Name: isolation of type i and type ii pericytes from mouse skeletal muscles
Uploaded: 2017-05-26T15:00:00.000+00:00
Duration: 10 min 7 s
Description: Watch this Scientific Journal Video about Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles at JoVE.com

这项工作得到了强直性营养不良基金会（MDF-FF-2014-0013）和美国心脏协会（16SDG29320001）的科学家发展拨款的基金A-Fellow资助部分支持。

野生型和Nestin-GFP转基因小鼠被安置在明尼苏达大学的动物设施中。所有实验程序均由明尼苏达大学体制动物护理和使用委员会批准，并符合NIH"实验动物护理和使用指南"。 肌肉解剖和单细胞隔离<ol><li>用三溴乙醇（250mg / kg，ip）将成年小鼠（6-10周，男性和女性）安乐死，并用70％乙醇对其腹部皮肤进行消毒。 注意:这里使用三溴乙醇代替氯胺酮进行麻醉/安乐死，因为已知氯胺酮与NMDA受体相互作用，这可能对研究有影响。 </li><li>将小鼠放置在仰卧位，使用手术刀在腹部皮肤上进行水平切口。用手剥离皮肤，拉动相反方向以暴露后肢肌肉。 </li><li> COLLEC使用镊子和剪刀从两个后肢的肌肉。将它们储存在补充有1％青霉素 - 链霉素（P / S）的无菌磷酸盐缓冲盐水（PBS）中。 </li><li>在补充有1％P / S的冰冷PBS中洗涤解剖后肢肌肉两次，并将其转移到无菌10cm板上。 </li><li>使用镊子和剪刀，在2倍放大倍数的夹层显微镜下，从肌肉中仔细剖析神经，血管和结缔组织。 </li><li>使用无菌剪刀和刀片将肌肉切碎并切碎成小块（1-2毫米3 ）。如有必要，加入少量Dulbecco&#39;s Modified Eagle Medium（DMEM），以确保肌肉不会变干。 </li><li>通过10毫升血清移液管上下移液10次机械分解组织。 </li><li>加入新鲜消化溶液（补充有0.2％2型胶原酶的DMEM）。在37℃下用香茅孵育2小时以35转/分钟搅拌。 </li><li>使用18 G针头研磨以均匀化混合物。然后，以500×g离心5分钟。弃去上清液，然后将沉淀重悬于0.25％胰蛋白酶/ EDTA。在37℃孵育10分钟。重复离心步骤两次。 </li><li>加入10 mL补充有20％胎牛血清（FBS）的DMEM溶液，并以500 xg离心5 min。弃去上清并将沉淀重悬于红细胞裂解缓冲液（155mM NH 4 Cl，10mM KHCO 3和0.1mM EDTA）中。 </li><li>以500 xg离心5分钟。弃去上清液并将沉淀物重新悬浮在分选缓冲液（20mM HEPES，pH 7.0; 1mM EDTA;和1％BSA，不含Ca / Mg 2+的 PBS，pH 7.0）中。通过40μm的细胞过滤器过滤混合物，以获得单细胞悬液。 </li><li>以500 xg离心5分钟。弃去上清液，并将沉淀重悬于1 mL分选缓冲液中。 </li><li>算了细胞数用血细胞计数器，并在分选缓冲液中将单细胞悬浮液稀释至5×10 6 / mL。 </li></ol>细胞染色和分选<ol><li>按照表1所述准备对照和样品。如表1所述，用冰上各自的抗体将单细胞悬浮液染色30分钟。 </li><li>以500×g离心5分钟，用分选缓冲液洗涤两次。 </li><li>将DAPI添加到单细胞溶液中，如表1所示。对于PDGFRβ-PE-FMO对照和样品，使用5μg/ mL DAPI（终浓度）作为DAPI单色对照和1μg/ mL DAPI。在整个实验过程中将所有管保持在冰上。 </li><li>打开分拣机和软件。提示时，扫描并插入100μm的分选芯片。 </li><li>执行自动设置（ 即，芯片对齐，液滴校准，侧流校准和排序延迟校准on）通过在提示时加载自动设置珠。 </li><li>自动设置完成后，进入"实验"选项卡，点击"新建"，从"公共模板"中选择"空白模板"。 </li><li>在"测量设置"下，输入"FL1"，"FL2"的"Nestin-GFP"和"FL3"的"PDGFRβ"的"DAPI"。取消选中"FL4" - "FL6"的框。 </li><li>勾选方框激活405,448和561激光，然后点击"创建新实验"。 </li><li> 选择"开始补偿向导"选项，并按照"补偿向导"软件提示设置补偿。 <ol><li>加载未染色的控件，然后单击"开始"。点击"检测器和阈值设置"，并调整FSC和BSC检测器的传感器增益，使人口达到规模。 </li><li>广告只是FL1-FL3荧光通道的增益水平将负个体放在直方图的左侧。点击"记录"按钮记录数据。 </li><li>出现提示时逐个加载单色控件。单击"开始和记录"以记录数据。调整直方图上阳性人群的门。点击下一步。" </li><li>转到"补偿"选项卡上的"计算矩阵"，然后单击"计算补偿设置"面板中的"计算"，执行补偿。单击"完成"退出"补偿向导"。 </li></ol></li><li>加载PDGFRβ-PE-FMO控件，然后单击"开始"。在"所有事件"图下的感兴趣单元周围绘制多边形门（门A）。 </li><li>双击门A内创建一个小孩绘图。将Y轴更改为DAPI并在活（DAPI 低 ）单元周围绘制多边形门（Gate B）。双击我N侧门B创建一个小孩情节。将X轴更改为FSC-H，将Y轴更改为FSC-W，并在单体周围绘制多边形门（Gate C）以消除双峰。 </li><li>双击门C内创建一个小孩图。将X轴更改为Nestin-GFP，将Y轴更改为PDGFRβ-PE。点击"记录"按钮记录数据。 </li><li>加载样品并重复步骤2.11-2.12。录制后，点击"暂停"保存样品。 </li><li>基于PDGFRβ-PE-FMO对照定义PDGFRβ +和Nestin-GFP +细胞的门控边界。绘制PDGFRβ + Nestin-GFP 和 PDGFRβ + Nestin-GFP +群体的门。 </li><li>在"排序方法"下，选择"双向管"，将"PDGFRβ + Nestin-GFP"和"PDGFRβ + Nestin-GFP + "细胞分配到左右收集管，spectively。将分选缓冲液填充的15 mL收集管装入收集台上，然后单击"加载收集"按钮。 </li><li>单击"恢复"按钮保持样品运行。单击"开始排序"以收集PDGFRβ + Nestin-GFP - （I型周细胞）和PDGFRβ + Nestin-GFP + （II型周细胞）细胞。 </li></ol>后排序分析<ol><li>将分选的细胞以500×g离心5分钟，将沉淀重悬于1mL周细胞培养基中（参见材料表 ），并使用血细胞计数器计数细胞密度。 </li><li>在约1×10 4个细胞/ cm 2的聚-D-赖氨酸（PDL）涂覆的盖玻片上的种子I型和II型周细胞。在37℃下用5％CO 2在周细胞培养基中生长3天。 </li><li>在第3天，使用荧光微观检查周细胞形态（相位对比）和内源性巢蛋白-GFP表达范围（激发激光:488nm，激发滤光片:470/40nm，发射滤光片:515 / 30nm）。以20X物镜（0.45 NA）拍摄图像。 </li><li>如前所述，分别用脂肪生成（小鼠MSC基础培养基+脂肪形成刺激性补充剂）和肌原性（DMEM + 2％马血清）培养基替代周细胞培养基，以启动脂肪形成和肌原性分化。每2-3天更换培养基。 </li><li>在室温下，在4％多聚甲醛（PFA）中，在17天（脂肪形成/肌原成分分化后14天）将细胞固定20分钟。 注意:注意，PFA是致癌物质。 </li><li> 如先前的出版物19,20所述，对Perilipin（脂肪细胞标记）和S-肌球蛋白（成熟肌管/肌纤维标记）进行免疫细胞化学。 <ol><li>在PBS中将固定细胞洗涤3次，室温10分钟。 </li><li>加入封闭缓冲液（PBS补充5％驴血清，3％BSA和0.3％Triton X-100），并在室温下孵育1小时。 </li><li>在4℃下将细胞与抗Perilipin（2μg/ mL）和/或抗S-肌球蛋白（2μg/ mL）抗体孵育过夜。 </li><li>在PBS中将细胞洗涤3次，室温10分钟。 </li><li>在室温下使用Alexa 555驴抗兔（4μg/ mL）和/或Alexa555驴抗小鼠（4μg/ mL）抗体孵育细胞1小时。 </li><li>在PBS中将细胞洗涤3次，室温10分钟。 </li><li>用含有DAPI的安装介质装载免疫染色细胞（参见材料表）。使用荧光显微镜（激发激光:543nm; 540 / 45nm激发和600/50nm发射）检查肾上腺素和S-肌球蛋白表达，并在40X物镜（0.60NA）下拍摄图像。 </li></ol></li></ol>

