我们感激 Clemire Cimper 的出色技术支持。在实验室工作的资金由巴斯德研究所, 中心全国研究科学, 和国家研究 (Laboratoire 卓越复兴, Investissement d 艾文莉;ANR-10-LABX-73), 研究 (ANR-16-CE13-0017-01) 和基金会弧 (PJA 20161205028)。cc 和 ac 是由复兴财团的博士和博士后奖学金资助的。

动物按照欧洲共同体的准则和巴斯德研究所 (CETEA) 批准的议定书的伦理委员会处理. 
 1. 库存解决方案的准备工作 <ol> <li> 准备用于肌肉标本固定的材料。将0.5 克的粉胶与20毫升的水溶于 RT, 用于肌肉固定的冷冻埋入培养基. </li>
	<li> 准备 SA 和 #946 的解决方案;-Gal 染色. 
	<ol> <li> 通过在蒸馏水中溶解各自的粉末, 准备 k 3 Fe (cn) 6 (100 mm)、k 4 Fe (CN) 6 (100 mm)、氯化镁 2 (1M) 的库存解决方案. </li>
		<li> 准备 0.2% C 的库存解决方案 20 H 6 Br 4 Na 2 O 5 (曙红) 稀释它在水中。
		<li> 准备 c 14 H 15 BrClNO 6 (x gal) (50 毫克/毫升) 的库存解决方案, 方法是在 c 3 H 7 NO (胺, DMF) 中溶解 X gal 粉末. </li>
		<li> 存储 K 3 fe (cn) 6 和 k 4 fe (cn) 6 解决方案位于4和 #176; C 和氯化镁 2 在 RT 上. 
		注意: X gal 可存储在分-20 和 #176; C, 最多6月。红红溶液可在 RT 中保存, 必要时可在过滤后再使用。k 3 Fe (cn) 6 和 k 4 Fe (cn) 6 解决方案是 protectedfromthe 光。X gal 解决方案在水中不稳定, 必须保护不受光照. </li>
	</ol> </li> <li> Nanog 染色解决方案的准备: 性解决方案包含 0.1% Na 3 c 6 h 5 o 7 (柠檬酸三钠), 0.1% c 14 h 22 o (c 2 h 4 O) n (n = 9-10) (海卫 X-100) 在蒸馏水中, 应储存在4和 #176; c. 制备含有5% 胎牛血清 (FBS) 的含磷酸盐缓冲盐水 (PBS) 的阻断溶液, 应在 RT 中保存. </li>
</ol> 2。SA 和 #946;-Gal 染色在冰冻的 ta 肌肉部分 <ol> <li> 为 TA 肌肉准备固定材料, 将少量的粉胶放在软木片上. </li>
	<li> 受伤的小鼠 (2 月大, C57/B6) 在10天前与心脏 (CDX) 一起受伤, 如前面所描述的 13 。使用受害 (PBS 注入) TA 从同一个鼠标的负控制。如果 在体内 需要重新编程, 则在 Dox (1 毫克/毫升) 在饮用水中的同一天 (保护免受光照), 在酰损伤后. 
	注: Dox 解决方案需要每3天更换一次, 总处理时间为7天。
	<ol> <li> 将两个 TA 肌肉 (受伤和控制) 从小鼠中分离出来, 如前所述 ( 图 1A ) 13 。为了确保横断面, 将 TA 肌肉的远端肌腱插入粉胶中, 并 #190; 部分肌肉外 ( 图 1B ), 并直接冻结在液氮冷却戊的 #60; 1最小 
		注意: 确保 TA 肌肉处于垂直位置, 并在软木塞的中心。样品可存储在-80 和 #176; C 或直接 cryosectioned 在10和 #181; m 部分. </li>
	</ol> </li> <li> 处理如 图 1B 中所述的 TA 肌肉。
	<ol> <li> 将 1 st -10 th 节按正确的顺序分发到每张幻灯片左上角的十张不同的幻灯片上。将 11 th 节放在第一张幻灯片的 1 st 部分的旁边;除第二张幻灯片上的 2 nd 部分外, 12 th 节将按照相同的顺序放置。重复此过程直到10节/幻灯片获得十张幻灯片 (总共100节), 以确保第一节和最后一节之间的最小 1 mm 距离. </li>
