Isolation, Kultur og transplantation af Muskel satellit celler
Summary
Isoleringen og kultur af en ren population af hvilende satellit-celler, en muskel stamcellepopulation, er afgørende for forståelsen af muskel stamcellebiologiske og regenerering, samt stamcelletransplantation terapier i muskelsvind og andre degenerative sygdomme.
Abstract
Muskel satellit celler er en stamcelle population kræves for postnatal skeletmuskulatur udvikling og revitalisering, der tegner sig for 2-5% af sublaminal kerner i muskelfibrene. Hos voksne muskel, satellit-celler normalt mitotisk hvilende. Efter skaden dog satellit celler indlede cellulær proliferation at producere myoblaster, deres afkom, for at mægle regenerering af musklen. Transplantation af satellitbaserede celleafledte myoblaster er blevet bredt undersøgt som en mulig behandling for flere regenerative sygdomme herunder muskeldystrofi, hjertesvigt og urologiske dysfunktion. Myoblast transplantation i dystrofisk skeletmuskulatur, infarktramte hjerte, og funktionsforstyrrelser urin kanaler har vist, at transplanteret myoblaster kan differentiere i muskelfibre i værtsvævene og vise delvis funktionelle forbedringer på disse sygdomme. Derfor er udviklingen af effektive oprensningsmetoder af hvilende satellit celler fra skelet muscle, samt etablering af satellit celleafledte myoblast kulturer og transplantationscentre metoder til myoblaster, er afgørende for at forstå de molekylære mekanismer bag satellit celle selvfornyelse, aktivering og differentiering. Derudover udviklingen af cellebaserede behandlingsformer til muskelsvind og andre regenerative sygdomme er også afhængig af disse faktorer.
De nuværende potentielle oprensningsmetoder af hvilende satellit celler kræver anvendelse af dyre fluorescens-aktiveret cellesortering (FACS) maskiner. Her præsenterer vi en ny metode til hurtig, økonomisk og pålidelig rensning af hvilende satellit celler fra voksne mus skeletmuskulatur ved enzymatisk dissociation efterfulgt af magnetisk cellesortering (MACS). Efter isolering af rene hvilende satellit-celler, kan disse celler dyrkes for at opnå store mængder af myoblaster efter flere passager. Disse frisk isolerethvilende satellit celler eller ex vivo ekspanderede myoblaster kan transplanteres ind i cardiotoxin (CTX)-induceret regenererende mus skeletmuskulatur at undersøge bidraget fra donor-afledte celler til at regenerere muskelfibre samt satellit celle rum til undersøgelse af selv-fornyelse aktiviteter.
Introduction
Muskel satellit-celler er en lille population af myogene stamceller placeret under den basale lamina af skelet muskelfibre. De er kendetegnet ved ekspressionen af Pax7, PAX3, c-Met, M-cadherin, CD34, Syndecan-3 og calcitonin 1-3. Satellit celler har vist sig at være ansvarlig for muskelregenerationen som muskel stamceller. Hos voksne muskel, satellit-celler normalt mitotisk hvilende 4-8. Efter skaden er satellit celler aktiveres, indlede udtryk for MyoD, og indtast cellecyklus at udvide deres afkom, betegnes myogene præcursorceller eller myoblaster 3. Efter adskillige runder af celledeling, myoblaster forlade cellecyklen og sikring til hinanden med henblik på at undergå differentiering til multi-kerneholdige myorør, efterfulgt af modne muskelfibre. Myoblaster isoleret fra voksne muskel kan let udvides ex vivo. Kapacitet til myoblaster blive muskelfibre i regenererende muskler ogdanner ektopiske muskelfibre i nonmuscle væv udnyttes af myoblast transplantation, en potentiel terapeutisk tilgang til Duchennes muskeldystrofi (DMD) 4, urologisk dysfunktion 9, og hjertesvigt 10. Faktisk har myoblaster blevet transplanteret i musklen af både mdx (DMD model) mus og DMD patienter 11-14. De injicerede normale myoblaster fusionerer med værten muskelfibre at forbedre histologi og funktion af syge muskler. Tidligere arbejde viste, at subpopulationer af myoblaster er mere stamcelle-lignende og forbliver i en udifferentieret tilstand længere muskel under muskelregenerationen 5. Nyligt arbejde har vist, at frisk isolerede satellit celler fra voksne muskel indeholde en stamcelle-lignende befolkning, der udstiller mere effektiv transplantation og selv-fornyelse aktivitet i regenererende muskler 5-8. Derfor oprensning af en ren population af hvilende satellit celler fra voksne skelet muscle er væsentlige for forståelsen af biologi, myoblaster og muskelregenerationen og til udviklingen af cellebaserede behandlingsformer.
