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Skeletal Myogenesis in vitro
体外骨骼肌发生   

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Abstract

Mature skeletal myofibers are elongated and multinucleated cells. Many stem/progenitor cell types, including committed muscle stem (satellite cells) and progenitor (myoblasts) cells, muscle-derived stem cells, myogenic endothelial cells, and mesenchymal stem/stromal cells, have been shown to exhibit skeletal myogenesis under appropriate inductive conditions. Committed muscle stem/progenitor cells and multipotent stem/progenitor cells which have skeletal myogenic capacity can typically be differentiated into skeletal myofibers in vitro following extended low-serum exposure. Differentiated cells exhibit distinct fiber-like elongated morphology with multiple nuclei and express unique muscle molecular markers indicating myogenesis, including desmin (early) and fast- and/or slow-myosin heavy chain (mature).

Materials and Reagents

  1. Collagen type-I coated plates (sterilized by UV overnight after coating) (the protocol for coating plates is provided by Sigma-Aldrich in the associated product information)
  2. Myogenic cells in sterile conditions (Refer to Chen et al., 2014 and Gharaibeh et al., 2008 for primary human and mouse cell isolation respectively)
    Note: For mouse cells, C2C12 cell line (ATCC, catalog number: CRL-1772) may be used as a positive control.
  3. DMEM high-glucose (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11995 )
  4. Fetal bovine serum (FBS) (Invitrogen, catalog number: 10437-028 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 10437-028”.
  5. Horse serum (HS) (Invitrogen, catalog number: 26050-088 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 26050-088”.
  6. Chicken embryo extract (CEE) (Accurate Chemical & Scientific Corporation,  catalog number: MD-004-D )
  7. Penicillin-Streptomycin (P/S) (Invitrogen, catalog number: 15140-122 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122”.
  8. Monoclonal Anti-Desmin antibody produced in mouse (1:100) (Sigma-Aldrich, catalog number: D1033 ), Mouse anti-fast-MyHC (1:250) (Sigma-Aldrich, catalog number: M4276 ) or Monoclonal Anti-Myosin (Skeletal, Slow) antibody produced in mouse (1:250) (Sigma-Aldrich, catalog number: M8421 )
  9. Normal donkey serum (Jackson Immuno Research, catalog number: 017-000-121 )
  10. Alexa 594-conjugated anti-mouse IgG antibody (1:500) (Invitrogen, Molecular Probes, catalog number: A-21203 ) or Alexa 488-conjugated anti-mouse IgG antibody (1:500) (Invitrogen, Molecular Probes, catalog number: A-21202 )
    Note: Currently, it is “Thermo Fisher Scientific, NovexTM, catalog numbers: A-21203 and A-21202”.
  11. 4',6-diamidino-2-phenylindole (DAPI) (100 ng/ml, diluted with DPBS from the stock) (Sigma-Aldrich, catalog number: D9542 )
  12. Mouse-on-mouse (M.O.M) Basic kit (staining kit) (Vector Labs, catalog number: BMK-2202 )
  13. Collagen from calf skin (non-sterile) (Sigma-Aldrich, catalog number: C9791 )
  14. Dulbecco’s DPBS without calcium and magnesium (DPBS) (Invitrogen, catalog number: 14190-250 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: A-21203 and A-21202”.
  15. Methanol (Sigma-Aldrich, catalog number: 322415 )
  16. Acetone (Sigma-Aldrich, catalog number: 270725 )
  17. Formalin (10%) (Sigma-Aldrich, catalog number: HT501128 )
  18. Trypsin-EDTA (1x, diluted from the 10x stock with sterile DPBS, no phenol red) (Invitrogen, catalog number: 15400-054 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM catalog number: 15400-054”.
  19. Collagen type-I (Sigma-Aldrich, catalog number: C9791)
  20. Muscle proliferation medium (see Recipes)
  21. Muscle fusion medium (see Recipes)

Equipment

  1. Cell culture incubator
  2. Fluorescence microscope

Procedure

  1. Skeletal myogenic priming
    1. For cells not natively committed to skeletal myogenesis (for example, cells isolated from non-muscle organs), a priming step may facilitate myogenic differentiation in vitro.
    2. To prime cells of interest for skeletal myogenesis, cells are plated on collagen type-I coated plates at low density (2 x 103 cells/cm2) for 5-7 days with muscle proliferation medium.
    3. Medium should be completely changed every 2 days.
    4. To further increase their myogenic efficiency, passage cells by typical trypsinization for 5 min at 37 °C and then repeat the priming step one or more times.

