这项工作是由一个试点赠款, 从联盟的再生康复研究和培训 (AR3T) 和教员启动包从韦恩州立大学到 JAR。AR3T 由国家儿童健康和人类发展研究所 (研究院)、国家神经紊乱和中风研究所 (一) 和国家生物医学成像和生物工程研究所 (NIBIB) 支持。) 的国家卫生研究院奖编号 P2CHD086843。内容完全是作者的责任, 不一定代表国家卫生研究院的官方观点。

所有涉及活体动物的研究都由美国密歇根州立大学的机构动物护理和使用委员会 (IACUC) 批准, 并符合实验室动物看护和使用指南 (第八版, 2011, 出版的国家科学院出版社, 500 第五街, NW, 密码箱 285, 华盛顿, 华盛顿特区 20055, 美国)。根据批准的 IACUC 协议, 涉及疼痛和/或窘迫的程序在全身麻醉下进行, 这是由异氟醚吸入引起和维持的 (1.5-5%)。麻醉被证实由于缺乏撤退到脚趾捏, 并且缺乏眼睑或触须反应 (异氟醚的百分比增加, 因为需要保持效果)。由于协议的微创性, a 和 #34; 无菌提示和 #34; 技术被跟随。当动物被麻醉时, 用石油果冻来防止干燥。不需要特殊治疗;然而, 饮食凝胶是提供给动物的 24 h 以下程序, 涉及全身麻醉. 和 #160; 调查员被 IACUC 批准在哑剧之后停止镇痛, 因为该手术不涉及手术暴露宿主肌肉, 因为它是限制在一个后肢和不影响正常功能, 因为许多常见的镇痛药物是已知的影响正常肌肉再生。和 #160; 1。动物模型 <ol> <li> 宿主小鼠用于 MIME <ol> <li> 使用核荧光蛋白小鼠 (8-10 周, 男性) 作为肌肉移植研究的宿主. 
		注意: 这些小鼠是同种异体肌移植的理想宿主, 因为它们缺乏成熟的 T、B 淋巴细胞和功能性自然杀伤细胞, 在细胞因子信号传导方面存在缺陷, 并且被其他人用于异体或 xenografting 肌肉细胞。类 = "xref" > 14 。无处不在的 gfp 表达可以方便地识别寄主 (gfp +) 和捐赠细胞衍生 (gfp) 肌纤维. </li>
	</ol> </li> <li> 供小鼠用于 MIME <ol> <li> 使用 C57BL/6J 鼠 (12-16 周) 作为供鼠者. 
		注意: 这些老鼠是合适的供体小鼠, 因为他们有一个完全映射的基因组, 不知道有任何骨骼肌肉病理学, 是容易获得和经济的, 并且不表达 GFP. </li>
	</ol> </li> </ol> 2。准备供体肌肉组织 <ol> <li> 捆绑引导缝合和收割捐献者的 EDL 肌肉 <ol> <li> 安乐供者小鼠颈椎脱位和开胸手术在一般麻醉下执行的桌面由医用氧驱动的异氟醚蒸发器 (吸入异氟醚; 2-5% 到效果). </li>
		<li> 要访问 EDL 肌肉, 请使用一对外科剪刀打开腿部前表面的皮肤。找到前腿部的 ta 肌肉, 并将其移除, 以显示位于 ta 肌肉后面的 EDL 肌肉。在 edl 肌肉后面滑动一副镊子的尖端, 用轻柔的牵引来观察脚趾是否伸展 (这证实了肌肉是 edl). #160; #160; #160; #160; #160; #160; #160; #160; #160; #160; #160; #160; #160; #160;..。#160、#160、#160、#160、#160、#160、#160、#160、#160、#160、#160、#160、#160、#160; #160;...... #160、#160、#160、#160、#160、#160、#160、#160、#160、#160、#160、#160、#160、#160; #160; #160;.....。#160、#160、#160、#160、#160、#160、#160、#160、#160、#160、#160、#160、#160、#160; #160;...... #160、#160、#160、#160、#160、#160、#160、#160、#160、#160、#160、#160、#160、#160; #160; #160;.....。#160、#160、#160、#160、#160、#160、#160、#160、#160、#160、#160、#160、#160、#160; #160;...... #160、#160、#160、#160、#160、#160、#160、#160、#160、#160、#160、#160、#160、#160; #160; #160;.....。#160、#160、#160、#160、#160、#160、#160、#160、#160、#160、#160、#160、#160、#160; #160;...... #160、#160、#160、#160、#160、#160、#160、#160、#160、#160、#160、#160、#160、#160; #160; #160;.....。#160; #160; #160; #160; #160; #160; #160; #160; #160、#160、#160、#160、#160、#160、#160、#160、#160; 和 #160;、#160、、、、#160、#160、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、
		<li> 使用〜10厘米4-0 丝, 编织, 吸收, 无菌缝合线, 并使两个双结的指导缝合应用的近端和远端肌腱的 EDL 肌肉。( 图 1A ). </li>