	</ol> </li> <li> 在 PBS 中固定4分钟, 其中包含1% 甲醛和0.2% 戊二醛。用 PBS、2x 和 #160 冲洗; 10 分钟接下来, 在 PBS (pH = 5.5) 中孵育30分钟的部分, 执行 RT 中的所有步骤. 
	注意: 固定必须是温和的, 以保持酶活性。在引擎盖下面执行此步骤。PBS 的 pH 值是至关重要的, 必须保护 X gal 解决方案免受光照. </li>
	<li> 在 x gal 解决方案中孵育部分, 其中包含: 4 mm K3Fe (cn) 6 、4 mm K4Fe (cn) 6 、2 mm MgCl2 和400和 #181; PBS 中的 g/mL X gal, pH = 5.5。在黑暗中孵育37和 #176; C 至少24小时. 用 PBS 冲洗幻灯片, 3x 10 分钟, 在 RT 上. 
	注意: 孵化需要至少24小时, 并可持续48小时, 以最大化 SA 和 #946;-Gal 信号。该解决方案需要在24小时的孵化后改变。应保护幻灯片不受光照。如果只需要 SA 和 #946;-Gal 染色, 请继续执行步骤2.5。如果需要 co-staining 与 Nanog, 请跳到步骤 3.1. </li>
	<li> 在 pbs 中修复1% 甲醛中的幻灯片, 用 pbs 进行30分钟的洗涤, 3x 10 min. Counterstain 与0.2% 曙红。将这些幻灯片浸泡在红红溶液中1分钟, 并简单地用蒸馏水冲洗。最后, 用水性 non-fluorescing 安装介质安装幻灯片 (请参阅 材料表 ). 
	注意: 必须执行 post-fixation。在引擎盖下面执行此步骤。所有步骤都在 RT 上执行. </li>
</ol> 3。抗 Nanog 抗体的免疫组织化学研究 <ol> <li> 将带有 pbs 的4% 甲醛的幻灯片修复为 10 min. 2x 10 分钟添加200和 #181; 性溶液直接放在幻灯片上, 孵育5分钟。 <ol> <li> 用 pbs 清洗幻灯片, 2x 5 分钟, 最后一次洗涤, 使用200和 #181; L PBS 在幻灯片上直接包含 0.25% BSA。在 RT 上执行所有这些步骤. 
		注意: 在引擎盖下执行固定步骤. </li>
	</ol> </li> <li> 将具有主要抗 Nanog 抗体的幻灯片 (最终浓度: 1.25 微克/毫升) 过夜于4和 #176; PBS 中含有 5% FBS。用 pbs 清洗幻灯片, 2x 10 分钟, 最后一次洗涤, 使用200和 #181; L PBS 含有 0.25% BSA, 用于5分钟. 在 RT 上执行所有洗涤步骤. 
	注: 用湿纸巾在盒子里孵育幻灯片, 以防蒸发. </li>
	<li> 孵育100和 #181 的幻灯片; 从准备使用的工具包中的二次抗体的 L, (请参见 材料表 ) 为45分钟, 用 PBS 冲洗幻灯片, 3x 5 分钟, 去除二次抗体。在 RT 中执行所有步骤, 保护不受光照. </li>
	<li> 可视化 <ol> <li> 首先, 稀释33和 #39;-diaminobenzidine (c 12 h 14 N 4 c 12 h 18 Cl 4 N 4 4 HCl) 在基板缓冲区中由准备使用的工具包 (20 和 #181; L 为1毫升的基板缓冲液) 提供。在每张幻灯片上直接添加100和 #181, 并在 RT 中孵育最多10分钟. 
		注: 稀释后的民建联解决方案应该是新鲜的, 可以在4和 #176 的一周内储存。孵化时间可以调整到最小化背景信号, 但必须保持相同的所有幻灯片. </li>
		<li> 通过用水冲洗消除民建联的解决方案。Counterstain 快速红色解决方案 (准备使用, 请参阅 材料表 ) 的幻灯片, 20 分钟. 简要地再用水洗涤幻灯片. </li>