  2. Skeletal myogenic differentiation
    1. For human cells, cells are plated on collagen type-I coated 24- or 48-well plates at high confluency (>2.5 x 104 cells/cm2) for 7-14 days with human muscle fusion (myogenic) medium: DMEM high-glucose supplemented with 1% FBS, 1% HS, 0.5% CEE, and 1% P/S.
    2. Half of the fusion medium is renewed every 2-3 days until elongated, multinucleated skeletal myofibers appear (Crisan et al., 2008).
    3. For mouse cells, cells near or at confluency (>1.5 x 104 cells/cm2) are cultured for 3-7 days in mouse muscle fusion (myogenic) medium: DMEM high-glucose supplemented with 2% FBS and 1% P/S.
    4. Half of the fusion medium is renewed every 2 days until elongated, multinucleated skeletal myofibers appear (Lu et al., 2014).
    5. For human and mouse cells not tolerating sudden switch to low-serum fusion media with resultant cell death, skeletal myogenesis can be induced by gradually lowering serum concentration from 20% to 2% (for example, 20%-10%-5%-2%, each stage for 3-7 days) until elongated, multinucleated skeletal myofibers appear.
    6. A positive myogenic cell control (e.g., skeletal myoblast) can be included in the experiment with cells of interest to confirm the efficacy of myogenic media.

  3. Skeletal myofiber detection
    1. Skeletal myogenic differentiation can be first identified by the distinct morphology of elongated, multinucleated skeletal myofibers (Figure 1).
    2. To precisely determine the myogenic differentiation, immunofluorescent staining for muscle cell markers: desmin (early myogenesis), fast- and slow-myosin heavy chain (fast- and slow-MyHC; mature myogenesis) is performed.
    3. For human cells, 7-14 days after culturing in muscle fusion medium, cells are rinsed two times with DPBS and then fixed for 5 min in cold methanol/acetone mixture (1:1, -20 °C) (Chen et al., 2015).
    4. For mouse cells, 3-7 days after culturing in muscle fusion medium, cells are rinsed two times with DPBS and then fixed for 5 min in cold methanol (-20 °C) (Lu et al., 2014).
    5. Alternatively, cells can be fixed with 10% formalin for 8 min at room temperature (RT) (Sohn et al., 2015).
    6. After washing 3 times with DPBS for 5 min each, fixed cells are blocked with 10% normal donkey serum for 1-2 h and then incubated, without washing, with the primary mouse anti-desmin, mouse anti-fast-MyHC, or mouse anti-slow-MyHC antibody (diluted in 5% normal donkey serum) for 2 h at RT or at 4 °C overnight.
    7. After washing 3 times with DPBS for 5 min each, the cells are then incubated with the secondary Alexa 594-conjugated anti-mouse IgG antibody or Alexa 488-conjugated anti-mouse IgG antibody (diluted with 5% normal donkey serum) for 30 min to 1 h at RT.
    8. After washing 3 times with DPBS for 5 min each, nuclei are stained by DAPI for 5 min.
    9. After washing 2 times with DPBS for 5 min each, stained cells can be observed using a fluorescent microscope.
    10. The percentage of differentiated myotubes can be quantified as the number of nuclei in MyHC-positive myotubes relative to the total number of nuclei.
    11. Stained cells can be preserved in sterile DPBS at 4 °C in the dark for up to 1 week.
    12. If staining background persists after washing, the primary antibody can be detected using a mouse-on-mouse (M.O.M) staining kit to reduce signal noise, according to the manufacturer's directions.

Representative data


Figure 1. Representative data. A representative picture of human multi-nuclei skeletal myotube formation after myogenic induction for 7 days.

Notes

  1. We observed variability in skeletal myogenesis between early- and late-passage cells. Early-passage cells had higher skeletal myogenesis (with or without priming) in general.
  2. This protocol is not suitable for cells that could not proliferate to near confluency in high-glucose DMEM with high serum concentration.

Recipes

  1. Muscle proliferation medium
    DMEM high-glucose supplemented with 10% fetal bovine serum (FBS), 10% horse serum (HS), 1% chicken embryo extract (CEE), and 1% Penicillin-Streptomycin (P/S) before switching to muscle fusion medium (Chen et al., 2015)
  2. Muscle fusion medium
    Human muscle fusion medium: DMEM high-glucose supplemented with 1% FBS, 1% HS, 0.5% CEE, and 1% P/S (Chen et al., 2015)
    Mouse muscle fusion medium: DMEM high-glucose supplemented with 2% FBS and 1% P/S (Lu et al., 2014)

Acknowledgments

This protocol was adapted with modification from Chen et al. (2015). This work was supported by grants from the Medical Research Council (to B. P.), British Heart Foundation (to B. P.), National Institute of Health (R21HL083057 to B. P.).

