作者要感谢艾莉森洛加尔她用流式细胞仪卓越的技术援助。这项工作是由美国国防部（JH），亨利·J·曼金基金全人教育教授（JH），以及教育和哈萨克斯坦共和国科学部（AS）的资助。 CWC在由美国心脏协会博士前奖学金（11PRE7490001）部分得到了支持。 M.Corselli是由美国加州再生医学研究所的训练津贴（TG2-01169）的支持。

1，肌肉活检处理<ol><li>保存在冰上人骨骼肌活检中的Dulbecco改良的Eagle培养基（DMEM），补充有5％胎牛血清（FBS）和1％青霉素 - 链霉素运输过程中（P / S）。 </li><li>在接收到肌肉活检后，除去从运输容器中的检体和在磷酸盐缓冲盐水（PBS）中补充有在无菌条件下的2％抗生素 - 抗真菌溶液（A / A）的两次洗涤。 </li><li>除去附着的脂肪和结缔组织，用无菌剪刀和镊子的DMEM补充有2％A / A。 </li><li>解剖显微镜下去掉大的血管，并随后切开肌肉标本切成小块（&lt;1cm 2的面积）。 </li><li>保持切口肌片（&lt;5克），在20毫升的保存介质（PM; DMEM补充有10％FBS和1％P / S）在4℃至5天。 </li></ol> 2，肌肉Dissociat离子与细胞分离<ol><li>上的细胞分离的日子，从PM去除肌肉块，在PBS中洗两次，补充有2％P / S。为了使消化液，加I型，II型和IV型胶原酶（100毫克/毫升）新鲜到下午。 </li><li>切碎并机械剁碎肌肉块，用无菌剪刀和镊子在培养皿用少量的PM，直到溶液通过10毫升血清吸液管与无凝块。在此过程中除去残留的脂肪和结缔组织。至少使用8-10克，成人的肌肉或2克胎儿肌肉的每一个细胞分离的。 </li><li>转4-5克剁碎成肌〜20毫升（或2克剁碎胎儿肌肉成10毫升）与国税发10毫升血清移液管消解液。消化为50 - 60分钟，在37℃在轨道摇床上于70 - 80转。通过调节消化时间和/或搅拌速度相应地基于所述量优化细胞产率和表面抗原的保存组织。观察消化状况每次15-20分钟，直到浊度几乎清除。 </li><li>大力吸取消化组织3-5次10毫升血清吸管。加入相等量的PM，使反应停止，然后离心分离，在400×g离心4分钟。 </li><li>小心取出上清液;重悬沉淀用10毫升PM洗，然后通过一个100微米的细胞过滤网过滤。 </li><li>离心400×g离心4分钟，小心取出上清液。重悬粒料在红细胞裂解缓冲液（155 mM的氯化铵 ，10mM的KHCO 3，0,1mM EDTA）中，过滤通过70微米的细胞过滤网，以获得单细胞悬浮液，然后下室温温育10分钟。经过70m的细胞过滤器再次过滤，如果观察到任何沉淀。 </li><li>离心机在400×g离心4分钟，并重新悬浮于0.5ml PBS中的细胞沉淀。计数细胞的数目。得到总共至少5百万个细胞的细胞分选。淡化SINGLë细胞悬液，以每毫升少于5百万个细胞，用PBS进行染色。