		<li> 脱水他们与95% 乙醇为 5 m其次是100% 乙醇, 2x 5 分钟。最后, 用快速硬化安装介质装载幻灯片 (请参阅 材料表 ). </li>
		<li> 在20X 的明亮场中观察显微镜下的幻灯片以避免背景. 
		注: 快速红色溶液可在 RT 中保存并在过滤后重复用. </li>
	</ol> </li> </ol> 4。分析和量化 <ol> <li> SA 和 #946;-Gal 量化 <ol> <li> 扫描幻灯片, 并根据获得的图像选择每个幻灯片上的最佳分区。使用至少2节/TA 的量化。根据组织完整性、染色质量和 counterstaining, 判断切片和 #39 质量。对于量化, 选择两个最高质量的部分, 最大可能的距离在样品之间, 这使得 SA 和 #946 更好的代表性;-Gal 量化整个 TA 肌肉. </li>
		<li> 量化 SA 和 #946;-Gal 阳性单元格 ( 图 2 )。
		<ol> <li> 通过手动选择最小和最大的正向 SA 和 #946;-Gal 单元格来确定像素大小的秩。
			<ol> <li> 使用 ImageJ 软件打开扫描的幻灯片的数字图像. </li>
				<li> 在界面中, 单击并 #39; 分析、#39、#62、#39、工具、#39、#62、#39、#39、#62、#39、#39、#62、#39、#39、#62 等; 选择区域和 #39;使用选择工具, 并围绕最小和最大的积极 SA 和 #946;-Gal 单元格, 并通过单击和 #39 将其添加到 ROI 管理器; 添加和 #39;。使用 and #39 测量大小; 测量和 #39; 按钮并保存这些值以备以后使用. </li>
			</ol> </li> <li> 调整阈值参数以确保选中所有可见的正单元格。
			<ol> <li> 通过单击和 #39; 图像和 #39; #62; #39; 类型和 #39; #62; #39; 8 位和 #39; ( 图 2A ), 将图像转换为灰度级。接下来, 去 #39; 图像和 #39; #62; #39; 调整 #39; #62; #39; 阈值和 #39;, 移动第二个游标, 直到所有正的 SA 和 #946;-Gal 单元格以红色覆盖 ( 图 2B ), 然后单击和 #39; 应用和 #39; </li>
				<li> 通过单击和 #39 分析粒子; 分析和 #39; #62; #39; 分析粒子和 #39; #62; #39; 大小 (pixel^2) 和 #39;, 并应用在步骤4.1.2.1 中定义的值, 输入和 #39; 循环和 #39;: #39; 0.00-1. 00 和 #39;, 单击和 #39; ok 和 #39;; 所有的摘要计数的粒子显示在 ROI 管理器中 ( 图 2D ). </li>
			</ol> </li> <li> 将所有选定的粒子转移到原始图像。手动调整选择以确保精确的量化。添加正单元格 (绿色箭头, 图 2E ) 和/或删除假阳性单元格 (红色箭头, 图 2E )。最后, 通过使用 and #39 对区域 ( 图 2D ) 进行分级, 从而测量其面积; 选择工具和 #39;。单击和 #39; ROI 管理器和 #39; #62; #39; 添加和 #39; #62; #39; 度量和 #39;. </li>
			<li> 重要的是, 按节的区域规范化正单元格的数目. </li>
		</ol> </li> </ol> </li> <li> Nanog 量化 <ol> <li> 在明亮的字段中, 在显微镜下以20X 倍的比例手动计数 Nanog 阳性细胞. 
		注: 快速红 counterstaining 可以很好地评价 TA 肌肉的形态。然而, 染色是非常轻的, 缺乏一个明确的部分轮廓。扫描仪通常无法识别剖面的边界, 无法正确对焦。在扫描之前, 可以使用标记来圈出剖面的边缘, 或者使用更灵敏的扫描仪来检测剖面。对于当前协议, 最有效的方法是手动计算 Nanog 的正单元格. </li>
	</ol>
	</li>
</ol>