<ol>
	<li>Rennert, R. C., Sorkin, M., Garg, R. K., Gurtner, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+recruitment+after+injury:+lessons+for+regenerative+medicine.">Stem cell recruitment after injury: lessons for regenerative medicine.</a> Regenerative medicine. 7 (6), 833-850 (2012).</li><li>Sambasivan, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pax7-expressing+satellite+cells+are+indispensable+for+adult+skeletal+muscle+regeneration.">Pax7-expressing satellite cells are indispensable for adult skeletal muscle regeneration.</a> Development. 138 (17), 3647-3656 (2011).</li><li>Relaix, F., Zammit, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Satellite+cells+are+essential+for+skeletal+muscle+regeneration:+the+cell+on+the+edge+returns+centre+stage.">Satellite cells are essential for skeletal muscle regeneration: the cell on the edge returns centre stage.</a> Development. 139 (16), 2845-2856 (2012).</li><li>von Maltzahn, J., Jones, A. E., Parks, R. J., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pax7+is+critical+for+the+normal+function+of+satellite+cells+in+adult+skeletal+muscle.">Pax7 is critical for the normal function of satellite cells in adult skeletal muscle.</a> Proc Natl Acad Sci U S A. 110 (41), 16474-16479 (2013).</li><li>Lepper, C., Partridge, T. A., Fan, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+absolute+requirement+for+Pax7-positive+satellite+cells+in+acute+injury-induced+skeletal+muscle+regeneration.">An absolute requirement for Pax7-positive satellite cells in acute injury-induced skeletal muscle regeneration.</a> Development. 138 (17), 3639-3646 (2011).</li><li>Kuang, S., Charge, S. B., Seale, P., Huh, M., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+roles+for+Pax7+and+Pax3+in+adult+regenerative+myogenesis.">Distinct roles for Pax7 and Pax3 in adult regenerative myogenesis.</a> J Cell Biol. 172 (1), 103-113 (2006).</li><li>Murphy, M. M., Lawson, J. A., Mathew, S. J., Hutcheson, D. A., Kardon, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Satellite+cells,+connective+tissue+fibroblasts+and+their+interactions+are+crucial+for+muscle+regeneration.">Satellite cells, connective tissue fibroblasts and their interactions are crucial for muscle regeneration.</a> Development. 138 (17), 3625-3637 (2011).</li><li>Morgan, J. E., Pagel, C. N., Sherratt, T., Partridge, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+persistence+and+migration+of+myogenic+cells+injected+into+pre-irradiated+muscles+of+mdx+mice.">Long-term persistence and migration of myogenic cells injected into pre-irradiated muscles of mdx mice.</a> J Neurol Sci. 115 (2), 191-200 (1993).</li><li>Beauchamp, J. R., Morgan, J. E., Pagel, C. N., Partridge, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+myoblast+transplantation+reveal+a+discrete+minority+of+precursors+with+stem+cell-like+properties+as+the+myogenic+source.">Dynamics of myoblast transplantation reveal a discrete minority of precursors with stem cell-like properties as the myogenic source.</a> J Cell Biol. 144 (6), 1113-1122 (1999).</li><li>Partridge, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invited+review:+myoblast+transfer:+a+possible+therapy+for+inherited+myopathies.">Invited review: myoblast transfer: a possible therapy for inherited myopathies.</a> Muscle Nerve. 14 (3), 197-212 (1991).</li><li>Montarras, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+isolation+of+satellite+cells+for+skeletal+muscle+regeneration.">Direct isolation of satellite cells for skeletal muscle regeneration.</a> Science. 309 (5743), 2064-2067 (2005).</li><li>Asakura, A., Seale, P., Girgis-Gabardo, A., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myogenic+specification+of+side+population+cells+in+skeletal+muscle.">Myogenic specification of side population cells in skeletal muscle.</a> J Cell Biol. 159 (1), 123-134 (2002).