounisen Laboratorieudstyr A/S | 1606.327 | Straight forceps with fine tips |
| Dumont nr. 7, 115 mm | Dumont, Hounisen Laboratorieudstyr A/S | 1606.335 | Curved forceps |
| F-10 Nutrient mixture (Ham) (1x), +L-glutamine | Gibco, Fisher Scientific | Lot. 2453614 (cat# 31550-023) | |
| FITC anti-mouse CD31 | BioLegend, NordicBioSite | MEC13.3 (Cat # 102506) | |
| FITC Anti-mouse CD45 | BioLegend, NordicBioSite | 30-F11 (Cat# 103108) | |
| Glacial acetic acid (100%) | EMSURE, Merck | K44104563 9Cat # 1000631000) | |
| Head over head mini-tube rotator | Fisher Scientific | 15534080 (Model no. 88861052) | Head over head mini-tube rotator |
| Horse serum | Gibco, Fisher Scientific | Lot. 2482639 (cat# 10368902 ) | |
| Isotemp SWB 15 | FisherBrand, Fisher Scientific | 15325887 | Shaking water bath |
| MS2 mini-shaker | IKA | Vortex unit | |
| Needle 20 G (0.9 mm x 25 mm) | BD microlance, Fisher Scientific | 304827 | 20G needle |
| Neutral formalin buffer 10% | CellPath, Hounisen Laboratorieudstyr A/S | Lot: 03822014 (Cat # HOU/1000.1002) | |
| Non-pyrogenic cell strainer (40 µM) | Sarstedt AG & Co. KG, Hounisen Laboratorieudstyr A/S | 83.3945.040 | Cell strainer |
| Pacific Blue anti-mouse Ly-6A/E (Sca-1) | BioLegend, NordicBioSite | D7 (Cat# 108120) | |
| Pax7 primary antibody | DSHB | Lot: 2/3/22-282ug/mL (Cat# AB 528428) | |
| PBS 10x powder concentrate | Fisher BioReagents, Fisher Scientific | BP665-1 | |
| PE/Cy7 anti-mouse CD106 (VCAM1) | BioLegend, NordicBioSite | 429 (MVCAM.A) (Cat # 105720) | |
| Pen/strep | Gibco, Fisher Scientific | Lot. 163589 (cat# 11548876 ) | |
| Pipette tips p10 | Art tips, self sealing barrier, Thermo Scientific | 2140-05 | Low retention, pre-sterilized, filter tips |
| Pipette tips p1000 | Art tips, self sealing barrier, Thermo Scientific | 2279-05 | Low retention, pre-sterilized, filter tips |
| Pipette tips p20 | Art tips, self sealing barrier, Thermo Scientific | 2149P-05 | Low retention, pre-sterilized, filter tips |
| Pipette tips p200 | Art tips, self sealing barrier, Thermo Scientific | 2069-05 | Low retention, pre-sterilized, filter tips |
| Protective underpad | Abena | ACTC-7712 | 60 x 40cm, 8 layers |
| Rainin, pipet-lite XLS | Mettler Toledo, Thermo Scientific | 2140-05, 2149P-05, 2279-05, 2069-05 | Pipettes (P10, P20, P200, P1000) |
| Recombinant anti-PDGFR-alpha | RabMAb, abcam | AB134123 | |
| Scalpel (shaft no. 3) | Hounisen, Hounisen Laboratorieudstyr A/S | 1902.502 | Scalpel |
| Scalpel blade no. 11 | Heinz Herenz, Hounisen Laboratorieudstyr A/S | 1902.0911 | Scalpel |
| Scanlaf mars | Labogene | class 2 cabinet: Mars | Flow bench |
| ScanR | Olympus | Microscope, Core facility Aarhus University | |
| Scissors | FST | 14568-09 | |
| Series 8000 DH | Thermo Scientific | 3540-MAR | Incubator |
| Serological pipette 10 mL | VWR | 612-3700 | Sterile, non-pyrogenic |
| Serological pipette 5 mL | VWR, Avantor delivered by VWR | 612-3702 | Sterile, non-pyrogenic |
| Syringe 5 mL, Luer tip (6%), sterile | BD Emerald, Fisher Scientific | 307731 | Syringe |
| TC Dish 100, standard | Sarstedt AG & Co. KG, Hounisen Laboratorieudstyr A/S | 83.3902 | Petri dish |
| Tissue Culture (TC)-treated surface, black polystyrene, flat bottom, sterile, lid, pack of 20 | Corning, Sigma Aldrich | 3764 | 96-well Half bottom plate |
| Triton X-100 | Sigma Aldrich, Merck | Source: SLCJ6163 (Cat # T8787) |
Riferimenti
- Mauro, A. Satellite cell of skeletal muscle fibers. The Journal of Biophysical and Biochemical Cytology. 9 (2), 493-495 (1961).