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作者声明他们没有竞争的金融利益。

在这里, 我们提出了一种检测编程小鼠骨骼肌中的衰老和多能干细胞的方法。该方法可用于评价和量化衰老和诱导细胞可塑性在体内, 并检查衰老在组织修复和再生中的作用。
在目前的协议, 衰老相关的β-乳糖 (SA β加仑) 检测被用来探测体内衰老细胞的骨骼肌肉。这种检测检测到增加的溶酶体β-乳糖活性在 suboptimum ph 值 (6.0 或 5.5), 特别是与衰老细胞相关, 而这酵?...

细胞衰老是一种以稳定的细胞周期阻滞为特征的应激反应形式。在过去的十年中, 研究已经确定衰老与各种生物学和病理过程有关, 包括胚胎发育、纤维化和生物体老化1,2。细胞衰老首先被确定在人成纤维细胞在其复制寿命结束时由端粒缩短3触发。除了复制的压力, 还有许多其他刺激, 可以诱导衰老, 包括 DNA 损伤, 氧化应激, 致癌信号, 和基因组/表的改变, 其中任何可能最终激活 p53/p21 和/或 pRB 途径建立并加强永久性增长逮捕1。衰老细胞的一个重要特征是它们保持新陈代谢活性, 并表现出衰老相关的分泌表型 (SASP): 许多炎症细胞因子、生长因子和细胞外基质的分泌因素4。SASP 因子在调节和增强衰老作用方面起着重要作用, 因为它们对吸引免疫细胞和改变局部和全身组织环境1有很强的影响。有趣的是, 衰老最近被提出是重要的组织修复和再生5,6。此外, 包括我们在内的几个实验室的数据表明, 组织损伤导致的衰老可能增强细胞的可塑性, 通过 SASPs, 促进再生7-9。因此, 所有涌现的数据突出了研究衰老的重要性在体内。
在后诱导多能干细胞 (iPSC) 时代, 细胞可塑性是一个细胞获得一个新的身份和采取替代的命运时, 暴露在不同的刺激, 在文化和在体内10的能力。众所周知, 完全重新编程可以实现在体内11,12, 其中包含四 Yamanaka 因素的卡带的表达: Oct4, Sox2, Klf4, 和c-myc(OSKM) 可诱导体内促进多器官畸胎瘤的形成。因此, 一个编程的小鼠模型 (i4F) 可以被用来作为一个强大的系统, 以确定关键的调节器和路径, 是重要的细胞可塑性11。
一个合适的和敏感的在体内系统是必要的, 以了解如何细胞衰老调节细胞可塑性的背景下组织再生。在这里, 我们提出了一个健壮的系统和详细的协议, 以评估在组织再生的背景下衰老和细胞可塑性之间的联系。心脏 (酰) 诱导的肌肉损伤的胫骨前 (TA) 肌肉组, 一个行之有效的系统研究组织再生, 和 i4F 小鼠模型, 允许检测细胞衰老和在体内在肌肉再生过程中重新编程。
为了评估细胞可塑性和衰老之间的联系, i4F 小鼠受酰损伤, 用强力霉素 (0.2 毫克/毫升) 在7天内诱发急性肌肉损伤, 以诱导体内重编程。虽然酰引起的急性肌肉损伤和再生协议最近已发布13, 出于道德上的原因, 此过程将在当前协议中省略。TA 肌肉样本将收集在10天后受伤的13, 当衰老细胞的峰值已经被观察到之前,14。在这里, 这个详细的协议描述了所有的步骤来评估衰老的水平 (通过 SA β-加仑) 和重新编程 (通过 IHC 染色 Nanog)。
衰老相关的β-乳糖 (SA-β-加仑) 检测是最常用的检测方法, 可以在培养和体内15中发现衰老细胞。与其他检测方法相比, SA β-加仑法可以在其本机环境中使用完整的组织结构来鉴定衰老细胞, 这对体内研究尤为重要。此外, 它是可能的夫妇的 SA β加仑测定与其他标记使用 IHC。然而, SA β-加仑检测确实需要新鲜或冰冻样本, 这仍然是一个主要的限制。当新鲜或冰冻的组织是常规可用的, 如冰冻的 TA 肌肉样本, SA β-加仑显然是最合适的检测衰老细胞的方法。Nanog 是用于检测 reprogramed 细胞的标记, 有两个原因: 1) 是多的重要标志;2) 更重要的是, 它的表达不是由强力霉素 (dox) 驱动的, 因此它检测诱导的多而不是 Yamanaka 盒的强迫表达。
值得注意的是, 本研究中提出的染色协议可以单独进行, 以简化量化程序, 但也可以在 co-staining 过程中进行, 以在同一剖面上显示衰老和多能干细胞。