</li><li>Tamaki, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle-derived+CD34+/45-+and+CD34-/45-+stem+cells+are+situated+hierarchically+upstream+of+Pax7++cells.">Skeletal muscle-derived CD34+/45- and CD34-/45- stem cells are situated hierarchically upstream of Pax7+ cells.</a> Stem Cells Dev. 17 (4), 653-667 (2008).</li><li>Dellavalle, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes+resident+in+postnatal+skeletal+muscle+differentiate+into+muscle+fibres+and+generate+satellite+cells.">Pericytes resident in postnatal skeletal muscle differentiate into muscle fibres and generate satellite cells.</a> Nat Commun. 2, 499 (2011).</li><li>Mitchell, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+a+non-satellite+cell+muscle+resident+progenitor+during+postnatal+development.">Identification and characterization of a non-satellite cell muscle resident progenitor during postnatal development.</a> Nat Cell Biol. 12 (3), 257-266 (2010).</li><li>Pannerec, A., Formicola, L., Besson, V., Marazzi, G., Sassoon, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+skeletal+muscle+resident+progenitors+and+their+cell+fate+potentials.">Defining skeletal muscle resident progenitors and their cell fate potentials.</a> Development. 140 (14), 2879-2891 (2013).</li><li>Dellavalle, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes+of+human+skeletal+muscle+are+myogenic+precursors+distinct+from+satellite+cells.">Pericytes of human skeletal muscle are myogenic precursors distinct from satellite cells.</a> Nat Cell Biol. 9 (3), 255-267 (2007).</li><li>Kostallari, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes+in+the+myovascular+niche+promote+post-natal+myofiber+growth+and+satellite+cell+quiescence.">Pericytes in the myovascular niche promote post-natal myofiber growth and satellite cell quiescence.</a> Development. 142 (7), 1242-1253 (2015).</li><li>Birbrair, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+pericytes+in+skeletal+muscle+regeneration+and+fat+accumulation.">Role of pericytes in skeletal muscle regeneration and fat accumulation.</a> Stem Cells Dev. 22 (16), 2298-2314 (2013).</li><li>Yao, Y., Norris, E. H., Mason, C. E., Strickland, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminin+regulates+PDGFRbeta(+)+cell+stemness+and+muscle+development.">Laminin regulates PDGFRbeta(+) cell stemness and muscle development.</a> Nat Commun. 7, 11415 (2016).</li><li>Armulik, A., Genove, G., Betsholtz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes:+developmental,+physiological,+and+pathological+perspectives,+problems,+and+promises.">Pericytes: developmental, physiological, and pathological perspectives, problems, and promises.</a> Dev Cell. 21 (2), 193-215 (2011).</li><li>Dore-Duffy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes:+pluripotent+cells+of+the+blood+brain+barrier.">Pericytes: pluripotent cells of the blood brain barrier.</a> Curr Pharm Des. 14 (16), 1581-1593 (2008).</li><li>Dore-Duffy, P., Katychev, A., Wang, X., Van Buren, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CNS+microvascular+pericytes+exhibit+multipotential+stem+cell+activity.">CNS microvascular pericytes exhibit multipotential stem cell activity.</a> J Cereb Blood Flow Metab. 26 (5), 613-624 (2006).</li><li>Birbrair, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+pericyte+subtypes+differ+in+their+differentiation+potential.">Skeletal muscle pericyte subtypes differ in their differentiation potential.</a> Stem Cell Res. 10 (1), 67-84 (2013).</li><li>Gautam, J., Nirwane, A., Yao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminin+differentially+regulates+the+stemness+of+type+I+and+type+II+pericytes.">Laminin differentially regulates the stemness of type I and type II pericytes.</a> Stem Cell Research &amp; Therapy. 8 (1), 28 (2017).</li><li>Birbrair, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes:+multitasking+cells+in+the+regeneration+of+injured,+diseased,+and+aged+skeletal+muscle.">Pericytes: multitasking cells in the regeneration of injured, diseased, and aged skeletal muscle.</a> Front Aging Neurosci. 6, 245 (2014).</li></ol>