取出50-100微升的细胞悬浮液和分割成11个管用于控制染色（未染色的对照，阴性对照，与单色阳性对照）。 </li></ol> 3，细胞标记和排序<ol><li>孵育的单细胞悬浮液10分钟，在小鼠血清（在PBS中按1:10稀释）4℃阻断目的，如果有必要的。 </li><li>加入CD34-APC，CD45-APC-Cy7的，CD56-PE-Cy7的，CD144-PE，和CD146-FITC（所有1:100）成单细胞悬浮液，并温育20分钟，在4℃。为阴性对照，添加当量浓度APC-，APC-Cy7-，PE-Cy7-，PE-和FITC缀合的同种型IgG抗体，孵育20分钟，在4℃。对于单色阳性对照，加入等量浓度的CD34-APC，CD45-APC-Cy7的，CD56-PE-Cy7的，CD144-PE，和CD146-FITC的单独向每个管中，孵育20分钟，在4℃。 </li><li>孵化后，离心机在400×g离心4分钟，洗。重悬在1细胞沉淀 - 2毫升的DMEM补充有5％FBS和1％P / S。将细胞悬浮液的最终浓度应为每毫升少于5百万个细胞。加入7-AAD（1:100）孵育15分钟，在RT下死细胞排除。为阴性对照和单色阳性对照，以0.5毫升的DMEM补充有5％FBS和1％P / S的重悬细胞沉淀。 </li><li>转移所有细胞悬液，以圆底聚苯乙烯流式细胞仪管。制备细胞收集管（每管是预填充用500μl适当的培养基:MPM对的MECs; EGM-2的个人电脑;交流介质为ACS）。运输的细胞悬浮液在冰上的细胞分选器。 </li><li>同时调节激光强度，信道补偿和细胞群门控严格最大化单元P上的未染色的对照，阴性对照，单色阳性对照，而主要的细胞悬浮液的顺序进行细胞分选仪上运行的细胞悬浮液urity（请参考由朱庇特和其他刊物的流动细节仪发表的文章）。 </li><li>收集在合适的收集管所需的细胞群。商店收集到的细胞在4℃，如果不立即播种。 </li></ol> 4，邮政分拣细胞培养<ol><li>种子新鲜排序的MECs（P0）在&lt;10,000细胞/ cm 2在MPM上板预涂覆有I型胶原蛋白。对于的MECs随后传代，种子3,500 - 4,000个细胞/厘米2的MPM到板/瓶预包被I型胶原蛋白。 </li><li>种子在EGM-2刚整理电脑（P0），在&lt;20000个/ cm 2的平板上新涂0.2％明胶。 3比在PC培养基（DMEM，用20％FBS和1％P / S补充）到正规的聚苯乙烯培养板，直到P2:用于PC的随后的传代，以1分裂细胞。从P3以后，种子细胞在6,500 - 7,000个细胞/厘米2的PC媒体上经常聚苯乙烯培养板/瓶。 </li><li>种子新鲜排序的AC（P0）在&lt;20000细胞/ cm 2在AC培养基（DMEM补充有20％FBS和1％P / S）到正规聚苯乙烯培养板中。对于随后的传代AC的种子细胞在6,500 - 7,000个细胞/厘米2的交流媒介上经常聚苯乙烯培养板/瓶。 </li></ol>