<table><tbody><tr><td>K&lt;sub&gt;3&lt;/sub&gt;Fe(CN)&lt;sub&gt;6&lt;/sub&gt;</td><td>Sigma</td><td>13746-66-2</td><td>For SA-&amp;beta; Gal staining solution</td></tr><tr><td>K&lt;sub&gt;4&lt;/sub&gt;Fe(CN)&lt;sub&gt;6&lt;/sub&gt;</td><td>Sigma</td><td>14459-95-1</td><td>For SA-&amp;beta; Gal staining solution</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>7786-30-3</td><td>For SA-&amp;beta; Gal staining solution</td></tr><tr><td>X-Gal</td><td>Sigma</td><td>B4252</td><td>For SA-&amp;beta; Gal staining solution</td></tr><tr><td>Doxycycline</td><td>Sigma</td><td>D3447</td><td>For inducing in vivo reprogramming</td></tr><tr><td>Cardiotoxin</td><td>Lotaxan Valence, France</td><td>L8102</td><td>For muscle injury</td></tr><tr><td>Glutaraldehyde</td><td>Sigma</td><td>111-30-8</td><td>For Fixation solution</td></tr><tr><td>Paraformaldehyde</td><td>Electron microscopy science</td><td>50-980-487</td><td>For Fixation solution</td></tr><tr><td>NaCitrate : &lt;br/&gt;Sodium Citrate monobasic bioxtra, anhydre</td><td>Sigma</td><td>18996-35-5</td><td>For permeabilization solution</td></tr><tr><td>Triton</td><td>Sigma</td><td>93443</td><td>For permeabilization solution</td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A3608</td><td>Washing solution</td></tr><tr><td>Antibody anti- Nanog</td><td>Cell signalling</td><td>8822S</td><td>Rabbit monoclonal antibody</td></tr><tr><td>EnVision+ Kits (HRP. Rabbit. DAB+)</td><td>Dako</td><td>K4010</td><td>For Nanog revelation</td></tr><tr><td>Eosin 1%</td><td>Leica</td><td>380159EOF</td><td>Counterstainning</td></tr><tr><td>Fast red</td><td>Vector Laboratories</td><td>H-3403</td><td>Counterstainning</td></tr><tr><td>Thermo Scientific Shandon Immu-Mount</td><td>Fisher scientific</td><td>9990402</td><td>Mounting solution</td></tr><tr><td>Quick-hardening mounting medium for microscopy : Eukitt&amp;reg;</td><td>Sigma</td><td>25608-33-7</td><td>Mounting solution</td></tr><tr><td>Microscope Phase Contrast Brightfield CKX41: 10X-20X-40X objectives</td><td>Olympus</td><td>CKX41</td><td>Microscope for Nanog quantification</td></tr><tr><td>Mouse: i4F-A</td><td>Abad et al., 2013</td><td>N/A</td><td>Reprogrammable mouse model</td></tr><tr><td>Skeletal muscle, Tibialis Anterior</td><td></td><td></td><td></td></tr><tr><td>Slide Scanner</td><td>Zeiss</td><td>Axio Scan Z1</td><td>slides scanning</td></tr></tbody></table>

evaluation of injury-induced senescence and in vivo reprogramming in the skeletal muscle

细胞衰老是一种应激反应, 其特点是稳定的细胞生长阻滞, 这是重要的许多生理和病理过程, 如癌症和衰老。最近, 衰老也牵涉到组织修复和再生。因此, 识别衰老细胞在体内变得越来越重要。衰老相关的β-乳糖 (SA β-加仑) 检测是最广泛使用的检测方法, 在培养和体内的衰老细胞。这种方法是基于在衰老细胞中增加溶酶体的含量, 这使得在 suboptimum pH 值 (6 或 5.5) 上, 组织化学检测溶体乳糖活性。与其他的检测方法, 如流式细胞仪, 这使得在其居住环境中的衰老细胞的鉴定, 这提供了宝贵的信息, 如与组织结构的位置, 形态学, 和通过免疫组织化学 (IHC) 与其他标记物耦合的可能性。对新鲜的或冰冻的样品的要求是对 SA β-加仑测定的主要限制。
在这里, 我们提出了一个详细的协议, 以了解如何细胞衰老促进细胞可塑性和组织再生在体内。我们使用 SA β-加仑来检测骨骼肌损伤后的衰老细胞, 这是一个行之有效的组织再生研究系统。此外, 我们使用 IHC 检测 Nanog, 一个多能干细胞的标志, 在转基因小鼠模型。该协议使我们能够检查和量化细胞衰老的背景下诱导细胞可塑性和体内重新编程。