De nuværende potentielle oprensningsmetoder af hvilende satellit celler kræver anvendelse af et dyrt fluorescens-aktiveret cellesortering (FACS) maskine 1,2,6-8. Desuden FACS laserstråling tendens til at inducere celledød under adskillelse, hvilket medfører lavere udbytte af hvilende satellit celler 15. Her præsenterer vi en ny metode til hurtig, økonomisk og pålidelig rensning af hvilende satellit celler fra voksne mus skeletmuskulatur. Denne metode udnytter enzymatisk dissociation efterfulgt af magnetisk cellesortering (MACS). Efter isolering af rene hvilende satellit-celler, kan disse celler dyrkes for at opnå store mængder af myoblaster efter flere passager. Vi viser også, at intramuskulær injektion af disse frisk isolerede hvilende satellit celler eller ex vivo udvidet myoblaster kan transplanteres ind i cardiotoxin (CTX)-induceret regenererende mus skeletmuskulatur at undersøge bidraget fra donor-afledte celler til at regenerere muskelfibre, samt satellit celle rum til undersøgelse af selvfornyelse aktiviteter.
Protocol
Representative Results
Discussion
I denne protokol, kan hvilende satellit celler let oprenses fra voksen skeletmuskulatur af mus ved collagenasefordøjelse og overflade antistof-medieret MACS separation. Denne metode tager cirka 6 timer og behøver ikke noget dyrt udstyr, såsom en FACS-maskine. Desuden er denne metode relativt billig i forhold til overfladen antistofmedieret FACS adskillelse. Et højere udbytte af hvilende satellit celler forventes også i forhold til FACS for denne metode, da FACS laserstråling tendens til at inducere celledød under…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Vi takker Dr. Shahragim Tajbakhsh for at levere Myf5 + / nLacZ mus. Vi takker også Alexander Hron og Michael Baumrucker for kritisk læsning af dette manuskript. Dette arbejde blev støttet af tilskud fra Muskelsvindfonden Association (MDA) og Gregory Marzolf Jr. MD center Award.
Materials
| Materials | |||
| Collagenase Type 2 | Worthington | CLS-2 | 100 mg |
| Marigel | BD Biosciences | 356234 | 5 ml |
| DMEM | Gibco-Invitrogen | 10569010 | 500 ml |
| Collagen (Rat Tail) | BD Biosciences | 354236 | 100 mg (3-4 mg/ml) |
| Acetic Acid | Sigma-Aldrich | 320099-500ML | 500 ml |
| bFGF, human, Recombinant | Gibco-Invitrogen | PHG0263 | 1 mg |
| Bovine Serum Albumin (BSA) | Sigma-Aldrich | A5611-1G | 1 g |
| Ham’s F10 Medium | Gibco-Invitrogen | 11550-043 | 500 ml |
| Fetal Bovine Serum (FBS) | Fisher Scientific | 3600511 | 500 ml |
| Horse Serum | Gibco-Invitrogen | 26050088 | 500 ml |
| Penicillin/Streptmycin | Gibco-Invitrogen | 15640055 | 100 ml |
| Phosphate Buffered Saline | Gibco-Invitrogen | 14190144 | 500 ml |
| 0.25% Trypsin/EDTA | Gibco-Invitrogen | 25200072 | 500 ml |
| 18G needle with 12cc Syringe | Fisher Scientific | 22-256-563 | |
| Cell strainer (70 μm) | Fisher Scientific | 22-363-548 | |
| Falcon 50 ml tube | BD Biosciences | 352098 | |
| Falcon 15 ml tube | BD Biosciences | 352097 | |
| 10 cm tissue culture plate | BD Biosciences | 353003 | |
| 6 cm tissue culture plate | BD Biosciences | 353004 | |
| Falcon 10 ml disposable pipet | BD Biosciences | 357551 | |
| Anti-CD31 antibody-PE | eBiosciences | 12-0311 | |
| Anti-CD45 antibody-PE | eBiosciences | 30-F11 | |
| Anti-Sca1 antibody-PE | eBiosciences | Dec-81 | |
| Anti-Integrin α7 antibody | MBL International | ABIN487462 | |
| Anti-PE MicroBeads | Miltenyi Biotec | 130-048-801 | |
| Anti-Mouse IgG MicroBeads | Miltenyi Biotec | 130-048-402 | |