所有作者没有利益冲突的披露。

周细胞是位于毛细血管21,26的外侧表面上的多能血管周围细胞22,23 。在骨骼肌中，周细胞能够沿着脂肪生成和/或造血途径19,20,24区分。最近的研究揭示了两个亚细胞周期细胞，具有不同的标记表达和不同的分化潜力19,24,25。 I型（NG2 + Nestin - ）周细胞是脂肪形成的，而II型（NG2 + Nestin + ）周细胞是肌原性的。这些亚群体外研究的分离需...

肌营养不良是一种肌肉退行性疾病，目前尚无有效治疗。促进组织再生的疗法的发展一直是非常有意义的。损伤后组织再生和修复取决于常驻干细胞/祖细胞1 。卫星细胞承诺造血前体细胞有助于肌肉再生2,3,4,5,6,7。然而，他们的临床使用受到其有限的迁移和注射后的低存活率以及在体外扩增后其分化能力降低8,9,10,11的阻碍。除了satellite细胞，骨骼肌也含有许多其他具有肌原性潜能12,13,14,15,16等细胞群，如血小板衍生生长因子受体-β（PDGFRβ） - 阳性间质细胞。有证据表明，肌肉衍生的PDGFRβ +细胞能够分化成肌原细胞，改善肌肉病理和功能14,17,18,19,20。 PDGFRβ主要标记周细胞21 ，它们是多能性的血管周围细胞22,23 。除PDGFRβ之外，许多其他标志物，包括神经元胶质细胞2（NG2）和CD146也被用于齿状细胞21 。然而，应该注意的是，这些标记物都不是percyte特异性21 。最近的研究揭示了肌肉周细胞的两种亚型，称为I型和II型，其表达不同的分子标记并实现不同的功能19,24,25。生物化学上，I型周细胞是NG2 + Nestin，而II型周细胞是NG2 + Nestin + 19,24。功能上，I型周细胞可能经历脂肪分化，有助于脂肪堆积和/或纤维化，而II型周细胞可以沿着肌原性途径分化，从而有助于肌肉再生19,24,25。这些结果表明:（1）I型周细胞可能be针对脂肪退化性疾病/纤维化的治疗，（2）II型周细胞对肌营养不良具有很大的治疗潜力。这些人群的进一步调查和表征需要能够以高纯度分离I型和II型周细胞的分离方案。 目前，周细胞亚群的分离取决于NG2-DsRed和Nestin-GFP双转基因小鼠19,24。 NG2-DsRed小鼠的可用性和大多数NG2抗体的质量限制了该方法的广泛使用。鉴于所有NG2 +周细胞也在骨骼肌19,20,24中表达PDGFRβ，我们假设NG2可被PDGFRβ替代以分离周细胞及其亚群。这项工作描述了基于FACS的协议使用PDGFRβ染色和Nestin-GFP信号。这种方法对于研究者来说要求较低，因为:（1）它不需要NG2-DsRed背景，（2）它使用市售的PDGFRβ抗体，其被良好表征。此外，它能够以高纯度同时分离I型和II型周细胞，从而进一步研究这些周细胞亚群体的生物学和治疗潜力。纯化后，这些细胞可以在培养物中生长，并且它们的形态可以被可视化。这项工作还表明，使用这种方法分离的I型和II型周细胞，如从双转基因小鼠纯化的那些，分别是脂肪形成和肌原性的。

