Name: isolation of blood-vessel-derived multipotent precursors from human skeletal muscle
Uploaded: 2014-08-21T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle at JoVE.com

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...

<ol>
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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 ...

Research

JoVE Journal

Biology

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

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

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

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

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

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

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

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

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

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

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 ...