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Preparation of Adult Mouse Muscle Stem Cells
成年小鼠肌肉干细胞的制备   

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Abstract

Muscle stem cells are adult stem cells responsible for muscle development, growth and regeneration. Current knowledge suggests those cells are heterogeneous population shared their position between the sarcolemma and basal lamina of muscle fibers. This protocol describes the technique to dissociate and collect the stem cells from skeletal muscle of adult mice, and separate them from other cells found in muscle (e.g. fat, connective tissue). To purify and preserve those myofiber associated muscle stem cells, we use two steps of enzyme digestion followed by cell pre-plating procedures.

Materials and Reagents

  1. Collagenase type II (Sigma-Aldrich, catalog number: C6885 )
  2. Penicillin Streptomycin (Gibco®)
  3. HEPES
  4. Dulbecco’s Modified Eagle Medium (DMEM)
  5. Dispase II (STEMCELL Technologies)
  6. Bovine Growth Serum (Hyclone)
  7. Collagenase type II solution (see Recipes)
  8. Collagenase/Dispase solution (see Recipes)
  9. Growth medium (see Recipes)

Equipment

  1. Scissor
  2. 50 ml conical tube
  3. Microscope
  4. 100 mm cell culture dish
  5. 40 μm filter
  6. Fluorescence Activated Cell Sorter (FACS)

Procedure

  1. Remove skeletal muscle of interest (tibialis anterior, Soleus/gastrocnemlus, or quadriceps) from adult mice, and clean the hair tendon and fat with a fine scissor.
  2. Place muscle into a 50 ml conical tube and add two volume of collagenase digestion medium (1 g of muscle into 2 ml of digestion medium). Gently agitate the tube at 37 °C for 30 min to 1.5 h. The digestion time depends on if the muscle is from neonatal, adult or aged animals. During the digestion, regularly check the muscle under microscope until the muscle start to lossen up and single myofibers are separated.
  3. After digestion, stop the collagenase with 5 ml of growth medium and directly transfer the dissociated muscle into 100 mm cell culture dishes.
  4. Triturate up and down to disrupt the muscle with broken end glass pipets (the pore size of the broken end is around 5 mm in diameter), until all the muscle can easily pass through the pipette. The dissociation of muscle tissue to single myofibers can be confirmed under microscope.
  5. Tilt the plate to pool the tissue suspension, and transfer it into new tubes.
  6. If muscle is not completely dissociated, add 5 ml of growth medium and repeat steps 5 and 6.
  7. Spin down dissociated muscle fibers suspension at 100 x g for 3 min. Muscle fibers will be settled and formed a pellet.
  8. Discard supernatant (or spin at 500 x g for 5 min to collect mononuclear cells), and re-suspend the myofibers in 5 ml of growth medium.
  9. Repeat step 7 and 8 twice to remove all the mononuclear cells and small fiber fragments.
  10. Add 15 ml of collagenase II/Dispase II solution to 5 ml of myofiber suspension, digest the fibers at 37 °C for 1 h with gentle agitation. The long incubation time will make sure the myofibers are completely digested.
  11. Votex the digested fiber pellet for at least 1 min, then pass all the digested cells through 40 μm filter.
  12. Centrifuge digested cells at 500 x g for 5 min to pellet satellite cells. Wash the cell pellet with PBS twice to remove all the enzyme.
  13. Resuspend satellite cells in growth medium (~10ml for 1,000,000 cells), and preplate cells in uncoated plate for 20 min to remove fibroblast contamination.
  14. Collect unattached cells in growth medium for further application, such as cell culture and FACS (Chapman et al., 2013; Conboy et al., 2010).
  15. The quality and purity of isolated muscle stem cells could be analyzed with immunostaining of myogenic markers, such as Pax7 and Myf5 (Figure 1).


    Figure 1. Immunostaining of isolated muscle stem cells

Recipes

  1. Collagenase digestion mediumCollagenase type II 250 U/ml
    Penn/Strip: 1%
    HEPES 10 mM (pH 6.4)
    Solved in DMEM
  2. Growth medium for muscle stem cells
    20% bovine growth serum
    1% Pen Strep
    Solved in DMEM medium
  3. Collagenase/Dispase solutionCollagenase type II (250 U/ml) 3.2 ml
    Dipase II (10 U/ml) 4 ml
    Growth medium 7.8 ml

Acknowledgments

This protocol is adapted from Chapman et al. (2013); Conboy et al. (2010) and Li et al. (2009).

References

  1. Chapman, M. R., Balakrishnan, K. R., Li, J., Conboy, M. J., Huang, H., Mohanty, S. K., Jabart, E., Hack, J., Conboy, I. M. and Sohn, L. L. (2013). Sorting single satellite cells from individual myofibers reveals heterogeneity in cell-surface markers and myogenic capacity. Integr Biol (Camb) 5(4): 692-702.
  2. Conboy, M. J. and Conboy, I. M. (2010). Preparation of adult muscle fiber-associated stem/precursor cells. Methods Mol Biol 621: 149-163.
  3. Conboy, M. J., Cerletti, M., Wajers, A. J., Conboy, I. M. (2010). Immuno-analysis and FACS sorting of adult muscle fiber-associated stem/precursor cells. Methods Mol Biol 621: 165-173.
  4. Li, J., Reed, S. A. and Johnson, S. E. (2009). Hepatocyte growth factor (HGF) signals through SHP2 to regulate primary mouse myoblast proliferation. Exp Cell Res 315(13): 2284-2292.