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J.H. received remuneration from Cook MyoSite, Inc. for consulting services and for royalties received from technology licensing during the period that the above research was performed. All other authors have no conflict of interest to disclose.

hBVSC亚群的鉴定和纯化代表海安个体发育的理解的一个重大进展。有越来越多的证据表明MSCs的血管周围的起源和组织特异性前体细胞和血管22-25之间的关联。此外，容量以分离均相亚hBVSCs的进一步有助于MSC异质性和血管细胞生物学26的理解。 在过去的几年中，的MECs，PC和无线控制器已经被单独识别并通过独立的研究不同的协议隔离。然而，没有尝试了同时净?...

人体骨骼肌肉一直被认为是一种临床上有吸引力的干/祖细胞的来源。骨骼肌包含不仅致力于肌祖细胞，成肌细胞，而且原始肌干细胞，包括卫星细胞和肌源性干细胞（肌源性干细胞）1。利用人体肌源性干细胞/祖细胞，自体或异体，在再生医学的已被广泛地研究在临床前动物模型和临床试验。肌肉干细胞/祖细胞的再生应用范围从再生在杜兴肌营养不良（DMD）患者的营养不良的肌肉来修复患者的受损心脏与心脏发作。 自间充质干细胞/基质细胞（MSC）和其它多能前体细胞群，其中包括骨髓来源的多能成体祖细胞（MAPC的）和脂肪来源的干细胞（ADSC中），成体干/的发现祖细胞已被广泛研究，以时间1-9。然而， 在原地家乡的身份和定位一直被掩盖着的回顾性分离方法。近年来，使用荧光激活细胞分选（FACS），我们和其他团体已前瞻性地确定和纯化3多能来自于人骨骼肌血管和其它几种器官的前体细胞群:肌源性内皮细胞（的MECs），周细胞（如PC），和外膜细胞（ACS）10。内膜，中膜和外膜:人类血管来源的干细胞（hBVSCs）这三个亚群可以在血管中的三个结构层可以分别找到。更具体地讲，的MECs和PC的微血管和毛细血管被检测，而AC均为定位于较大的动脉和静脉外膜层。每个前体细胞亚群表达的细胞表面抗原的独特组合:的MECs（CD34 + / 56 + / 144 + / 45 - ），个人电脑（CD146 + / 34 - / 45 - / 56 - ），和AC（CD34 + / 31 - / 45 - / 56 - / 146 - ）。 这些hBVSC亚群的进一步鉴定表明，所有三个前体细胞群具有类似于典型的干细胞，包括骨骼肌肉发育，成骨，软骨，脂肪形成和中胚层发育的潜力。所有hBVSC子集也表现出经典的MSC标志，包括CD44，CD73，CD90，和CD105，新鲜和文化。总的来说这些证据支持干细胞的血管起源。此外，的MECs，PC和无线控制器的治疗能力，最近被证明在独立的研究。排序的MECs从成人的肌肉活检显示再生受伤，营养不良骨骼肌和修复受损的心肌更有效地比骨骼肌成肌细胞和血管内切thelial细​​胞（EC）。从不同的人体器官纯化的电脑也已显示修复/再生损伤和营养不良骨骼肌和向卫星细胞池13-16。最近，我们已经证明，从人骨骼肌来源的电脑有效修复通过间接的旁分泌作用和直接的细胞相互作用17的梗塞心肌。 AC的，另一方面，已经可以直接分离自外植血管或通过FACS从人脂肪组织和骨骼肌纯化。 AC的一个显着的促血管生成作用表现在小鼠后肢缺血模型19。此外，无线控制器也被证明比传统的MSC更有效地修复心肌梗死，表示在局部缺血组织修复20 AC的鲁棒治疗潜力。 目前的纯化方案补助的同时，的MECs，个人电脑未来的净化和AC的从一个人类骨骼肌活检的血管。这使我们能够研究和/或选择最佳的hBVSC亚群的不同的治疗目的。此外，这种新技术进一步扩大，可从人骨骼肌衍生干细胞/祖细胞的剧目，使得它的多能前体细胞用于再生医学的一个理想的来源。