		<li> 切断远端 edl 肌腱, 反射 edl 肌肉并切断近端肌腱. </li>
		<li> 将施主 EDL 肌肉放置在填充了鼠标铃声解决方案的培养皿中. </li>
	</ol> </li> </ol> 3。准备用于 MIME <ol> <li> 麻醉的宿主小鼠 <ol> 将宿主鼠标置于全身麻醉下 (吸入异氟醚; 1.5-5% 至效果) 由医用氧气驱动的台式异氟醚蒸发器进行管理. <li>
		<li> 在诱导室内进行麻醉, 然后将鼠标转移到鼻锥, 以维持麻醉, 同时对动物执行其他操作。提供热支持与恒温凝胶加热垫, 而动物是麻醉. </li>
	</ol> </li> <li> 皮肤准备 <ol> 要移除动物和 #39 的毛皮, 请在左后腿的前部涂上一脱毛霜。右腿作为后续过程的控制腿. </li>
		<li> 将脱毛霜放在2分钟内, 将脱毛奶油与分离的毛皮一起清除, 并将湿巾浸泡在磷酸盐缓冲盐水 (PBS) 中。经过毛皮去除, 擦洗皮肤与三交替湿巾的聚维酮碘洗涤液和70% 乙醇. </li>
	</ol> </li> <li> 在主机 ta 肌肉中创建针轨 <ol> <li> 通过18口径 (1 长) 的外科针通过主 ta 肌肉中心沿肌肉和 #39; s 长轴 ( 图 1B )。通过针在一个头的方向, 沿长度的 TA 肌肉, 并通过中心的肌肉腹部 ( 图 1B ). 
		注: 这一步的目的是创建一个针轨, 其中一块捐赠的肌肉可能被嵌入。将捐献者组织植入 ta 肌肉中心, 将有可能允许来自捐献组织的 SCs 迁移并促进肌整个宿主的肌肉. </li>
	</ol> </li> <li> 在主机 ta 肌肉的针轨中嵌入供体组织 <ol> <li> 一旦手术针在主 ta 肌肉内, 通过手术针的管腔在捐献者组织的一端通过引导缝合caudo 头方向 ( 图 1C ). </li>
		<li> 通过针迹绘制供体组织, 因为针头从 caudo 的主 TA 肌肉中取出. </li>
		<li> 在供体组织的尾部和头端使用引导缝合线, 以调整捐献组织的位置。确保供体组织位于宿主 TA 肌肉内 ( 图 1D ). </li>
<li> 一旦对供体组织放置进行了优化, 切断了导向缝合线, 用细尖钳对供体组织放置进行了最后的调整. </li>
		<li> 密封皮肤针伤口 ( 图 1E -f ), 用镊子逼近伤口边缘, 用27口径外科针的尖端使用兽医组织粘合剂. </li>
	</ol> </li> </ol> 4。肌注 Myotoxin 注射液诱导协同肌肉变性和再生 <ol> <li> 在 MIME 之后, 注入 50-60 和 #181; L 1.2% 氯化 2 (Myotoxin) 到宿主 TA 肌肉中, 以诱发肌肉纤维在宿主肌肉中的广泛损伤, 并在它内嵌入的捐赠组织 15 。
	<ol> <li> 将氯化 2 插入到3站点, 沿主机 TA 肌肉的长度 (近、中和远端1/3 的肌肉腹部). 
		注: 注射 myotoxins 像氯化 2 , cardiotoxins 和 notexin 成骨骼肌诱导协同肌肉纤维损伤, 然后再生 16 . </li>
	</ol> </li> </ol> 5。后程序动物护理 <ol> <li> MIME 和氯化 2 注入后, 清洗主机的鼠标和 #39; 左后腿与乙醇和应用一层薄薄的石油果冻, 以保护皮肤. </li>
	<li> 在 post-procedural 皮肤护理后, 从机头锥中取出宿主鼠标. </li>
	<li> 将鼠标放在不带寝具的单独恢复笼中。通过置于回收笼的一半以下的恒温凝胶加热垫, 为恢复中的动物提供热量支持. 
	注意: 一半不加热, 以便动物可能离开加热垫, 如果需要. </li>
	<li> 在宿主鼠完全从麻醉中恢复后, 将动物恢复到原来的笼子。将动物放回动物设施, 直到进行后续实验。每天监视宿主鼠标, 直到它准备好跟进和 #160; #8211; 示例: 在3或14天后 MIME。在哑剧之后, 动物由实验室人员以及兽医人员和 #8211 监测; 这主要涉及评估动物是否聪明、警觉和反应灵敏;还要评估动物是否显示出明显的疼痛迹象, 或者在移动、进食、饮水和美容方面有困难. </li>
</ol> 6。组织集合 <ol> <li> 收获主机肌肉 <ol> <li> 安乐由医用氧气驱动的台式异氟醚蒸发器在全麻下执行的颈椎脱位和开胸手术的宿主小鼠 (吸入异氟醚;2-5% 的效果). 和 #160; 研究人员还 IACUC 同意在安乐死之前, 在全身麻醉下收获 ta 肌肉. 和 #160; </li> <li> 若要访问主机 ta 肌肉, 请使用一对外科剪刀打开腿部前表面的皮肤。定位在前腿部的 TA 肌肉, 切断远端肌腱, 反映肌肉和削减近端肌肉附件 (见2.1 节). </li>
		<li> 收集左 (实验) 和右 (控制) TA 肌肉. </li>
	</ol> </li> <li> 和 #160; 对齐冻结收获的肌肉 <ol> <li> 将所收获的肌肉放在重纸和称重秤上称重. </li>
		<li> 简要地浸洗矿物油中的肌肉冻。把多余的油涂抹掉. </li>
		<li> 将肌肉放在铝箔上, 并将肌肉快速地浸泡在一个杜瓦瓶中的液氮中。将样品转移到标签 cryovials, 并将样品存放在-80 和 #176; C 冷冻以备日后研究之用. </li>