肌损伤诱导细胞衰老的检测
最近已经证明, 肌肉损伤诱发短暂的细胞衰老14。在10天后 (DPI), 大部分受损的纤维正在进行再生与中央定位核, 一个标志, 再生纤维, 并重建肌肉的结构。在某些地区, 浸润性炎症细胞明显减少, 但仍可见。10 DPI 是一个很好的时间点来检测衰老细胞的 SA β-Gal, 因为有较少的坏死和炎症细?...

Watch this Scientific Journal Video about Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle at JoVE.com

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

骨骼肌损伤诱导衰老和体内重编程的评价

在这里, 我们提出了一个详细的协议, 以检测两个衰老和多能干细胞的骨骼肌肉损伤时, 诱导在体内重新编程。该方法适用于评价细胞衰老在组织再生过程中的作用和重新编程体内。

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

evaluation-injury-induced-senescence-vivo-reprogramming-skeletal

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

9.5K 次观看.  Institut Pasteur. 在这里, 我们提出了一个详细的协议, 以检测两个衰老和多能干细胞的骨骼肌肉损伤时, 诱导在体内重新编程。该方法适用于评价细胞衰老在组织再生过程中的作用和重新编程体内。

视频: Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced skeletal muscle regeneration is a widely used, powerful model system to study the events involved in muscle regeneration as well as the mechanisms and different players. Indeed, a detailed knowledge of this process is essential for a better understanding of the pathological conditions that lead to skeletal muscle degeneration, and it aids in identifying new targeted therapeutic strategies. The present work describes a detailed and reproducible protocol to induce acute skeletal muscle regeneration in mice through a single intramuscular injection of cardiotoxin (CTX). CTX belongs to the family of snake venom toxins and causes myolysis of myofibers, which eventually triggers the regeneration events. The dynamics of skeletal muscle regeneration is evaluated by histological analysis of muscle sections. The protocol also illustrates the experimental procedures for dissecting, freezing, and cutting the Tibialis Anterior muscle, as well as the routine Hematoxylin &#38; Eosin staining that is widely used for subsequent morphological and morphometric analysis.

This manuscript describes a detailed protocol to induce acute skeletal muscle regeneration in adult mice and subsequent manipulations of the muscles, such as dissection, freezing, cutting, routine staining, and myofiber cross-sectional area analysis.

通过注射心脏毒素急性骨骼肌再生的诱导

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

科研

发育生物学

Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting occurs. Loss of muscle mass and function is associated with muscle fiber type atrophy, fiber type switching, defective muscle regeneration associated with dysfunction of satellite cells, muscle stem cells, and other pathophysiological processes. These changes are associated with changes in intracellular as well as local and systemic niches. In addition to most commonly used rodent models of muscle ageing, there is a need to study muscle homeostasis and wasting using human models, which due to ethical implications, consist predominantly of in vitro cultures. Despite the wide use of human Myogenic Progenitor Cells (MPCs) and primary myoblasts in myogenesis, there is limited data on using human primary myoblast and myotube cultures to study molecular mechanisms regulating different aspects of age-associated muscle wasting, aiding in the validation of mechanisms of ageing proposed in rodent muscle. The use of human MPCs, primary myoblasts and myotubes isolated from adult and aged people, provides a physiologically relevant model of molecular mechanisms of processes associated with muscle growth, atrophy and regeneration. Here we describe in detail a robust, inexpensive, reproducible and efficient protocol for the isolation and maintenance of human MPCs&#160;and their progeny&#160;&#8212; myoblasts and myotubes from human muscle samples using enzymatic digestion. Furthermore, we have determined the passage number at which primary myoblasts from adult and aged people undergo senescence in an in vitro culture. Finally, we show the ability to transfect these myoblasts and the ability to characterize their proliferative and differentiation capacity and propose their suitability for performing functional studies of molecular mechanisms of myogenesis and muscle wasting in vitro.