<table border="0" cellpadding="0" cellspacing="0" width="697">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Collagenase Type 1</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:148px;">C5894</td>
			<td style="width:193px;">Sterile vial</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Collagenase Type 2</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:148px;">C1764</td>
			<td style="width:193px;">Sterile vial</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Collagenase Type 4</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:148px;">C1889</td>
			<td style="width:193px;">Sterile vial</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Anti-human CD34 APC</td>
			<td style="width:99px;">BD Pharmingen</td>
			<td style="width:148px;">555824</td>
			<td style="width:193px;">Keep sterile</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Anti-human CD45 APC-Cy7</td>
			<td style="width:99px;">BD Pharmingen</td>
			<td style="width:148px;">557833</td>
			<td style="width:193px;">Keep sterile</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Anti-human CD56 PE-Cy7</td>
			<td style="width:99px;">BD Pharmingen&#160;</td>
			<td style="width:148px;">557747</td>
			<td style="width:193px;">Keep sterile</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Anti-human CD144 PE</td>
			<td style="width:99px;">Beckman Coulter</td>
			<td style="width:148px;">A07481</td>
			<td style="width:193px;">Keep sterile</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Anti-human CD146 FITC</td>
			<td style="width:99px;">AbD Serotec</td>
			<td style="width:148px;">MCA2141F</td>
			<td style="width:193px;">Keep sterile</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">FACSAria II Flow Cytometer</td>
			<td style="width:99px;">Becton-Dickinson</td>
			<td style="width:148px;"></td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">EGM-2 Complete Medium</td>
			<td style="width:99px;">Lonza</td>
			<td style="width:148px;">CC-3162</td>
			<td style="width:193px;">For culturing PCs (only P0)</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:257px;">DMEM high glucose (1X), liquid, with L-glutamine, without sodium pyruvate</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:148px;">11965</td>
			<td style="width:193px;">For culturing PCs</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">DMEM high glucose (1X), liquid, with L-glutamine, with sodium pyruvate</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:148px;">11995</td>
			<td style="width:193px;">For culturing MECs and ACs</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Fetal Bovine Serum</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:148px;">10437-028</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Heat-inactivated horse serum</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:148px;">26050-088</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Penicillin/Streptomycin</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:148px;">15140-122</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Antibiotic-Antimycotic (100X)</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:148px;">15240-062</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Trypsin-EDTA 0.5%(10X)</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:148px;">15400-054</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Dulbecco&#8217;s PBS without calcium and magnesium</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:148px;">14190-250</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Chick embryo extract</td>
			<td style="width:99px;">Accurate Chemical</td>
			<td style="width:148px;">CE650T-10</td>
			<td style="width:193px;">Filter before use</td>
		</tr>
	</tbody>
</table>

isolation of blood-vessel-derived multipotent precursors from human skeletal muscle

由于间充质干细胞/基质细胞（MSCs）的发现，本土身份和干细胞的定位一直被掩盖着他们的文化追溯隔离。近年来，使用荧光激活细胞分选（FACS），我们和其他研究人员前瞻性地识别和纯化的人类骨骼肌的脉管系统相关联的多能前体细胞三个亚群。这三个细胞群:肌内皮细胞（的MECs），周细胞（PCS）和外膜细胞（ACS），分别定位于血管的三个结构层次:内膜，媒体和外膜。所有这些人的血管源性干细胞（hBVSC）的人群，不仅表达经典的MSC标志，而且具有类似于典型的中胚层干细胞的发育潜能。此前，的MECs，PC和无线控制器已经通过不同的协议隔离，随后其特征在于，独立的研究。当前隔离普罗特ocol，通过修改隔离工艺和调整的选择性细胞表面标志物，可以让我们同时净化三个hBVSC亚群流式细胞仪从一个单一的人体肌肉活检。这种新方法不仅简化多个BVSC亚群的隔离也方便hBVSCs为不同的治疗目的，未来的临床应用。

基于来自未染色的对照，阴性对照，以及单色阳性对照而获得的数据的FACS参数被首先校正。排除死细胞后，荧光标记的细胞悬浮液进行了一系列的阴性和阳性的细胞表面标记物的选择。首先，CD45 +细胞被选通的CD56 +和CD56前-细胞从CD45分离的-部分。的CD56 +级分进一步进行CD34-CD144的选择，其中只有CD34 + / CD144 +（双阳性）细胞中被标记为的MECs...

Watch this Scientific Journal Video about Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle at JoVE.com

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

人体骨骼肌肉的海港几个多系前驱体的人口，是理想的可再生应用血管内。这种隔离方法允许3多能前体细胞群的同时纯化分别从血管三个结构层次:从内膜肌内皮细胞，从媒体的周细胞，并从外膜外膜细胞。

从人骨骼肌隔离血管源性多能前体

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

isolation-blood-vessel-derived-multipotent-precursors-from-human

Research

JoVE Journal

Biology

11.6K 次观看.  University of Pittsburgh. 人体骨骼肌肉的海港几个多系前驱体的人口，是理想的可再生应用血管内。这种隔离方法允许3多能前体细胞群的同时纯化分别从血管三个结构层次:从内膜肌内皮细胞，从媒体的周细胞，并从外膜外膜细胞。 

视频: Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

胚胎干细胞分化成少突胶质前体

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

科研

生物学

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 ...