	</ol> </li> </ol> 7。组织学研究 <ol> <li> Cryosectioning 肌肉组织收集串行横截面 <ol> <li> 将样本从-80 和 #176; C 冷冻机放在含有液氮的杜瓦瓶中低温。
		<li> 在低温中, 一次处理一个示例。用一把冷剃刀刀片 (低温), 在 TA 肌肉的 mid-belly 上切割两次样品, 以产生大约 1-2 毫米厚的肌肉段。保持这个部分, 使低温部分, 并返回其余部分的肌肉, 以其 cryovial. </li>
		<li> 使用最佳切削温度 (OCT) 的冷冻化合物, 将组织的厚段固定在低温试样盘上。将试样盘放在标本架上. </li>
		<li> 粗糙-通过切割20和 #181; m 节来面对样品。收集一些20和 #181; m 部分的玻璃幻灯片为低温节. </li>
		<li> 如果20和 #181 的质量, m 部分是令人满意的, 改变切割厚度为5和 #181; m 和收集几个部分, 以评估质量。如果5和 #181; m 部分质量令人满意, 请收集串行部分。每张幻灯片收集 2 3 部分, 并为3张幻灯片收集足够的稿件. </li>
		<li> 收集每个动物的实验 (左) 和控制 (右) TA 肌肉的部分. </li>
	</ol> </li> <li> 研究 gfp 表达式 注: 指定一组准备好的幻灯片 (第7.1 节) 来研究肌纤维中 gfp 的表达。
	<ol> <li> 应用〜50和 #181; 防介质在剖面上, 并应用玻璃片。用清晰的指甲油封住片的边缘. </li>
		<li> 使用光和荧光显微镜 (参见 材料表 ), 研究具有10X 目标的剖面和用于可视化 GFP 的适当过滤器。
		<ol> <li> 收集重叠的可视字段的数字图像 (约 15), 以覆盖 TA 肌肉的整个横截面。通过在显微镜下从荧光到相衬模式的转换, 收集各视场的相衬图像. </li>
		</ol> </li> <li> 使用合适的映像软件打开图像, 可以平铺单个图像以生成单个复合图像; 例如 , 请使用 材料表 中列出的图像处理软件中的 "文件" 菜单下的 "#34;p hotomerge 和 #34" 选项. </li>
		<li> 使用合适的图像分析软件 ( 如 、ImageJ) 打开 GFP 荧光图像。用 #34; ROI 工具和 #34 对单个肌纤维进行循环, 并记录每个纤维的平均荧光强度。
		<ol> <li> 通过取显示高 gfp 表达的10纤维的平均荧光强度平均值来计算最大 gfp (荧光) 强度, 并具有正常的形态学。通过将每种纤维的平均花期强度除以 gfp 最大强度并乘以 100, 计算每种纤维的最大 gfp 强度。对每个 TA 肌肉的 GFP 分析结果进行制表, 如 图 3E 所示. </li>
		</ol> </li> </ol> </li> <li> 通过荧光蛋白和4和 #39 的标记来研究肌纤维完整性和 myonuclear 位置; 6-diamidino-2-吲 (DAPI), 分别. 
	注: 指定一组准备好的幻灯片, 如上图所示, 研究肌肉纤维中的蛋白标记。
	<ol> <li> 使用 pbs 中的2% 甲醛修复节10分钟. 使用 pbs 清洗该部分三次. 
		注意: 在处理甲醛时, 应穿戴适当的个人防护设备 (PPE). </li>
		<li> 在 PBS 中稀释3% 牛血清白蛋白, 含 0.01% Triton-X100 30 分钟。此阻塞解决方案称为以下主要稀释剂. </li>
		<li> 稀释兔 anti-desmin 免疫球蛋白 G (IgG) 在初级稀释剂 (1:200). </li>
		<li> 阻断完成后, 将稀释的主抗体涂抹在切片上, 然后在4和 #176 的冰箱中过夜; c. 用 PBS 清洗部分 (5 分钟), 其中含有0.1% 个海卫 X-100。此解决方案称为 "第一个洗涤缓冲区". </li>
		<li> 使用 PBS 清洗3次 (每次洗涤5分钟). </li>
		<li> 在含有0.01% 海卫 X-100 的 PBS 中, 通过稀释山羊抗兔 IgG (共轭红色荧光染料) 制备二次抗体. </li>
		<li> 将稀释的二级抗体应用于切片上, 并在室温 (〜23和 #176; C) 上孵育60分钟. </li>
		<li> 使用第一个洗涤缓冲区清洗分区 (5 分钟)。用 PBS 清洗3次 (每次洗涤5分钟). </li>
		<li> 应用 DAPI 3 分钟清洗3次 (每洗1分钟) 与 PBS。应用〜50和 #181; L 防培养基上的部分和应用玻璃片。用清晰的指甲油封住片的边缘. </li>
		<li> 使用光和荧光显微镜 (参见 材料表 ), 研究带有10X 目标的串行部分和用于可视化红色荧光染料的适当过滤器. </li>
		<li> 收集重叠视场的数字图像 (约 15), 以覆盖 TA 肌肉的整个横截面。通过切换到相应的筛选器设置, 收集每个可视字段的 DAPI 标记的荧光图像. </li>
		<li> 使用适当的成像软件打开和分析图像, 如步骤 7.2.3-7.2.4 中所述. 
		注意: 研究图像来识别, 哪些肌肉纤维被损坏 (蛋白阴性), 哪些肌肉纤维有中央成核 (蛋白正面纤维有 DAPI 标记的核位于内部而不是外围), 并且, 部分的区域有炎症和/或纤维化 (有许多 DAPI 标记的细胞核聚集在一起, 但缺乏肌肉纤维). </li>
	</ol>
	</li>
</ol>