- Relaix, F., et al. Perspectives on skeletal muscle stem cells. Nature Communications. 12 (1), 692 (2021).
- Cheung, T. H., Rando, T. A. Molecular regulation of stem cell quiescence. Nature Reviews. Molecular Cell Biology. 14 (6), 329-340 (2013).
- Kann, A. P., Hung, M., Krauss, R. S. Cell-cell contact and signaling in the muscle stem cell niche. Current Opinion in Cell Biology. 73, 78-83 (2021).
- Tedesco, F. S., Dellavalle, A., Diaz-Manera, J., Messina, G., Cossu, G. Repairing skeletal muscle: regenerative potential of skeletal muscle stem cells. Journal of Clinical Investigation. 120 (1), 11-19 (2010).
- Murphy, M. M., Lawson, J. A., Mathew, S. J., Hutcheson, D. A., Kardon, G. Satellite cells, connective tissue fibroblasts and their interactions are crucial for muscle regeneration. Development. 138 (17), 3625-3637 (2011).
- Lepper, C., Partridge, T. A., Fan, C. -. M. An absolute requirement for Pax7-positive satellite cells in acute injury-induced skeletal muscle regeneration. Development. 138 (17), 3639-3646 (2011).
- Sambasivan, R., et al. Pax7-expressing satellite cells are indispensable for adult skeletal muscle regeneration. Development. 138 (17), 3647-3656 (2011).
- Sacco, A., Doyonnas, R., Kraft, P., Vitorovic, S., Blau, H. M. Self-renewal and expansion of single transplanted muscle stem cells. Nature. 456 (7221), 502-506 (2008).
- Joe, A. W. B., et al. Muscle injury activates resident fibro/adipogenic progenitors that facilitate myogenesis. Nature Cell Biology. 12 (2), 153-163 (2010).
- Wosczyna, M. N., et al. Mesenchymal stromal cells are required for regeneration and homeostatic maintenance of skeletal muscle. Cell Reports. 27 (7), 2029-2035 (2019).
- Uezumi, A., Fukada, S. -. I., Yamamoto, N., Takeda, S., Tsuchida, K. Mesenchymal progenitors distinct from satellite cells contribute to ectopic fat cell formation in skeletal muscle. Nature Cell Biology. 12 (2), 143-152 (2010).
- Seale, P., Sabourin, L. A., Girgis-Gabardo, A., Mansouri, A., Gruss, P., Rudnicki, M. A. Pax7 Is Required for the Specification of Myogenic Satellite Cells. Cell. 102 (6), 777-786 (2000).
- Shea, K. L., et al. Sprouty1 regulates reversible quiescence of a self-renewing adult muscle stem cell pool during regeneration. Cell Stem Cell. 6 (2), 117-129 (2010).
- Fukada, S. -. I., et al. Molecular signature of quiescent satellite cells in adult skeletal muscle. Stem Cells. 25 (10), 2448-2459 (2007).
- Liu, L., Cheung, T. H., Charville, G. W., Rando, T. A. Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting. Nature Protocols. 10 (10), 1612-1624 (2015).
- Joe, A., Wang, J., Rossi, F. Prospective isolation of adipogenic progenitors from skeletal muscle. Journal of Investigative Medicine. 55 (1), 124 (2007).