References

  1. Chen, W. C., Baily, J. E., Corselli, M., Diaz, M. E., Sun, B., Xiang, G., Gray, G. A., Huard, J. and Péault, B. (2015). Human myocardial pericytes: multipotent mesodermal precursors exhibiting cardiac specificity. Stem Cells 33(2): 557-573.
  2. Chen, W. C., Saparov, A., Corselli, M., Crisan, M., Zheng, B., Péault, B. and Huard, J. (2014). Isolation of blood-vessel-derived multipotent precursors from human skeletal muscle. J Vis Exp(90): e51195.
  3. Crisan, M., Yap, S., Casteilla, L., Chen, C. W., Corselli, M., Park, T. S., Andriolo, G., Sun, B., Zheng, B., Zhang, L., Norotte, C., Teng, P. N., Traas, J., Schugar, R., Deasy, B. M., Badylak, S., Buhring, H. J., Giacobino, J. P., Lazzari, L., Huard, J. and Péault, B. (2008). A perivascular origin for mesenchymal stem cells in multiple human organs. Cell Stem Cell 3(3): 301-313.
  4. Gharaibeh, B., Lu, A., Tebbets, J., Zheng, B., Feduska, J., Crisan, M., Péault, B., Cummins, J. and Huard, J. (2008). Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique. Nat Protoc 3(9): 1501-1509.
  5. Lu, A., Poddar, M., Tang, Y., Proto, J. D., Sohn, J., Mu, X., Oyster, N., Wang, B. and Huard, J. (2014). Rapid depletion of muscle progenitor cells in dystrophic mdx/utrophin-/- mice. Hum Mol Genet 23(18): 4786-4800.
  6. Sohn, J., Lu, A., Tang, Y., Wang, B. and Huard, J. (2015). Activation of non-myogenic mesenchymal stem cells during the disease progression in dystrophic dystrophin/utrophin knockout mice. Hum Mol Genet 24(13): 3814-3829.

简介

成熟骨骼肌纤维是细长和多核细胞。 许多干/祖细胞类型,包括定型肌干(卫星细胞)和祖细胞(成肌细胞)细胞,肌肉衍生的干细胞,肌肉内皮细胞和间充质干/基质细胞,已显示在适当的诱导条件下表现出骨骼肌发生 。 具有骨骼肌生成能力的承诺的肌肉干/祖细胞和多能干/祖细胞可以在延长的低血清暴露后通常在体外分化成骨骼肌纤维。 分化的细胞表现出不同的纤维样细长形态与多个核,并表示独特的肌肉分子标记指示肌发生,包括结蛋白(早)和快和/或慢肌球蛋白重链(成熟)。

材料和试剂

  1. 胶原I型包被的板(在包被后通过UV灭菌过夜)(用于包被板的方案由Sigma-Aldrich在相关产品信息中提供)
  2. 在无菌条件下的生原细胞(分别参见Chen等人,2014和Gharaibeh等人,2008)用于原代人和小鼠细胞分离)
    注意:对于小鼠细胞,可以使用C2C12细胞系(ATCC,目录号:CRL-1772)作为阳性对照。
  3. DMEM高葡萄糖(Thermo Fisher Scientific,Invitrogen TM ,目录号:11995)
  4. 胎牛血清(FBS)(Invitrogen,目录号:10437-028)
    注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:10437-028"
  5. 马血清(HS)(Invitrogen,目录号:26050-088)
    注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:26050-088"。
  6. 鸡胚提取物(CEE)(Accurate Chemical& Scientific Corporation,MD-004-D)
  7. 青霉素 - 链霉素(P/S)(Invitrogen,目录号:15140-122) 注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:15140-122"。
  8. 在小鼠(1:100)(Sigma-Aldrich,目录号:D1033),小鼠抗Fast-MyHC(1:250)(Sigma-Aldrich,目录号:M4276)或单克隆抗肌球蛋白(1:250)(Sigma-Aldrich,目录号:M8421)中产生的(骨骼,慢)抗体
  9. 正常驴血清(Jackson Immuno Research,目录号:017-000-121)
  10. Alexa 594-缀合的抗小鼠IgG抗体(1:500)(Invitrogen,Molecular Probes,目录号:A-21203)或Alexa 488-缀合的抗小鼠IgG抗体(1:500)(Invitrogen,Molecular Probes, :A-21202)
    注意:目前,"Thermo Fisher Scientific,Novex TM ,目录号:A-21203和A-21202"。
  11. 4',6-二脒基-2-苯基吲哚(DAPI)(100ng/ml,用来自原种的DPBS稀释)(Sigma-Aldrich,目录号:D9542)
  12. 小鼠鼠标(M.O.M)基本试剂盒(染色试剂盒)(Vector Labs,目录号:BMK-2202)
  13. 来自小牛皮肤(非无菌)的胶原(Sigma-Aldrich,目录号:C9791)
  14. 不含钙和镁的Dulbecco's DPBS(DPBS)(Invitrogen,目录号:14190-250)
    注意:目前,是"Thermo Fisher Scientific,Gibco TM ,目录号:A-21203和A-21202。
  15. 甲醇(Sigma-Aldrich,目录号:322415)
  16. 丙酮(Sigma-Aldrich,目录号:270725)
  17. 福尔马林(10%)(Sigma-Aldrich,目录号:HT501128)
  18. 胰蛋白酶-EDTA(1x,用无菌DPBS稀释的10x储备液,无酚红)(Invitrogen,目录号:15400-054)
    注意:目前,"Thermo Fisher Scientific,Gibco TM 目录号:15400-054" br />
  19. 胶原I型(Sigma-Aldrich,目录号:C9791)
  20. 肌肉增殖培养基(见配方)
  21. 肌肉融合培养基(见配方)