<ol>
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作者没有相互竞争的金融利益。

在这里, 我们提出了一个详细的协议为新的实验技术称为哑剧, 开发在我们的实验室, 以植入供体肌肉组织的宿主肌肉组织。这是一个开放的肌肉移植技术的适应, 已经证明是有效的促进捐赠细胞介导的肌在主机肌肉9,10,17。
哑剧的目的是不使植入的捐赠肌肉组织本身的宿主肌肉 (我们目前不知道是否发生这种情况), 而是提供一个来源的 SCs, 可以促进肌在宿主肌肉的条件下, 刺激 muscle 再生。我们希望, 在基础和临床前研究中对 MIME 进行了优化和测试后, 它可以提供有价值的洞察力, 指导临床治疗, 以增加骨骼肌的肌肉再生, 这已经经历了肌力肌肉的丧失。
关于如何将 MIME 转化为临床治疗, 我们还有许多问题有待回答, 例如: 我们如何控制捐赠组织的质量？我们如何控制捐赠组织和细胞的免疫排斥？捐献的组织在为肌提供细胞后是否被清除, 还是留下了纤维化的疤痕？纤维类型的供体和/或宿主肌肉影响捐赠细胞介导的肌？哪些肌肉能从哑剧中获益？我们目前正在扩大我们的研究, 以评估是否 MIME 是安全和有效的, 找出辅助治疗, 可以增加捐助者派生的肌, 并回答上述的许多问题。
在完成我们的测试 mouse-to-mouse 异体移植与哑剧, 我们下一步是执行 human-to-mouse 的哑剧与尸体人体组织, 以评估的肌源性潜力的尸体肌肉组织。我们预计, 这些实验将导致一个新的基础和转化研究, 其中包括来自捐赠者的肌肉组织, 谁是注册的倡议, 如身体遗赠计划的教育和生命的礼物计划捐赠器官.
这份手稿中的代表性数据表明, 在14天后的 MIME, 有几个可行的纤维, 要么缺乏 gfp 或低含量的 gfp。我们对这些数据的解释是, GFP-施主卫星细胞促成了宿主肌肉中的肌。我们进行了这项实验, 准备将人类尸体组织植入宿主鼠的肌肉中。为了明确地证明, 捐献的卫星细胞在肌后的宿主肌肉中起到了促进作用, 因此, 植入表达荧光报告的捐献组织是很有用的, 它可以很容易地与 GFP 区别开来 (例如,红色荧光蛋白, 表达供体组织植入 GFP + 宿主肌)。
这种技术主要是受捐赠组织中的 SCs 的性质的限制。如果供体组织中的 SCs 是可行的, 该技术很可能促进供体细胞介导的肌, 而如果它们不可行, 肌就无法发生。然而, 由于 SCs 是非常有弹性的, 并且在大约2周后是可行的, 因此很有可能是为了实验目的, 在收获后几分钟内植入的捐赠组织将促进供体细胞介导的肌13.此外, 如上所述, 这是可能的, 因为整个肌肉组织 (包括 SCs, 成熟的肌肉纤维, 以及肌肉驻留成纤维细胞) 嵌入到宿主肌肉, 纤维化可能发生。然而, 这一假设需要经过实证检验。关于损伤性收缩、冷冻和实验 myotoxins 所引起的肌肉损害的文献表明, 免疫细胞 (主要是噬细胞) 能够有效地清除受损纤维中的细胞碎片并重塑胞外基质18。因此, 在 MIME 和氯化2注入后, 只要宿主免疫细胞能够接触到退化的供肌纤维, 它们就可以清除碎片, 只留下捐献者 SCs。最后, 供体来源的肌的程度取决于在宿主肌肉中嵌入的供体肌肉组织的数量。为了使宿主肌室完全重新由供体细胞衍生的纤维, 它需要一个方法, 如 X 或伽玛辐照蚀主机肌肉 SCs, 也需要重复的 MIME 程序。
已经证明, 手术暴露的主机肌肉和缝合一块捐赠组织的主机肌肉可以促进捐赠细胞介导的肌9,10,17。这种开放式的手术方法已经被用来追踪肌的进展, 并产生人类肌肉疾病的小鼠模型。MIME 技术的创新之处在于它是微创的, 不涉及手术暴露宿主肌肉。这降低了医源性感染的风险和宿主动物的不适程度, 因此, 如果需要的话, 在同一宿主肌肉上重复执行 MIME 更可行。
我们预计, MIME 技术可能是一个适当的细化, 开放的手术方法, 目前正在采取的植入供体肌肉组织的宿主鼠标。通过在宿主小鼠肌肉中嵌入活检的人的肌肉, 这可以加快产生人性化的小鼠模型。此外, 根据我们的初步数据从 mouse-to-mouse 嫁接, 我们预计, MIME 将是有效的, 以获得捐助者细胞介导的肌从人类尸体捐献组织, 只要捐助者 SCs 是可行的。最后, 通过额外的测试和验证, 我们希望, MIME 技术将有助于开发新的疗法, 以促进供体细胞介导的肌在人类肌肉疾病。
成功的 MIME 技术是关键依赖于准确地嵌入的捐赠组织在筋膜室 (epimysium) 的主机肌肉。只有当捐献者的组织被放置在寄主的肌肉舱内, 它才能够以精确的方式向宿主肌肉提供 SCs。如果捐献者的组织被放置在宿主肌肉之外, 那么它的命运还不清楚。在我们的哑剧实验模型中, 我们使用 TA 肌肉作为宿主肌肉, 因为它是突出的, 表面上放置在腿的前外侧。ta 肌肉的大小, 方向和解剖位置, 使它很容易证实, 捐赠组织是正确地放置在主机 ta 肌肉后哑剧。在本文中, 我们提供了实验证据, 供体组织 re在主机 TA 肌肉内嵌入的电源在3天后, 哑剧, 并有捐助者细胞介导的肌在主机肌肉在14天后, 哑剧。