| Mini & MidiMACS Starting Kit | Miltenyi Biotec | 130-091-632 | |
| MS Column | Miltenyi Biotec | 130-042-201 | |
| LD Column | Miltenyi Biotec | 130-042-901 | |
| Cardiotoxin | Sigma Aldrich | C9759-1MG | Stock 10 μM in PBS |
| 31G Insulin syringe | BD Biosciences | 328438 | |
| Refrigerated Microcentrifuge (Microfuge 22R) | Beckman Coulter | 368826 | |
| S241.5 Swinging Bucket Rotor | Beckman Coulter | 368882 | |
| Refrigerated Centrifuge (Allegra X-22R) | Beckman Coulter | 392187 | |
| Nod/Scid immunodeficient mice | Charles River Laboratories | Strain Code 394 | Use 2 months old mice |
| Reagents | |||
| Name of the reagent | Recipie | ||
| 10% and 2% FBS DMEM | DMEM (Gibco-Invitrogen #10569010) with 10% or 2% FBS (Fisher Scientific #03600511) and 1% Penicillin/Streptomycin (Gibco-Invitrogen #15640055). | ||
| 0.2% Collagenase solution | Collagenase Type 2 (Worthington, #CLS-2), Stock: 50 ml: 100 mg Collagenase Type 2 in 10% FBS DMEM. | ||
| 10% Matrigel solution | Matrigel (BD Biosciences: #356234) is placed on ice for thawing overnight. Five ml Matrigel is dilute by 45 ml DMEM and 5 ml aliquots are stored at -20°C until use. | ||
| Matrigel-coated plate | Five ml of 10% Matrigel solution is placed on ice for thawing and is used for coating 10 cm plate at room temprature for 1 minutes. The plate is placed in 5% CO2 incubator at 37°C for 30 minute after removing Matrigel solution, and let the plate dry in culture hood for another 30 minutes. Removed 10% Matrigel solution is stored at -20°C for reusing. | ||
| 0.01% Collagen solution | Mix to final: 0.01% Collagen (Collagen, Rat Tail: BD Biosciences #354236) in 0.2% acetic acid (320099-500ML) in ddH2O. | ||
| Collagen-coated plate | Add 5 ml or 2 ml of Collagen solution to a 10 cm or 6 cm tissue culture plate and let sit at room temperature for three hours. Then, aspirate off liquid and allow to dry in culture hood for 30 min to overnight. Plates can be stored at room temperature for several months. | ||
| bFGF stock solution | bFGF, Human, Recombinant (Gibco-Invitrogen #PHG0263, 1 mg) is dissolved with 0.1% BSA solution consisting of 1 mg BSA (Sigma-Aldrich #A5611-1G) and 2 ml ddH2O (0.5 mg/ml bFGF). Aliquot 20 μl in 500 μl microcentrifuge tubes and kept in -80°C. | ||
| Myoblast medium | 500 mL HAM’S F10 Medium (Gibco-Invitrogen #11550-043) supplemented with 20% FBS (Fisher Scientific #03600511), Penicillin/streptomycin (Gibco-Invitrogen #15640055), and 10 μg of bFGF (20 μl of bFGF stock). | ||
| Differentiation medium | 500 mL DMEM (Gibco-Invitrogen #10569010) supplemented with 5% Horse serum (Gibco-Invitrogen #26050088) and 1% Penicillin/streptomycin (Gibco-Invitrogen #15640055). | ||
| 10 μM Cardiotoxin stock | 1 mg Cardiotoxin (EMD Millipore #217504-1MG) is dissolved with 13.9 ml PBS. |
References
- Fukada, S., et al. Purification and cell-surface marker characterization of quiescent satellite cells from murine skeletal muscle by a novel monoclonal antibody. Exp. Cell Res. 296, 245-255 (2004).
- Hirai, H., Verma, M., Watanabe, S. C. T., Asakura, Y., Asakura, A. MyoD regulates apoptosis of myoblasts through microRNA-mediated down-regulation of Pax3. J. Cell Biol. (191), 347-365 (2010).
- Asakura, A. Stem cells in adult skeletal muscle. Trends Cardiovasc. Med. 13, 123-128 (2003).
- Partridge, T. A. Cells that participate in regeneration of skeletal muscle. Gene Ther. 9, 752-753 (2002).
- Collins, C. A., et al. Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche. Cell. 122, 289-301 (2005).