<table><tbody><tr><td>Cell Sorter</td><td>Sony</td><td>SH800</td><td></td></tr><tr><td>Automatic Setup Beads</td><td>Sony</td><td>LE-B3001</td><td></td></tr><tr><td>DMEM</td><td>Gibco</td><td>11995</td><td></td></tr><tr><td>Avertin&amp;nbsp;</td><td>Sigma</td><td>T48402</td><td></td></tr><tr><td>Pericyte Growth Medium</td><td>ScienCell</td><td>1201</td><td></td></tr><tr><td>MSC Basal Medium (Mouse)</td><td>Stemcell Technologies</td><td>5501</td><td></td></tr><tr><td>Adipogenic Stimulatory Supplement (Mouse)</td><td>Stemcell Technologies</td><td>5503</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>16000</td><td></td></tr><tr><td>Horse Serum</td><td>Sigma</td><td>H1270</td><td></td></tr><tr><td>Collagenase Type 2</td><td>Worthington</td><td>LS004176</td><td></td></tr><tr><td>0.25% Trypsin/EDTA&amp;nbsp;</td><td>Gibco</td><td>25200</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco</td><td>15140</td><td></td></tr><tr><td>PDL</td><td>Sigma</td><td>P6407</td><td></td></tr><tr><td>PDGFR&amp;beta;-PE Antibody</td><td>eBioscience</td><td>12-1402</td><td></td></tr><tr><td>Perilipin Antibody</td><td>Sigma</td><td>P1998</td><td></td></tr><tr><td>S-Myosin Antibody</td><td>DSHB</td><td>MF-20</td><td></td></tr><tr><td>Alexa 555-anti-rabbit antibody&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>A-31572</td><td></td></tr><tr><td>Alexa 555-anti-mouse antibody</td><td>ThermoFisher Scientific</td><td>A-31570</td><td></td></tr><tr><td>Mounting Medium with DAPI</td><td>Vector Laboratories</td><td>H-1200</td><td></td></tr><tr><td>DAPI</td><td>ThermoFisher Scientific</td><td>D1306</td><td></td></tr><tr><td>HEPES</td><td>Gibco</td><td>15630</td><td></td></tr><tr><td>EDTA</td><td>Fisher</td><td>BP120</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A2058</td><td></td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;Cl</td><td>Fisher Scientific</td><td>A661</td><td></td></tr><tr><td>KHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Fisher Scientific</td><td>P184</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>14190</td><td></td></tr><tr><td>18 G Needles</td><td>BD</td><td>305196</td><td></td></tr><tr><td>10 mL Serological Pipette</td><td>BD</td><td>357551</td><td></td></tr><tr><td>OneComp eBeads</td><td>eBioscience</td><td>01-1111</td><td></td></tr></tbody></table>

isolation of type i and type ii pericytes from mouse skeletal muscles

周细胞是血管内多能细胞，其表现出不同器官甚至在相同组织内的异质性。在骨骼肌中，至少有两个周细胞亚群（称为I型和II型），其表达不同的分子标记并具有明显的分化能力。使用NG2-DsRed和Nestin-GFP双转基因小鼠，成功分离了I型（NG2-DsRed + Nestin-GFP）和II型（NG2-DsRed + Nestin-GFP + ）周细胞。然而，这些双转基因小鼠的可用性阻止了该纯化方法的广泛使用。这项工作描述了一种替代方案，允许从骨骼肌容易和同时隔离I型和II型周细胞。该协议采用荧光激活细胞分选（FACS）技术，并将靶蛋白GFP信号靶向PDGFRβ而不是NG2。隔离后，键入I和type II周细胞显示不同的形态。此外，用这种新方法分离的I型和II型周细胞，如分离自双转基因小鼠的那些，分别是脂肪形成和肌原性的。这些结果表明，该方案可用于从骨骼肌和可能来自其他组织中分离周细胞亚群。

FACS参数，包括激光强度和通道补偿，基于未染色控制和单色控制的结果进行校正。 PDGFRβ-PE-FMO对照用于设置PDGFRβ-PE +群体的门控（ 图1A ）。在PDGFRβ-PE-细胞中，代表巢蛋白-GFP +和巢蛋白 - GFP-细胞的两个群体被清楚地分离（ 图1A ）。基于PDGFRβ-PE +和巢蛋白-GFP +的门控设置PDGFRβ-PE +巢蛋?...

Watch this Scientific Journal Video about Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles at JoVE.com

Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles

这项工作描述了基于FACS的协议，允许从骨骼肌容易和同时地分离I型和II型周细胞。

从小鼠骨骼肌分离I型和II型周细胞

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at ...

isolation-of-type-i-and-type-ii-pericytes-from-mouse-skeletal-muscles

Research

JoVE Journal

Developmental Biology

9.1K 次观看.  University of Minnesota. 这项工作描述了基于FACS的协议，允许从骨骼肌容易和同时地分离I型和II型周细胞。 

视频: Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

从人类骨骼肌成肌原细胞和成纤维细胞的分离和定量免疫细胞化学表征

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

科研

发育生物学

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

小鼠骨骼肌肉外泄原发性肌基器的分离和分化

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...

Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal muscles regulating the exchange of fluids and nutrients as well as the immune response against infectious agents by controlling immune cell migration. For these functions, MMEC form a functional &#34;myovascular unit&#34; (MVU), with further cell types, such as fibroblasts, pericytes and skeletal muscle cells. Consequently, a dysfunction of MMEC and therefore the MVU contributes to a vast variety of myopathies. However, regulatory mechanisms of MMEC in health and disease remain insufficiently understood and their elucidation precedes more specific treatments for myopathies. The isolation and in-depth investigation of primary MMEC functions in the context of the MVU might facilitate a better understanding of these processes.
This article provides a protocol to isolate primary murine MMEC of the skeletal muscle by mechanical and enzymatic dissociation including purification and culture maintenance steps.

Microvascular endothelial cells of skeletal muscles (MMEC) shape the inner wall of muscle capillaries and regulate both, exchange of fluids/molecules and migration of (immune) cells between muscle tissue and blood. Isolation of primary murine MMEC, as described here, enables comprehensive in vitro investigations of the &#34;myovascular unit&#34;.

原代小鼠骨骼肌微血管内皮细胞的分离

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal ...

神经科学

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

单个肌纤维和成人骨骼肌卫星细胞的分离和培养

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

生物学

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

分离，培养和移植肌卫星细胞

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.26 that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 &#956;M in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

We have developed a novel and reproducible technique to isolate primary cultures of pulmonary artery smooth muscle cells (PASMC) from mice as young as P7, thereby allowing better study of the signaling pathways involved in neonatal smooth muscle cell contraction and relaxation.

肺动脉平滑肌细胞从新生小鼠隔离

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

Isolation and Purification of Murine Cardiac Pericytes

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier integrity. However, their tissue-specific functions in the heart are not well understood. Moreover, there is currently no protocol utilizing readily accessible materials to isolate and purify pericytes of cardiac origin. Our protocol focuses on using the widely used mammalian model, the mouse, as our source of cells. Using the enzymatic digestion and mechanical dissociation of heart tissue, we obtained a crude cell mixture that was further purified by fluorescence activating cell sorting (FACS) by a plethora of markers. Because there is no single unequivocal marker for pericytes, we gated for cells that were CD31-CD34-CD45-CD140b+NG2+CD146+. Following purification, these primary cells were cultured and passaged multiple times without any changes in morphology and marker expression. With the ability to regularly obtain primary murine cardiac pericytes using our protocol, we hope to further understand the role of pericytes in cardiovascular physiology and their therapeutic potential.

We have optimized a protocol to isolate and purify murine cardiac pericytes for basic research and investigation of their biology and therapeutic potential.

鼠心围细胞的分离与纯化

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier ...

医学

Single Myofiber Isolation and Culture from a Murine Model of Emery-Dreifuss Muscular Dystrophy in Early Post-Natal Development

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins that sustain the nuclear envelope and the components of the nucleoplasm. We recently reported that muscle wasting in EDMD can be ascribed to intrinsic epigenetic dysfunctions affecting muscle (satellite) stem cells regenerative capacity. Isolation and culture of single myofibers is one of the most physiological ex-vivo approaches to monitor satellite cells behavior within their niche, as they remain between the basal lamina surrounding the fiber and the sarcolemma. Therefore, it represents an invaluable experimental paradigm to study satellite cells from a variety of murine models. Here, we describe a re-adapted method to isolate intact and viable single myofibers from post-natal hindlimb muscles (Tibialis Anterior, Extensor Digitorum Longus, Gastrocnemius and Soleus). Following this protocol, we were able to study satellite cells from Lamin &#916;8-11 -/- mice, a severe EDMD murine model, at only 19 days after birth.
We detail the isolation procedure, as well as the culture conditions for obtaining a good amount of myofibers and their associated satellite-cells-derived progeny. When cultured in growth-factors rich medium, satellite cells derived from wild type mice activate, proliferate, and eventually differentiate or undergo self-renewal. In homozygous Lamin &#916;8-11 -/- mutant mice these capabilities are severely impaired.
This technique, if strictly followed, allows to study all processes linked to the myofiber-associated satellite cell even in early post-natal developmental stages and in fragile muscles.