健康的骨骼肌肉, 即使有丝分裂, 具有优良的再生能力, 由于存在组织驻留的肌细胞 (SCs)1,2;并在3,4中进行了审阅。然而, 在肌肉营养, 创伤或加速老化引起的病理条件下, 肌肉的再生可能与肌肉破裂不匹配, 因此, 渐进性肌纤维丢失发生在5。虽然已经开发出有效的方法来分离肌肉中的 SCs 和扩大他们的文化, 以产生大量的成肌细胞 (和随后管), 试图产生生理相关的肌肉纤维在主机肌肉有仅取得了最小的成功6。与许多其他细胞类型一样, 当成肌细胞被单独注射到宿主组织中时, 大多数单元格不嫁接7,8。我们已经表明, 神经肌肉电刺激 (NMES) 促进供体细胞衍生肌在再生不足的宿主小鼠肌肉注射与人成肌细胞8。其他人已经证明, 手术移植的活检肌或单肌纤维与连接的 scs, 促进适度肌即使没有 NMES, 建议植入全肌纤维与 scs 可能比植入更有利单肌细胞9,10。由于移植到宿主肌肉中的供体或工程化肌肉组织产生的结果比单独移栽细胞的效果好, 所以组织或组织样结构可能为供体细胞植入提供关键的线索;一个概念, 这是越来越明显的细胞治疗研究涉及各种细胞类型10,11,12。
最近的数据表明, 从人类获得的 SCs 超过2周后宰后生成管在文化13。因此, 我们打算评估, 如果植入的肌肉组织被收割后的活体主机将扭转肌肉纤维的损失。我们开发了一种新的技术叫哑剧, 将捐献者的肌肉组织植入活寄主的骨骼肌组织中, 以促进供体细胞衍生的肌。哑剧包括通过一根外科针通过宿主肌肉来创建一个针迹;通过针迹绘制一小段供体肌肉组织;将供体组织留在宿主肌肉中;用纸巾把针孔关上在其他实验中进行的安乐死小鼠的技术练习后, 我们已经在缺陷和无所不在表达绿色荧光蛋白 (GFP) 的活鼠中进行了哑剧, 并在3和14天后随访。在3天后的 MIME, 我们确认, 捐赠鼠 (gfp) EDL 肌肉植入宿主鼠 (gfp +) TA 肌肉, 仍然嵌入在主机肌肉。在14天后的 MIME, 在氯化2 myotoxin 损伤引起损伤和肌后, 我们确认在单个主机 TA 肌肉中, 大约5%、26%、26% 和43% 的纤维显示没有主机贡献, 最小主机贡献, 适度主机贡献, 和最大的主机贡献, 分别。在 myotoxin 注射后的大约95% 的 TA 肌纤维中, 出现了中央核化 (退化和随后再生的标志)。
我们研究的 TA 肌肉14天后, MIME + 氯化2 , 因为这个时间点捕捉再生的中间阶段, 当大多数再生纤维是中央有核的。我们研究 GFP + 小鼠作为宿主的哑剧, 因此, 当我们最终移植到宿主鼠的尸体肌肉, 我们将能够很容易地区分寄主和捐献者的肌肉纤维。我们使用鼠 EDL 肌肉作为实验性的供体组织, 因为从这个肌肉的单纤维显示出比 TA 肌肉更大的肌电位9。EDL 肌肉也与 TA 肌肉协同, 具有类似的纤维型成分。我们的初步数据表明, MIME 能够促进宿主肌肉中供体细胞衍生的肌。