This protocol describes a robust, reproducible and simple method of isolation and culture of myoblast progenitor cells from the skeletal muscle of adult and aged people. The muscles used here include foot and leg muscles. This approach enables the isolation of an enriched population of primary myoblasts for functional studies.

制备及文化活动从成年和老年人类骨骼肌成肌前体细胞/成肌细胞主要的

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting ...

Analyzing Satellite Cell Function During Skeletal Muscle Regeneration by Cardiotoxin Injury and Injection of Self-delivering siRNA In Vivo

Skeletal muscle possesses an enormous capacity to regenerate after injury. This process is mainly driven by muscle stem cells, also termed satellite cells. Satellite cells are characterized by the expression of the transcription factor Pax7 and their location underneath the basal lamina in the resting skeletal muscle. Upon injury, satellite cells get activated, undergo self-renewal or differentiation to either form new myofibers or to fuse with damaged ones. The functionality of satellite cells in vivo can be investigated using a cardiotoxin based injury model of skeletal muscle. To study the function of one gene during the regeneration of skeletal muscle, transgenic mouse models are mostly used. Here, we present an alternative method to transgenic mice, to investigate the gene function in satellite cells during regeneration, e.g., in cases where transgenic mice are not available. We combine the cardiotoxin mediated injury of a specific skeletal muscle with the injection of a self-delivering siRNA into the regenerating muscle which is then taken up by satellite cells among other cells. Thereby, we provide a method to analyze gene function in satellite cells during regeneration under physiological conditions without the need for transgenic mice.

We describe an in vivo method to investigate the functional loss of a specific gene in satellite cells by using a combination of cardiotoxin mediated injury of skeletal muscle and the injection of a self-delivering siRNA.

通过心肌毒素损伤和在 Vivo 中注射自传递 siRNA 在骨骼肌再生过程中的卫星细胞功能分析

Skeletal muscle possesses an enormous capacity to regenerate after injury. This process is mainly driven by muscle stem cells, also termed satellite ...

遗传学

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

中的应用<em&gt;在体内</em&gt;评定组织的鼠胫骨前的功能测试工程化骨骼肌修复

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

生物工程

Minimally Invasive Muscle Embedding (MIME) - A Novel Experimental Technique to Facilitate Donor-Cell-Mediated Myogenesis

Skeletal muscle possesses regenerative capacity due to tissue-resident, muscle-fiber-generating (myogenic) satellite cells (SCs), which can form new muscle fibers under the right conditions. Although SCs can be harvested from muscle tissue and cultured in vitro, the resulting myoblast cells are not very effective in promoting myogenesis when transplanted into host muscle. Surgically exposing the host muscle and grafting segments of donor muscle tissue, or the isolated muscle fibers with their SCs onto host muscle, promotes better myogenesis compared to myoblast transplantation. We have developed a novel technique that we call Minimally Invasive Muscle Embedding (MIME). MIME involves passing a surgical needle through the host muscle, drawing a piece of donor muscle tissue through the needle track, and then leaving the donor tissue embedded in the host muscle so that it may act as a source of SCs for the host muscle. Here we describe in detail the steps involved in performing MIME in an immunodeficient mouse model that expresses a green fluorescent protein (GFP) in all of its cells. Immunodeficiency in the host mouse reduces the risk of immune rejection of the donor tissue, and GFP expression enables easy identification of the host muscle fibers (GFP+) and donor-cell-derived muscle fibers (GFP-). Our pilot data suggest that MIME can be used to implant an extensor digitorum longus (EDL) muscle from a donor mouse into the tibialis anterior (TA) muscle of a host mouse. Our data also suggest that when a myotoxin (barium chloride, BaCl2) is injected into the host muscle after MIME, there is evidence of donor-cell-derived myogenesis in the host muscle, with approximately 5%, 26%, 26% and 43% of the fibers in a single host TA muscle showing no host contribution, minimal host contribution, moderate host contribution, and maximal host contribution, respectively.