- Yi, L., Rossi, F. Purification of progenitors from skeletal muscle. Journal of Visualized Experiments. (49), e2476 (2011).
- Sherwood, R. I., et al. Isolation of adult mouse myogenic progenitors: functional heterogeneity of cells within and engrafting skeletal muscle. Cell. 119 (4), 543-554 (2004).
- Montarras, D., et al. Direct isolation of satellite cells for skeletal muscle regeneration. Science. 309 (5743), 2064-2067 (2005).
- Conboy, M. J., Cerletti, M., Wagers, A. J., Conboy, I. M. Immuno-analysis and FACS sorting of adult muscle fiber-associated stem/precursor cells. Methods In Molecular Biology. 621, 165-173 (2010).
- de Morree, A., et al. Alternative polyadenylation of Pax3 controls muscle stem cell fate and muscle function. Science. 366 (6466), 734-738 (2019).
- Stuelsatz, P., et al. Extraocular muscle satellite cells are high performance myo-engines retaining efficient regenerative capacity in dystrophin deficiency. Biologia dello sviluppo. 397 (1), 31-44 (2015).
- Mookhtiar, K., Randall Steinbrink, D., Van Wart, H. E. Mode of hydrolysis of collagen-like peptides by class I and class II Clostridium histolyticum collagenases: evidence for both endopeptidase and tripeptidylcarboxypeptidase activities. Biochimica. 24 (23), 6527-6533 (1985).
- Stenn, K. S., Link, R., Moellmann, G., Madri, J., Kuklinska, E. Dispase, a neutral protease from Bacillus polymyxa, is a powerful fibronectinase and type IV collagenase. The Journal of Investigative Dermatology. 93 (2), 287-290 (1989).
- Baghdadi, M. B., et al. Reciprocal signalling by Notch-Collagen V-CALCR retains muscle stem cells in their niche. Nature. 557 (7707), 714-718 (2018).
- van Velthoven, C. T. J., de Morree, A., Egner, I. M., Brett, J. O., Rando, T. A. Transcriptional profiling of quiescent muscle stem cells in vivo. Cell Reports. 21 (7), 1994-2004 (2017).
- Machado, L., et al. In situ fixation redefines quiescence and early activation of skeletal muscle stem cells. Cell Reports. 21 (7), 1982-1993 (2017).
- Machado, L., et al. Tissue damage induces a conserved stress response that initiates quiescent muscle stem cell activation. Cell Stem Cell. 28 (6), 1125-1135 (2021).
- vanden Brink, S. C., et al. Single-cell sequencing reveals dissociation-induced gene expression in tissue subpopulations. Nature Methods. 14 (10), 935-936 (2017).
- Moore, D. K., Motaung, B., du Plessis, N., Shabangu, A. N., Loxton, A. G. SU-IRG consortium isolation of B-cells using Miltenyi MACS bead isolation kits. PloS One. 14 (3), 0213832 (2019).
- Liou, Y. -. R., Wang, Y. -. H., Lee, C. -. Y., Li, P. -. C. Buoyancy-activated cell sorting using targeted biotinylated albumin microbubbles. PloS One. 10 (5), 0125036 (2015).
- Brett, J. O., et al. Exercise rejuvenates quiescent skeletal muscle stem cells in old mice through restoration of Cyclin D1. Nature Metabolism. 2 (4), 307-317 (2020).
- Tabula Muris Consortium. A single-cell transcriptomic atlas characterizes ageing tissues in the mouse. Nature. 583 (7817), 590-595 (2020).
- de Morrée, A., et al. Staufen1 inhibits MyoD translation to actively maintain muscle stem cell quiescence. Proceedings of the National Academy of Sciences. 114 (43), 8996-9005 (2017).
Citazione di questo articolo
Frimand, Z., Das Barman, S., Kjær, T. R., Porpiglia, E., de Morrée, A. Isolation of Quiescent Stem Cell Populations from Individual Skeletal Muscles. J. Vis. Exp. (190), e64557, doi:10.3791/64557 (2022).