<table><tbody><tr><td>NSG-GFP mice</td><td>The Jackson Laboratory (Bar Harbor, ME)</td><td>Stock #021937</td><td>Immunodeficient host mice that ubiquitously express green fluorescent protein</td></tr><tr><td>C57BL/6J mice</td><td>The Jackson Laboratory (Bar Harbor, ME)&amp;nbsp;</td><td>Stock #000664</td><td>Control donor mice</td></tr><tr><td>Tabletop isoflurane vaporizer&amp;nbsp;</td><td>VetEquip (Livermore, CA)</td><td>Item #901801</td><td>Inhaled anesthesia system</td></tr><tr><td>Magic depilatory crea</td><td>Softsheen Carson (New York, NY)</td><td>N/A</td><td>Razorless hair removal cream</td></tr><tr><td>4-0 Silk, black, braided, non-absorbable sutures</td><td>Roboz Surgical Instrument Co, Inc. (Gaithersburg, MD)</td><td>SUT106631</td><td>Guiding sutures for donor tissue</td></tr><tr><td>VetBond veterinary tissue adhesive</td><td>3M (Maplewood, MN)</td><td>Catalog #1469Sb</td><td>Veterinary tissue adhesive for sutureless skin closure</td></tr><tr><td>Barium chloride&amp;nbsp;</td><td>Ricca Chemical Company (Arlington, TX)</td><td>Product #R0854000-500A 854-16&amp;nbsp;</td><td>Myotoxin to induce muscle damage and stimulate regeneration</td></tr><tr><td>Deltaphase isothermal gel heating pad&amp;nbsp;</td><td>Braintree Scientific (Braintree, MA)</td><td>&amp;nbsp;Item #39DP</td><td>Heating pad to provide thermal support to animals while under anesthesia</td></tr><tr><td>HM525NX cryostat&amp;nbsp;&amp;nbsp;</td><td>ThermoFisher (Waltham, MA)</td><td>Catalog #HM525NX&amp;nbsp;</td><td>Cryostat to make frozen sections of muscle</td></tr><tr><td>Vectashield Antifade Medium</td><td>Vector Laboratories, Inc. (Burlingame, CA)</td><td>Catalog Number: H-1000</td><td>Antifade medium to preserve fluorescence in immunofluorescently labeled samples</td></tr><tr><td>Rabbit anti Desmin Antibody</td><td>Labvision Thermo Scientific (Fremont, CA)</td><td>RB9014P</td><td>Rabbit anti desmin antibody for desmin immunofluorescent labeling</td></tr><tr><td>Goat anti Rabbit Alexa 568 Antibody</td><td>ThermoFisher (Waltham, MA)</td><td>Catalog#: A-21069</td><td>Goat anti rabbit secondary antibody for desmin immunofluorescent labeling</td></tr><tr><td>4&#039;,6-diamidino-2-phenylindole (DAPI)&amp;nbsp;</td><td>Seracare (Milford, MA)</td><td>Catalog #5930-0006 or 71-03-01</td><td>Reagent for fluorescent labeling of nuclei</td></tr><tr><td>Axio Scope.A1 microscope</td><td>Carl Zeiss (Peabody, MA)&amp;nbsp;</td><td>Product #Axio Scope.A1</td><td>Light and fluorescence microscope</td></tr><tr><td>Photoshop CS4</td><td>Adobe Systems (San Jose, CA)</td><td>Photoshop CS4</td><td>Imaging Software for perform image tiling</td></tr><tr><td>Image J</td><td>National Institutes of Health (Bethesda, MD)</td><td>Image J for Windows 64-bit Operating System</td><td>Imaging Software for quantitative fluorescence analysis</td></tr></tbody></table>

minimally invasive muscle embedding (mime) - a novel experimental technique to facilitate donor-cell-mediated myogenesis

骨骼肌具有再生能力, 由于组织驻留, 肌肉纤维生成 (肌) 卫星细胞 (SCs), 可以形成新的肌肉纤维在适当的条件。虽然 SCs 可以从肌肉组织和培养的体外, 产生的肌细胞在促进肌移植到宿主肌肉时并不十分有效。手术暴露的宿主肌肉和移植部分的捐赠肌肉组织, 或独立的肌肉纤维与他们的 SCs 到主机肌肉, 促进更好的肌相比, 肌移植。我们开发了一种新的技术, 我们称之为微创肌肉植入 (MIME)。哑剧包括通过一根外科针通过宿主肌肉, 通过针迹来绘制捐献者的肌肉组织, 然后将捐献者的组织植入宿主的肌肉中, 这样它就可以充当宿主肌肉的 SCs 的来源。在这里, 我们详细描述了在缺陷鼠标模型中执行 MIME 所涉及的步骤, 它在所有的细胞中表达绿色荧光蛋白 (GFP)。宿主小鼠的免疫缺陷降低了供体组织免疫排斥的风险, 而 gfp 表达可以方便地识别宿主肌纤维 (gfp +) 和供体细胞来源的肌纤维 (gfp)。我们的试验数据表明, MIME 可以用来植入一个伸肌腱长长 (EDL) 肌肉从一个供鼠到胫骨前 (TA) 肌肉的宿主鼠标。我们的数据还表明, 当 myotoxin (氯化钡, 氯化2) 被注射到宿主肌肉后, MIME, 有证据表明, 捐赠细胞衍生的肌在主机的肌肉, 约 5%, 26%, 26% 和43% 的纤维在一个单一的主机 TA肌肉显示没有主机的贡献, 最低的主机贡献, 适度的主机贡献, 和最大的主机贡献, 分别。