Here, we propose a method to efficiently obtain single muscle fibers at early post-natal developmental stages from homozygous mutant Lamin &#916;8-11 mouse model, a very severe model for Emery-Dreifuss muscular dystrophy (EDMD).

单肌纤维隔离与文化从埃默里-德雷福斯肌肉萎缩的早期后纳塔尔发展穆林模型

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate ...

A High Output Method to Isolate Cerebral Pericytes from Mouse

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood brain barrier formation and physiology is now demonstrated but its molecular basis remains largely unknown. As the pathophysiological role of cerebral pericytes in neurological disorders is intriguing and of great importance, the in vitro models are not only sufficiently appropriate but also able to incorporate different techniques for these studies. Several methods have been proposed as in vitro models for the extraction of cerebral pericytes, although an antibiotic-free protocol with high output is desirable. Most importantly, a method that has increased output per extraction reduces the usage of more animals.
Here, we propose a simple and efficient method for extracting cerebral pericytes with sufficiently high output. The mouse brain tissue homogenate is mixed with a BSA-dextran solution for the separation of the tissue debris and microvascular pellet. We propose a three-step separation followed by filtration to obtain a microvessel rich filtrate. With this method, the quantity of microvascular fragments obtained from 10 mice is sufficient to seed 9 wells (9.6 cm2 each) of a 6-well plate. Most interestingly with this protocol, the user can obtain 27 pericyte rich wells (9.6 cm2 each) in passage 2. The purity of the pericyte cultures are confirmed with the expression of classical pericyte markers: NG2, PDGFR-&#946; and CD146. This method demonstrates an efficient and feasible in vitro tool for physiological and pathophysiological studies on pericytes.

We present a protocol for the extraction of murine cerebral pericytes. Based on an antibiotic-free enrichment oriented pericyte extraction, this protocol is a valuable tool for in vitro studies providing high purity and high yield, thus decreasing the number of experimental animals used.

一种从小鼠中分离大脑围取体的高输出方法

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood ...

Isolation of Quiescent Stem Cell Populations from Individual Skeletal Muscles

Skeletal muscle harbors distinct populations of adult stem cells that contribute to the homeostasis and repair of the tissue. Skeletal muscle stem cells (MuSCs) have the ability to make new muscle, whereas fibro-adipogenic progenitors (FAPs) contribute to stromal supporting tissues and have the ability to make fibroblasts and adipocytes. Both MuSCs and FAPs reside in a state of prolonged reversible cell cycle exit, called quiescence. The quiescent state is key to their function. Quiescent stem cells are commonly purified from multiple muscle tissues pooled together in a single sample. However, recent studies have revealed distinct differences in the molecular profiles and quiescence depth of MuSCs isolated from different muscles. The present protocol describes the isolation and study of MuSCs and FAPs from individual skeletal muscles and presents strategies to perform molecular analysis of stem cell activation. It details how to isolate and digest muscles of different developmental origin, thicknesses, and functions, such as the diaphragm, triceps, gracilis, tibialis anterior (TA), gastrocnemius (GA), soleus, extensor digitorum longus (EDL), and the masseter muscles. MuSCs and FAPs are purified by fluorescence-activated cell sorting (FACS) and analyzed by immunofluorescence staining and 5-ethynyl-2&#180;-deoxyuridine (EdU) incorporation assay.

This protocol describes the isolation of muscle stem cells and fibro-adipogenic progenitors from individual skeletal muscles in mice. The protocol involves single muscle dissection, stem cell isolation by fluorescence activated cell sorting, purity assessment by immunofluorescence staining, and quantitative measurement of S-phase entry by 5-ethynyl-2&#180;-deoxyuridine incorporation assay.

从单个骨骼肌中分离静止干细胞群

Skeletal muscle harbors distinct populations of adult stem cells that contribute to the homeostasis and repair of the tissue. Skeletal muscle stem cells ...