Minimally Invasive Muscle Embedding MIME - A Novel Experimental Technique to Facilitate Donor-Cell-Mediated Myogenesis

We describe a novel experimental technique that we call Minimally Invasive Muscle Embedding (MIME), which is based on the evidence that skeletal muscle tissue contains viable myogenic cells that can facilitate donor-cell-mediated myogenesis when implanted into a host muscle.

微创肌肉植入 (MIME)--一种促进供体细胞介导的肌的新实验技术

Skeletal muscle possesses regenerative capacity due to tissue-resident, muscle-fiber-generating (myogenic) satellite cells (SCs), which can form new ...

医学

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and age-related muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration.

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Active autophagy is associated with productive muscle regeneration, which is essential for Muscle Stem Cell (MuSC) activation. Here, we provide a protocol for the in situ detection of LC3, an autophagy marker in MyoD-positive MuSCs of muscle tissue sections from control and injured mice.

肌肉干细胞中自噬的原位免疫荧光染色

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal ...

生物学

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells leave their quiescent state and activate the myogenic program ultimately leading to the repair of damaged tissue concomitant with the replenishment of the muscle stem cell pool. Various factors influence muscle stem cell activity, among them intrinsic stimuli but also signals from the direct muscle stem cell environment, the stem cell niche. The isolation and culture of single myofibers with their associated muscle stem cells preserves most of the interaction of the stem cell with its niche and is, therefore, the closest possibility to study muscle stem cell functionality ex vivo. Here, a protocol for the isolation, culture, siRNA transfection and immunostaining of muscle stem cells on their respective myofibers from mouse EDL (extensor digitorum longus) muscles is provided. The experimental conditions outlined here allow the study and manipulation of muscle stem cells ex vivo including investigation of myogenic activity without the inherent need for in vivo animal experiments.

In this protocol an in vitro culturing and functional analysis method for muscle stem cells is described, which preserves most of their interactions with their endogenous niche.

单一肌纤维培养测定法用于评估体外成人肌肉干细胞功能

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells ...

采集小鼠骨骼肌以培养干细胞及其生态位

Unfractionated Bulk Culture of Mouse Skeletal Muscle to Recapitulate Niche and Stem Cell Quiescence

Skeletal muscle is the largest tissue of the body and performs multiple functions, from locomotion to body temperature control. Its functionality and recovery from injuries depend on a multitude of cell types and on molecular signals between the core muscle cells (myofibers, muscle stem cells) and their niche. Most experimental settings do not preserve this complex physiological microenvironment, and neither do they allow the ex vivo study of muscle stem cells in quiescence, a cell state that is crucial for them. Here, a protocol is outlined for the ex vivo culture of muscle stem cells with cellular components of their niche. Through the mechanical and enzymatic breakdown of muscles, a mixture of cell types is obtained, which is put in 2D culture. Immunostaining shows that within 1 week, multiple niche cells are present in culture alongside myofibers and, importantly, Pax7-positive cells that display the characteristics of quiescent muscle stem cells. These unique properties make this protocol a powerful tool for cell amplification and the generation of quiescent-like stem cells that can be used to address fundamental and translational questions.

作者聚焦:使用生态位组分培养静止肌肉干细胞的离体方案 

Skeletal muscle comprises multiple cell types, including resident stem cells, each with a special contribution to muscle homeostasis and regeneration. Here, the 2D culture of muscle stem cells and the muscle cell niche in an ex vivo setting that preserves many of the physiological, in vivo, and environmental characteristics are described.

小鼠骨骼肌的普通散装培养物，以概括生态位和干细胞静止

Skeletal muscle is the largest tissue of the body and performs multiple functions, from locomotion to body temperature control. Its functionality and ...

骨骼肌损伤诱导衰老和体内重编程的评价

摘要

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此视频中的章节

通过注射心脏毒素急性骨骼肌再生的诱导

制备及文化活动从成年和老年人类骨骼肌成肌前体细胞/成肌细胞主要的

通过心肌毒素损伤和在 Vivo 中注射自传递 siRNA 在骨骼肌再生过程中的卫星细胞功能分析

中的应用

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