在3天后 mime 中, 由 mime 植入的施主 EDL 包含在主机 TA 肌肉舱内 (图 2A-C)。不出所料, 在荧光光学下研究的供体小鼠的 TA 肌肉的横断面不显示绿色荧光, 因为它们不表达 GFP (图 2D)。与此相反, 来自宿主小鼠的 ta 肌肉的横断面不与供体组织植入, 在 ta 肌肉纤维中显示出均匀的绿色荧光, 就像光纤表达 GFP (图 2E)。在由供体肌肉组织的哑剧中植入的肌肉的横断面上, 有一条在寄主肌纤维 (gfp +) 和供肌纤维 (gfp) 之间的分界线。图 2D-f中显示的可视字段的相位对比度图像出现在图 2E"f中, 表明在没有 GFP 的区域中确实存在肌纤维。信号.
在14天后的 MIME 和氯化2注入, 通过研究左无标记或标记为蛋白 (图 3) 的 TA 肌肉的串行横截面, 我们得知 GFP 信号是由所有的肌肉纤维表达的未经治疗的肌肉从宿主小鼠, 但不是在 MIME 治疗的肌肉 (红色蛋白标记检测到可行的肌肉纤维)。在使用 MIME 和氯化2注射液治疗的宿主 TA 肌肉中, 供体细胞衍生的肌在许多蛋白 (+) 肌纤维存在的情况下是明显的, 显示没有可检测的 GFP 信号 (图 3CD, 3 C " -D")。由宿主和供体肌细胞融合而产生的嵌合肌纤维可由这些纤维所展示的低至中度的 GFP 荧光检测到。大量的嵌合纤维出现在 TA 肌肉的整个直径上, 这意味着捐献者 SCs 能够在寄主肌肉的 epimysium 中迁移几百微米。荧光蛋白 + 纤维的定量显示在图 3E中。图 4显示了整个 MIME + 氯化2处理的 TA 肌肉的连续剖面的高放大图像。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55731/55731fig1.jpg"> 
图 1.介入微创肌肉植入 (MIME) 的步骤。 哑剧是在全身麻醉下进行的。它涉及在林格溶液中放置〜10毫克的供体肌 (供鼠 EDL), 并将导向缝合线与其两端(A)捆绑在一起。一个18口径的针通过长轴的主机肌肉(B)和捐赠组织是通过针轨道绘制的(C)。供体组织留在宿主肌肉(D)中, 切割导缝线, 针孔用组织粘合剂 (E)密封。密封针伤口用箭头(F)表示。<a href="http://ecsource.jove.com/files/ftp_upload/55731/55731fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55731/55731fig2.jpg"> 
图 2.研究的 TA 肌肉3天后, 哑剧。 在哑剧后3天收集的主机肌肉;注意主 TA (箭头上的针痕;A-b)。数据进一步确认嵌入的供体鼠标 EDL 位于 TA 肌肉舱内 (B-C)。ta 肌肉横断面的荧光图像在野生型 ta 肌(D)中无绿色荧光, 在核型细胞中的亮绿色荧光-gfp ta 肌(E), 并在3天内将寄主和捐献者的肌肉区别开来后 MIME (F)。d-f 中的视觉场的相衬图像分别显示在 d-f 中。"f" 和 "f" 面板中的红线显示了宿主和供体组织之间的分界线。缩放栏 = 50 µm.<a href="http://ecsource.jove.com/files/ftp_upload/55731/55731fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55731/55731fig3.jpg"> 
图 3.研究的 TA 肌肉14天后, 哑剧。 收集了14天后的数据。红色信号是从荧光标记蛋白 (一个正面信号为蛋白表示, 纤维是可行的), 蓝色信号是从 DAPI (污点核), 并且绿色信号是从 GFP。在控制 TA 肌肉 (右后肢) 从宿主鼠收获, 如预期, 几乎所有的纤维是积极的蛋白, 建议这些纤维是可行的 (A; 红色信号)。此外, 根据 DAPI 染色, 很明显, myonuclei 几乎所有的纤维都是外围定位, 预计在健康的肌肉 (A; 蓝色信号)。控制 TA 肌肉的连续部分显示, 几乎所有的肌肉纤维是明亮的绿色, 暗示他们是正面的 GFP。在 MIME + 氯化2治疗的 TA 肌肉 (左后肢), 捐助者细胞介导的肌是明显的存在, 许多蛋白 (+) 肌肉纤维, 显示没有可检测的 GFP 荧光 (c-d, c "-d")。蛋白 (+) 肌纤维, 低至中度 GFP 荧光 (嵌合肌纤维) 的整个 ta 肌肉的直径, 表明, MIME 提供的捐助者 SCs, 可以迁移在 epimysium 的 ta 肌肉和促进供体细胞衍生的肌。"-d"是a-d中的蓝色框内区域的高倍图像。在E中显示了 GFP (+) 纤维和中央有核纤维的定量。缩放条形图 = 100 µm (a-d) 和50µm (a "-d")。<a href="http://ecsource.jove.com/files/ftp_upload/55731/55731fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" s alt="Figure 4" class="xfigimg"></img alt="Figure 4" class="xfigimg" s>rc = "/文件/ftp_upload/55731/55731fig4. jpg"/> 
图 4.在 MIME 和氯化2注入后14天内广泛损坏和再生的证据。 高放大率, 整个 MIME + 氯化2处理的 TA 肌肉的串行横截面。数据显示, 许多肌纤维, 适度或强烈的 GFP + 有中央定位核 (a-b; 红色信号在 a 是从蛋白, 蓝色信号在 a 是从 DAPI, 并且绿色信号在 B 是从 GFP)。这些数据表明, 氯化2注入后的变性和再生不影响 GFP 表达式。在这种情况下, 在 MIME + 氯化2中有中心有核的肌纤维的存在治疗 TA 肌肉, 与到 GFP 表达, 表明这些纤维是可能的结果, 肌 (或再生) 有重大贡献GFP-肌细胞。这些观测可以在所选字段的高倍放大图像中进一步验证 (C-f;C 和 D、E 和 F 分别是从连续剖面上的重叠区域;图像边框的颜色编码表示这些区域在 A 和 B 中的位置。缩放条形图 = 100 µm (A-B) 和50µm (C-f)。<a href="http://ecsource.jove.com/files/ftp_upload/55731/55731fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Minimally Invasive Muscle Embedding MIME - A Novel Experimental Technique to Facilitate Donor-Cell-Mediated Myogenesis at JoVE.com

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

我们描述了一种新的实验技术, 我们称之为微创肌肉植入 (MIME), 这是基于证据表明, 骨骼肌肉组织含有可行的肌细胞, 可以促进捐赠细胞介导的肌时, 注入一个宿主肌肉。

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 ...