设备

  1. 细胞培养箱
  2. 荧光显微镜

程序

  1. 骨骼肌原始激活
    1. 对于非天然地致力于骨骼肌形成的细胞(例如, 从非肌肉器官分离的细胞),启动步骤可以促进 肌原性分化体外 。
    2. 以引发感兴趣的细胞 对于骨骼肌形成,将细胞接种在包被I型胶原上 平板以低密度(2×10 3个细胞/cm 2 )与肌肉一起培养5-7天 增殖培养基
    3. 中度应每2天完全更换一次。
    4. 为了进一步提高它们的生肌效率,通过细胞 在37℃下典型的胰蛋白酶消化5分钟,然后重复启动 步骤一次或多次。

  2. 骨骼肌分化
    1. 对于人细胞,将细胞接种在胶原I型包被的24-或 对于7-14的高汇合(> 2.5×10 4个细胞/cm 2 )的48-孔板 天用人类肌肉融合(肌原性)培养基:DMEM高葡萄糖 补充有1%FBS,1%HS,0.5%CEE和1%P/S。
    2. 一半 融合培养基每2-3天更新一次,直到伸长, 多核骨骼肌纤维出现(Crisan等人,2008)。
    3. 对于小鼠细胞,接近或处于汇合(> 1.5×10 4细胞/cm 2)的细胞, 在小鼠肌肉融合(生肌)培养基:DMEM中培养3-7天 ?高葡萄糖补充有2%FBS和1%P/S。
    4. 一半 融合培养基每2天更新一次,直到伸长,多核 骨骼肌纤维出现(Lu等人。,2014)。
    5. 对于人和鼠标 ?细胞不耐受突变转为低血清融合培养基 可导致细胞死亡,可逐渐诱导骨骼肌形成 降低血清浓度从20%降至2%(例如,20%-10%-5%-2% ?每个阶段3-7天),直到伸长的多核骨骼 肌纤维出现。
    6. 阳性肌细胞对照(例如骨骼肌成肌细胞)可以包括在实验中,其中细胞 感兴趣以确认肌原性培养基的功效。

  3. 骨骼肌纤维检测
    1. 骨骼肌分化可以首先通过鉴定 细长的多核骨骼肌纤维的不同形态 (图1)。
    2. 为了精确确定生肌分化, 免疫荧光染色的肌细胞标记:结蛋白(早期 肌细胞增生),快和慢肌球蛋白重链(快和慢-MyHC; 成熟肌发生)。
    3. 对于人类细胞,7-14天 在肌肉融合培养基中培养后,将细胞漂洗两次 DPBS,然后在冷甲醇/丙酮混合物(1:1,-20)中固定5分钟 ?°C)(Chen等人,2015)。
    4. 对于小鼠细胞,3-7天后 在肌肉融合培养基中培养,用DPBS漂洗细胞两次 然后在冷甲醇(-20℃)中固定5分钟(Lu等人,2014)。
    5. 或者,可以在室温(RT)下用10%福尔马林固定细胞8分钟(Sohn等人,2015)。
    6. 用DPBS洗涤3次,每次5分钟后,固定细胞 用10%正常驴血清封闭1-2小时,然后孵育, 无需洗涤,用原代小鼠抗结蛋白,小鼠 抗Fast-MyHC或小鼠抗 - 慢-MyHC抗体(稀释于5%正常 驴血清)在室温或4℃过夜2小时。
    7. 洗后3 ?用DPBS洗涤5次,每次5分钟,然后将细胞与 二抗Alexa 594缀合的抗小鼠IgG抗体或Alexa 488-缀合的抗小鼠IgG抗体(用5%正常驴稀释 血清)在室温下孵育30分钟至1小时。
    8. 用DPBS洗涤3次,每次5分钟后,通过DAPI将细胞核染色5分钟。
    9. 用DPBS洗涤2次,每次5分钟后,用荧光显微镜观察染色的细胞
    10. 分化的肌管的百分比可以量化为 MyHC阳性肌管中的核数相对于总数 的核。
    11. 染色的细胞可以在无菌DPBS中于4℃在黑暗中保存长达1周。
    12. 如果染色背景在洗涤后仍然存在,第一抗体 ?可以使用小鼠 - 小鼠(M.O.M)染色试剂盒进行检测以减少 信号噪声,根据制造商的说明。