简介

肌肉干细胞是负责肌肉发育,生长和再生的成体干细胞。 目前的知识表明这些细胞是异质群体共享他们在肌纤维和肌肉纤维的基底层之间的位置。 该方案描述了从成年小鼠的骨骼肌中分离和收集干细胞并将它们与在肌肉(例如脂肪,结缔组织)中发现的其他细胞分离的技术。 为了纯化和保存这些肌纤维相关的肌肉干细胞,我们使用两个步骤的酶消化,然后细胞预先铺板程序。

材料和试剂

  1. II型胶原酶(Sigma-Aldrich,目录号:C6885)
  2. 青霉素链霉素(Gibco )
  3. HEPES
  4. Dulbecco改良的Eagle培养基(DMEM)
  5. Dispase II(STEMCELL Technologies)
  6. 牛生长血清(Hyclone)
  7. II型胶原酶溶液(参见配方)
  8. 胶原酶/分散酶溶液(参见配方)
  9. 生长培养基(参见食谱)

设备

  1. 剪刀
  2. 50ml锥形管
  3. 显微镜
  4. 100 mm细胞培养皿
  5. 40μm过滤器
  6. 荧光活化细胞分选仪(FACS)

程序

  1. 从成年小鼠中取出感兴趣的骨骼肌(胫前肌,大疱/腓肠肌或四头肌),并用细剪刀清洁头发腱和脂肪。
  2. 将肌肉放入50毫升锥形管,加入两倍体积的胶原酶消化培养基(1克肌肉2毫升消化培养基)。在37°C轻轻搅拌试管30分钟至1.5小时。消化时间取决于肌肉是来自新生儿,成年或老年动物。在消化期间,定期检查显微镜下的肌肉,直到肌肉开始损失和单个肌纤维分离。
  3. 消化后,用5ml生长培养基终止胶原酶,并将解离的肌肉直接转移到100mm细胞培养皿中。
  4. 用破碎的末端玻璃移液管(破碎端的孔径大小为直径约5mm)破碎肌肉,直到所有的肌肉都能轻易地通过移液管。肌肉组织与单个肌纤维的解离可以在显微镜下确认
  5. 倾斜板以收集组织悬浮液,并将其转移到新管中
  6. 如果肌肉没有完全解离,加入5毫升生长培养基,并重复步骤5和6
  7. 在100×g下旋转离解的肌纤维悬浮液3分钟。 肌肉纤维将沉降并形成颗粒
  8. 弃去上清液(或以500×g旋转5分钟以收集单核细胞),并将肌纤维重悬于5ml生长培养基中。
  9. 重复步骤7和8两次,以去除所有单核细胞和小纤维碎片
  10. 加入15毫升的胶原酶II/Dispase II溶液到5ml的肌纤维悬浮液,消化纤维在37℃下1小时,轻轻地搅拌。 长的孵育时间将确保肌纤维完全消化
  11. Votex消化的纤维团块至少1分钟,然后将所有消化的细胞通过40μm过滤器
  12. 离心消化的细胞在500×g离心5分钟,以沉淀卫星细胞。 用PBS洗涤细胞沉淀两次,以去除所有的酶
  13. 将生长培养基中的卫星细胞重悬(对于1,000,000个细胞,约10ml),并在未涂布板中预培养板细胞20分钟以除去成纤维细胞污染。
  14. 在生长培养基中收集未附着的细胞用于进一步应用,例如细胞培养和FACS(Chapman等人,2013; Conboy等人,2010)。
  15. 分离的肌肉干细胞的质量和纯度可以通过肌肉生成标志物,如Pax7和Myf5(图1)的免疫染色进行分析。


    图1.分离的肌肉干细胞的免疫染色

食谱

  1. 胶原酶消化培养基II型胶原酶250U/ml
    HEPES 10mM(pH 6.4)
    解决在DMEM
  2. 肌肉干细胞的生长培养基
    20%牛生长血清
    1%Pen Strep
    溶于DMEM培养基中
  3. 胶原酶/分散酶溶液II型胶原酶(250U/ml)3.2ml
    Dipase II(10U/ml)4ml
    生长培养基7.8ml

致谢

该协议改编自Chapman等人(2013); Conboy等人(2010)和Li 等人(2009)。

参考文献

  1. Chapman,M.R.,Balakrishnan,K.R.,Li,J.,Conboy,M.J.,Huang,H.,Mohanty,S.K.,Jabart,E.,Hack,J.,Conboy,I.M.and Sohn,L.L。 从单个肌纤维分选单个卫星细胞揭示了细胞表面标记物和肌原生成能力的异质性。 Integr Biol (Camb) 5(4):692-702。
  2. Conboy,M.J.and Conboy,I.M。(2010)。 成人肌纤维相关干/前体细胞的制备 方法Mol Biol.61:149-163。
  3. Conboy,M.J.,Cerletti,M.,Wajers,A.J.,Conboy,I.M。(2010)。 成人肌纤维相关干/前体细胞的免疫分析和FACS分选。 Methods Mol Biol 621:165-173。
  4. Li,J.,Reed,S.A.and Johnson,S.E。(2009)。 肝细胞生长因子(HGF)通过SHP2信号调节原代小鼠成肌细胞增殖。 em> Exp Cell Res 315(13):2284-2292
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Li, J. (2013). Preparation of Adult Mouse Muscle Stem Cells. Bio-protocol 3(24): e1003. DOI: 10.21769/BioProtoc.1003.
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