minimally-invasive-muscle-embedding-mime-novel-experimental-technique

Research

JoVE Journal

Medicine

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

7.7K 次观看.  Wayne State University. 我们描述了一种新的实验技术, 我们称之为微创肌肉植入 (MIME), 这是基于证据表明, 骨骼肌肉组织含有可行的肌细胞, 可以促进捐赠细胞介导的肌时, 注入一个宿主肌肉。

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

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

工程骨骼肌组织中的小鼠成肌细胞的内皮祖细胞与应用电刺激

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

科研

生物工程

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

在肌肉再生的老鼠模型中移植诱导多能干细胞衍生的中生代细胞样肌原体

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

In Vitro Differentiation of Mature Myofibers for Live Imaging

Skeletal muscles are composed of myofibers, the biggest cells in the mammalian body and one of the few syncytia. How the complex and evolutionarily conserved structures that compose it are assembled remains under investigation. Their size and physiological features often constrain manipulation and imaging applications. The culture of immortalized cell lines is widely used, but it can only replicate the early steps of differentiation.
Here, we describe a protocol that enables easy genetic manipulation of myofibers originating from primary mouse myoblasts. After one week of differentiation, the myofibers display contractility, aligned sarcomeres and triads, as well as peripheral nuclei. The entire differentiation process can be followed by live imaging or immunofluorescence. This system combines the advantages of the existing ex vivo and in vitro protocols. The possibility of easy and efficient transfection as well as the ease of access to all differentiation stages broadens the potential applications. Myofibers can subsequently be used not only to address relevant developmental and cell biology questions, but also to reproduce muscle disease phenotypes for clinical applications.

In Vitro Differentiation of Mature Myofibers for Live Imaging

Muscle cells are among the most complex eukaryotic cells. We present a protocol for the in vitro differentiation of highly mature myofibers that allows for genetic manipulation and clear imaging during all developmental stages.

在成熟的肌纤维诱导分化实时成像

Skeletal muscles are composed of myofibers, the biggest cells in the mammalian body and one of the few syncytia. How the complex and evolutionarily ...

发育生物学

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration1, 2. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient4. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities9, 10. A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations11. Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse)12-14. Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living subject; given the tomographic nature, fine spatial resolution and ability to scale up to larger animals and humans10,11, PET will form the basis of future work that we suggest may facilitate rapid translation of methods developed in cells to preclinical models and to clinical applications.

A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging.

目标移植的肌肉祖细胞的分子成像

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle ...

医学

Performing Human Skeletal Muscle Xenografts in Immunodeficient Mice

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this failure is the use of inadequate laboratory models. It is challenging to model complex human diseases in traditional laboratory organisms, but this issue can be circumvented through the study of human xenografts. The surgical method we describe here allows for the creation of human skeletal muscle xenografts, which can be used to model muscle disease and to carry out preclinical therapeutic testing. Under an Institutional Review Board (IRB)-approved protocol, skeletal muscle specimens are acquired from patients and then transplanted into NOD-Rag1null IL2r&gamma;null (NRG) host mice. These mice are ideal hosts for transplantation studies due to their inability to make mature lymphocytes and are thus unable to develop cell-mediated and humoral adaptive immune responses. Host mice are anaesthetized with isoflurane, and the mouse tibialis anterior and extensor digitorum longus muscles are removed. A piece of human muscle is then placed in the empty tibial compartment and sutured to the proximal and distal tendons of the peroneus longus muscle. The xenografted muscle is spontaneously vascularized and innervated by the mouse host, resulting in robustly regenerated human muscle that can serve as a model for preclinical studies.

Complex human diseases can be challenging to model in traditional laboratory model systems. Here, we describe a surgical approach to model human muscle disease through the transplantation of human skeletal muscle biopsies into immunodeficient mice.

在免疫缺陷小鼠中表演人类骨骼肌异种移植物

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

从肌肉活检患者来源的细胞系疾病建模隔离和永生化

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

成人和胚胎骨骼的肌肉Microexplant文化和骨骼肌肉干细胞的分离

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

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 ...

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 ...

Dosage-Adjusted Resistance Training in Mice with a Reduced Risk of Muscle Damage

Progressive resistance training (PRT), which involves performing muscle contractions against progressively greater external loads, can increase muscle mass and strength in healthy individuals and in patient populations. There is a need for precision rehabilitation tools to test the safety and effectiveness of PRT to maintain and/or restore muscle mass and strength in preclinical studies on small and large animal models. The PRT methodology and device described in this article can be used to perform dosage-adjusted resistance training (DART). The DART device can be used as a standalone dynamometer to objectively assess the concentric contractile torque generated by the ankle dorsiflexors in mice or can be added to a pre-existing isokinetic dynamometry system. The DART device can be fabricated with a standard 3D printer based on the instructions and open-source 3D print files provided in this work. The article also describes the workflow for a study to compare contraction-induced muscle damage caused by a single bout of DART to muscle damage caused by a comparable bout of isometric contractions (ISOM) in a mouse model of limb-girdle muscular dystrophy type 2B/R2 (BLAJ mice). The data from eight BLAJ mice (four animals for each condition) suggest that less than 10% of the tibialis anterior (TA) muscle was damaged from a single bout of DART or ISOM, with DART being less damaging than ISOM.

The present protocol describes a unique technique called dosage-adjusted resistance training (DART), which can be incorporated into precision rehabilitation studies performed in small animals, such as mice.

降低肌肉损伤风险的小鼠剂量调整阻力训练

Progressive resistance training (PRT), which involves performing muscle contractions against progressively greater external loads, can increase muscle ...