Isolering af hvilende stamcellepopulationer fra individuelle skeletmuskler
Summary
Denne protokol beskriver isoleringen af muskelstamceller og fibro-adipogene forfædre fra individuelle skeletmuskler hos mus. Protokollen involverer enkelt muskeldissektion, stamcelleisolering ved fluorescensaktiveret cellesortering, renhedsvurdering ved immunofluorescensfarvning og kvantitativ måling af S-faseindgang ved 5-ethynyl-2′-deoxyuridininkorporeringsassay.
Abstract
Skeletmuskulatur har forskellige populationer af voksne stamceller, der bidrager til homeostase og reparation af vævet. Skeletmuskulaturstamceller (MuSC’er) har evnen til at lave nye muskler, mens fibro-adipogene stamceller (FAP’er) bidrager til stromale understøttende væv og har evnen til at fremstille fibroblaster og adipocytter. Både MuSC’er og FAP’er befinder sig i en tilstand af langvarig reversibel cellecyklusudgang, kaldet hvile. Den hvilende tilstand er nøglen til deres funktion. Hvilende stamceller renses almindeligvis fra flere muskelvæv samlet sammen i en enkelt prøve. Nylige undersøgelser har imidlertid afsløret tydelige forskelle i molekylære profiler og hviledybde af MuSC’er isoleret fra forskellige muskler. Denne protokol beskriver isolering og undersøgelse af MuSC’er og FAP’er fra individuelle skeletmuskler og præsenterer strategier til at udføre molekylær analyse af stamcelleaktivering. Det beskriver, hvordan man isolerer og fordøjer muskler af forskellig udviklingsmæssig oprindelse, tykkelser og funktioner, såsom membran, triceps, gracilis, tibialis anterior (TA), gastrocnemius (GA), soleus, extensor digitorum longus (EDL) og tyggemusklerne. MuSC’er og FAP’er renses ved fluorescensaktiveret cellesortering (FACS) og analyseres ved immunofluorescensfarvning og 5-ethynyl-2′-deoxyuridin (EdU) inkorporeringsassay.
Introduction
Skeletmuskulatur har en høj kapacitet til regenerering på grund af tilstedeværelsen af muskelstamceller (MuSC’er). MuSC’er er placeret på myofibrene under basallamina og befinder sig i en hvilende tilstand af langvarig, reversibel cellecyklusudgang 1,2,3,4. Ved skade aktiveres MuSC’er og kommer ind i cellecyklussen for at give anledning til forstærkende forfædre, der kan differentiere og smelte sammen til dannelse af nye myofibre 2,5. Tidligere arbejde har vist, at MuSC’er er absolut afgørende for muskelregenerering 6,7,8. Desuden kan en enkelt MuSC indpode og generere både nye stamceller og nye myofibre9. Skeletmuskulatur har også en population af mesenkymale stromale celler kaldet fibro-adipogene forfædre (FAP’er), som spiller en afgørende rolle i at understøtte MuSC-funktionen under muskelregenerering 6,10,11,12.
På grund af deres potentiale til at koordinere muskelregenerering har der været enorm interesse for at forstå, hvordan MuSC’er og FAP’er fungerer. Hvilende MuSC’er er kendetegnet ved ekspression af transkriptionsfaktorerne Pax7 og Sprouty1 og celleoverfladeproteinet calcitoninreceptor, mens hvilende FAP’er er markeret med celleoverfladeproteinblodpladeafledt vækstfaktorreceptor alfa (PDGFRa)10,12,13,14,15. Tidligere undersøgelser har vist, at MuSC’er og FAP’er kunne renses fra skeletmuskler ved hjælp af celleoverflademarkører og fluorescensaktiveret cellesortering (FACS)9,15,16,17,18,19,20,21. Mens disse protokoller i høj grad har avanceret evnen til at studere MuSC’er og FAP’er, er en ulempe, at de fleste af disse protokoller kræver isolering af MuSC’er fra en pulje af forskellige muskelvæv. Nyligt arbejde fra os og andre har afsløret forskelle i cellefænotype og genekspressionsniveauer mellem MuSC’er isoleret fra forskellige væv22,23. MuSC’er fra membranen, triceps og gracilis viser hurtigere aktivering end MuSC’er fra nedre bagbensmuskler22, mens MuSC’er fra ekstraokulær muskel viser hurtigere differentiering end MuSC’er fra membranen og nedre bagbenmuskler23.
Denne protokol beskriver isoleringen af MuSC’er og FAP’er fra individuelle skeletmuskler (figur 1). Dette omfatter dissektion af membranen, triceps, gracilis, tibialis anterior (TA), soleus, extensor digitorum longus (EDL), gastrocnemius (GA) og tyggemuskler. Dissekerede muskler dissocieres efterfølgende ved enzymatisk fordøjelse ved hjælp af collagenase II (en protease, der specifikt er rettet mod Pro-X-Gly-Pro-aminosekvensen i kollagen, hvilket muliggør nedbrydning af bindevæv og vævsdissociation24) og dispase (en protease, der spalter fibronectin og kollagen IV, hvilket muliggør yderligere celledissociation25). MuSC’er og FAP’er isoleres fra enkeltcellesuspensioner af FACS. Som eksempler på nedstrømsassays til celleanalyse bestemmes stamcelleaktivering ved assay af 5-ethynyl-2′-deoxyuridin (EdU) inkorporering, mens cellerenhed bestemmes ved immunofluorescensfarvning for de celletypespecifikke markører Pax7 og PDGFRa.
Protocol
Representative Results
Discussion
Flere trin er nøglen til udførelsen af denne protokol for at opnå gode udbytter. De enkelte muskler har et lille volumen sammenlignet med mængden af muskler, der anvendes i bulkisoleringsprotokoller. Dette resulterer i en risiko for, at musklen tørrer ud under dissektionen, hvilket reducerer udbyttet. For at forhindre dette er det vigtigt at tilføje medium til musklerne umiddelbart efter dissektion. Hertil kommer, at hvis dissektion tager længere tid, kan huden fjernes fra et lem ad gangen for at reducere musklern…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Cellesortering blev udført på FACS Core Facility, Aarhus Universitet, Danmark. Figurer blev oprettet ved hjælp af Biorender.com. Vi takker Dr. J. Farup for at dele kanin-anti-PDGFRa-antistoffet. Dette arbejde blev støttet af en AUFF Starting Grant til E.P. og Start Package bevillinger fra NovoNordiskFonden til E.P. (0071113) og til A.D.M. (0071116).
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) |
References
- 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. Developmental Biology. 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. Biochemistry. 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).