代表数据


图1.代表性数据。 肌肉性诱导7天后,人类多核心骨骼肌形成的代表性图片。

笔记

  1. 我们观察到早期和晚期传代细胞之间骨骼肌形成的变异性。早期传代细胞一般具有较高的骨骼肌形成(有或没有引发)
  2. 该方案不适用于在高血清浓度的高葡萄糖DMEM中不能增殖至接近汇合的细胞。

食谱

  1. 肌肉增殖培养基
    (FBS),10%马血清(HS),1%鸡胚胎提取物(CEE)和1%青霉素 - 链霉素(P/S)的DMEM高葡萄糖中,然后切换到肌肉融合培养基Chen 等人,2015)
  2. 肌肉融合介质
    人类肌肉融合培养基:补充有1%FBS,1%HS,0.5%CEE和1%P/S的DMEM高葡萄糖(Chen等人,2015) 小鼠肌肉融合培养基:补充有2%FBS和1%P/S的DMEM高葡萄糖(Lu等人,2014)

致谢

该协议改编自Chen等人(2015)的修改。这项工作是由医学研究委员会(B.P.),英国心脏基金会(B.P.),国家卫生研究所(R21HL083057到B.P。)的赠款支持。

参考文献

  1. Chen,W. C.,Baily,J. E.,Corselli,M.,Diaz,M. E.,Sun,B.,Xiang,G.,Gray,G.A.,Huard,J.andPéault, 人类心肌周细胞:显示心脏特异性的多能中胚层前体。干细胞 33(2):557-573。
  2. Chen,W. C.,Saparov,A.,Corselli,M.,Crisan,M.,Zheng,B.,Péault,B。和Huard,J.(2014)。 从人类骨骼肌中分离血管来源的多潜能前体。 J Vis Exp (90):e51195。
  3. Cris,M.,Yap,S.,Casteilla,L.,Chen,CW,Corselli,M.,Park,TS,Andriolo,G.,Sun,B.,Zheng,B.,Zhang,L.,Norotte, C.,Teng,PN,Traas,J.,Schugar,R.,Deasy,BM,Badylak,S.,Buhring,HJ,Giacobino,JP,Lazzari,L.,Huard,J。和Péault, )。 多种人体器官中的间充质干细胞的血管周围起源细胞干单元格 3(3):301-313。
  4. Gharaibeh,B.,Lu,A.,Tebbets,J.,Zheng,B.,Feduska,J.,Crisan,M.,Péault,B.,Cummins,J.and Huard,J。(2008)。 通过preplate技术分离含有来自鼠骨骼肌的干细胞的缓慢粘附的细胞级分。 a> Nat Protoc 3(9):1501-1509。
  5. Lu,A.,Poddar,M.,Tang,Y.,Proto,J. D.,Sohn,J.,Mu,X.,Oyster,N.,Wang,B.and Huard,J.(2014)。 营养不良mdx/utrophin -/- 小鼠中肌肉祖细胞的快速消耗 em> Hum Mol Genet 23(18):4786-4800。
  6. Sohn,J.,Lu,A.,Tang,Y.,Wang,B.and Huard,J.(2015)。 营养不良性肌营养不良蛋白/utrophin基因敲除小鼠在疾病进展期间激活非肌源性间充质干细胞。/a> Hum Mol Genet 24(13):3814-3829。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Chen, W. C. and Péault, B. (2015). Skeletal Myogenesis in vitro. Bio-protocol 5(21): e1645. DOI: 10.21769/BioProtoc.1645.
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