- Montarras, D., et al. Direct isolation of satellite cells for skeletal muscle regeneration. Science. 309, 2064-2067 (2005).
- Sacco, A., Doyonnas, R., Kraft, P., Vitorovic, S., Blau, H. M. Self-renewal and expansion of single transplanted muscle stem cells. Nature. 456, 502-506 (2008).
- 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 Mol. Biol. 621, 165-173 (2010).
- Yokoyama, T., Huard, J., Chancellor, M. B. Myoblast therapy for stress urinary incontinence and bladder dysfunction. World J. Urol. 18, 56-61 (2000).
- Menasche, P. Skeletal muscle satellite cell transplantation. Cardiovasc. Res. 58, 351-357 (2000).
- Huard, J., et al. Myoblast transplantation produced dystrophin-positive muscle fibres in a 16-year-old patient with Duchenne muscular dystrophy. Clin. Sci. 81, 287-288 (1991).
- Tremblay, J. P., et al. Results of a triple blind clinical study of myoblast transplantations without immunosuppressive treatment in young boys with Duchenne muscular dystrophy. Cell Transplant. 2, 99-112 (1993).
- Gussoni, E., Blau, H. M., Kunkel, L. M. The fate of individual myoblasts after transplantation into muscles of DMD patients. Nat. Med. 3, 970-977 (1997).
- Palmieri, B., Tremblay, J. P., Daniele, L. Past, present and future of myoblast transplantation in the treatment of Duchenne muscular dystrophy. Pediatr. Transplant. 14, 813-819 (2010).
- Mollet, M., Godoy-Silva, R., Berdugo, C., Chalmers, J. J. Acute hydrodynamic forces and apoptosis: a complex question. Biotechnol. Bioeng. 98, 772-788 (2007).
- Asakura, A., Rudnicki, M. A. Side population cells from diverse adult tissues are capable of in vitro hematopoietic differentiation. Exp. Hematol. 30, 1339-1345 (2002).
- Asakura, A., et al. Increased survival of muscle stem cells lacking the MyoD gene after transplantation into regenerating skeletal muscle. Proc. Natl. Acad. Sci. U.S.A. 104, 16552-16557 (2007).
- Gerard, X., et al. Real-time monitoring of cell transplantation in mouse dystrophic muscles by a secreted alkaline phosphatase reporter gene. Gene Ther. 16, 815-819 .
- Tajbakhsh, S., Rocancourt, D., Cossu, G., Buckingham, M. Redefining the genetic hierarchies controlling skeletal myogenesis Pax-3 and Myf-5 act upstream of MyoD. Cell. 89, 127-138 (1997).
- Beauchamp, J. R., et al. Expression of CD34 and Myf5 defines the majority of quiescent adult skeletal muscle satellite cells. J. Cell Biol. 151, 1221-1134 (2000).
- Sabourin, L. A., Girgis-Gabardo, A., Seale, P., Asakura, A., Rudnicki, M. A. Reduced differentiation potential of primary MyoD-/- myogenic cells derived from adult skeletal muscle. J. Cell Biol. 144, 631-643 (1999).
- Bischoff, R. Regeneration of single skeletal muscle fibers in vitro. Anat. Rec. 182, 215-235 (1975).
- Asakura, A., Seale, P., Girgis-Gabardo, A., Rudnicki, M. A. Myogenic specification of side population cells in skeletal muscle. J. Cell Biol. 159, 123-134 (2002).
- Kuang, S., Kuroda, K., Le Grand, F., Rudnicki, M. A. Asymmetric self-renewal and commitment of satellite stem cells in muscle. Cell. 129, 999-1010 (2007).
- Cerletti, M., et al. Highly efficient, functional engraftment of skeletal muscle stem cells in dystrophic muscles. Cell. 134, 37-47 (2008).
- Farina, N. H., et al. A role for RNA post-transcriptional regulation in satellite cell activation. Skelet. Muscle. 2, 21-21 (2012).
- Tanaka, K. K., Hall, J. K., Troy, A. A., Cornelison, D. D., Majka, S. M., Olwin, B. B. Syndecan-4-expressing muscle progenitor cells in the SP engraft as satellite cells during muscle regeneration. Cell Stem Cell. 4, 217-225 (2009).
- Pallafacchina, G., et al. An adult tissue-specific stem cell in its niche: a gene profiling analysis of in vivo quiescent and activated muscle satellite cells. Stem Cell Res. 4, 77-91 (2009).