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

骨骼肌的线粒体分离

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

纯化的骨骼肌的先觉者

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

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 ...

Tissue Triage and Freezing for Models of Skeletal Muscle Disease

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal results from functional, cellular, molecular, and pathological evaluations. Due to the subtlety of some pathological abnormalities seen in congenital muscle disorders and the potential for fixation to interfere with the recognition of these features, pathological evaluation of frozen muscle is preferable to fixed muscle when evaluating skeletal muscle for congenital muscle disease. Additionally, the potential to produce severe freezing artifacts in muscle requires specific precautions when freezing skeletal muscle for histological examination that are not commonly used when freezing other tissues. This manuscript describes a protocol for rapid freezing of skeletal muscle using isopentane (2-methylbutane) cooled with liquid nitrogen to preserve optimal skeletal muscle morphology. This procedure is also effective for freezing tissue intended for genetic or protein expression studies. Furthermore, we have integrated our freezing protocol into a broader procedure that also describes preferred methods for the short term triage of tissue for (1) single fiber functional studies and (2) myoblast cell culture, with a focus on the minimum effort necessary to collect tissue and transport it to specialized research or reference labs to complete these studies. Overall, this manuscript provides an outline of how fresh tissue can be effectively distributed for a variety of phenotypic studies and thereby provides standard operating procedures (SOPs) for pathological studies related to congenital muscle disease.

The analysis of skeletal muscle tissues to determine structural, functional, and biochemical properties is greatly facilitated by appropriate preparation. This protocol describes appropriate methods to prepare skeletal muscle tissue for a broad range of phenotyping studies.

组织分流和冻结的骨骼肌疾病模型

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

从小鼠骨骼肌高通量微孔板呼吸测量最低数量的隔离线粒体

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential centrifugation at low- and high-speed is commonly used to isolate mitochondria from tissues and cultured cells. Crude mitochondrial fractions obtained by differential centrifugation are used for respirometry measurements. The differential centrifugation technique is based on the separation of organelles according to their size and sedimentation velocity. The isolation of mitochondria is performed immediately after tissue harvesting. The tissue is immersed in an ice-cold homogenization medium, minced using scissors and homogenized in a glass homogenizer with a loose-fitting pestle. The differential centrifugation technique is efficient, fast and inexpensive and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.

Here, a quadriceps muscle specimen is taken from an anaesthetized pig and mitochondria are isolated by differential centrifugation. Then, the respiratory rates of mitochondrial respiratory chain complexes I, II and IV are determined using high-resolution respirometry.

骨骼肌完整的线粒体的分离差速离心高分辨率测量呼吸运动

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential ...

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or macrophage precursors. Excessive bone resorption is one the most significant cellular mechanisms leading to osteolytic diseases, including osteoporosis, periodontitis, and periprosthetic osteolysis. The main physiological function of osteoclasts is to absorb both the hydroxyapatite mineral component and the organic matrix of bone, generating the characteristic resorption appearance on the surface of bones. There are relatively few osteoclasts compared to other cells in the body, especially in adult bones. Recent studies have focused on how to obtain more mature osteoclasts in less time, which has always been a problem. Several improvements in the isolation and culture techniques have developed in laboratories in order to obtain more mature osteoclasts. Here, we introduce a method that isolates bone marrow in less time and with less effort compared to the traditional procedure, using a special and simple device. With the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts from rat bone marrow, which are identified by classical methods.

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.

大鼠骨髓破骨衣原体的分离、纯化及鉴别

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

骨骼肌成纤维细胞外泌体的分离和表征

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...