This work was supported by National Institute of Health (1R01DK103150-01A1, T32 DK007639, R01CA175803, K08CA125209) and the Institute for Translational Sciences at the University of Texas Medical Branch, and a Clinical and Translational Science Award (8UL1TR000071).

该协议获得从手术患者丢弃的人体组织，并建立原代培养的是经德克萨斯大学医学科和新墨西哥州机构审查委员会的大学大学。用于人体组织标本采购的一般要求如下所述。注意，由于隶属函数可以从冰冻组织，生物银行和商业供应商分离出来的也是可行的办法。 1.获取人体组织<ol><li>提交协议来获得人结肠组织的机构审查委员会批准。 </li><li>建立合作与普通外科医生和/或结直肠外科为了获得人体组织进行研究。与病理科协作也是强制性的，因为它们将被判定为不需要进行诊断和可被指定为研究的组织。 </li><li>奉劝病理学家从肿瘤提供组织，然后grosslŸ正常粘膜至少5厘米的肿瘤。立即放置在冰冷的洗涤介质的样品，并放置在冰上。病理学家将确定可以用于研究被安全地给定组织的量。需要MF隔离最小的组织为1.5 平方毫米。 </li><li>使用获得的组织或者立即建立原代培养物或冷冻并置于在-80℃下以供将来隔离。 <ol><li>如果冷冻购买隔离，切割组织成约1 - 2毫米2件 ，放入低温管与1ml冷冻介质（见下文），并储存在-80℃。注:利用此协议，隶属函数的自组织冻结了超过4年成功分离已被观察到。 </li></ol></li></ol> 2.准备试剂<ol><li>胶原酶的解决方案:准备100 U / ml的胶原酶I，II和IV在汉克的平衡盐溶液（HBSS）与钙 / 镁离子（ 见材料表 </strong&gt;的信息）。小心:胶原酶活性单位浓度和纯度可能变化从厂商而，这可能会妨碍组织消化在粘膜细胞表面表位的和/或保存的效率。为了防止这些潜在的缺陷，使用胶原酶具有最高纯度（≥95％）的，并准备基于活性单位浓度的储备溶液。 </li><li>制备水性DNA酶原液以10毫克/毫升（3.55 UA /毫克）。 </li><li>准备清洗介质:（MEM 89毫升，抗生素 - 抗真菌剂，100×1毫升，热失活的FCS 10毫升） </li><li>准备成纤维细胞生长培养基:MEM:一瓶500毫升; L-谷氨酰胺（200 mM计）:5毫升， MEM非必需氨基酸，100倍的溶液:5毫升，环丙沙星盐酸（10毫克/毫升）:570微升;抗生素 - 抗真菌剂，100倍溶液10毫升，丙酮酸钠（100毫摩尔）:5毫升，热灭活的FCS 50 ml的</li><li>制备冻结介质:成纤维细胞生长培养基（试剂2.4）用加入了10％的二甲亚砜（DMSO）中，然后过滤，韦伯斯特ilize用0.22微米的过滤器。 </li></ol> 3.酶促文摘人体组织<ol><li>如果组织被冻结在冷冻媒体，地方离心管中的温暖（〜37℃）的水，直到组织和媒体解冻。 </li><li>为4 - 5 2 2毫米组织片，两次用10ml的HBSS洗组织不含Ca 2+ / Mg 2 +的。 </li><li>一旦组织已落户在重力作用，精心弃上清没有旋转样品。 </li><li>加入10 mL胶原酶溶液的样品，并在组织的溶液混合物与转移到无菌的，DNA酶/无RNase管内置转子样品解离。用胶原酶以解离细胞外基质包围的上皮细胞的基础方面，形成在粘膜固有层中的非细胞部分。 </li><li>紧紧贴着管颠倒其安装到离解器（ 例如 ，GentleMACs）的袖子。如果实验室不具有接入到t他上述的设备，增加每个消化步骤的时间，以获得比较的结果。 </li><li>运行程序h_tumor_01，预先设定的信息包括与机器（36秒的总的持续时间，与间歇脉冲范围从1000 - 4000转，以268转每运行）。 </li><li>终止程序结束后，取下管从离解。 </li><li>在37℃下连续旋转孵育样品45分钟在摇床上，在140转。注意:对于不同的组织样本的量，消化的时间可以按比例减少或增加（ 即 ，更大的组织的量可能需要额外的时间）。 </li><li>连接管倒到离解的袖子。 </li><li>选择并运行程序h_tumor_02（37秒的总时间，间歇性脉冲，从1000 - 4000转，235发每运行）。 </li><li>终止程序结束后，取下管从离解。 </li><li>加入50微升DNA酶原液。使用DNA酶以解离/去除死细胞和细胞碎片的DNA的级分导致细胞结块。 </li><li>在37℃下在摇床上连续旋转孵育样品30分钟以60rpm。 </li><li>再次将试管倒置到离解的袖子。 </li><li>选择并运行程序h_tumor_03（37秒的总时间，间歇性脉冲，从1000 - 4000转，168发每运行）。 注意:如果组织未完全消化，离心机在250×g的10分钟，在20℃，弃上清，重悬在2毫升细胞解​​离溶液和在室温下孵育10分钟。 </li><li>通过无菌70微米的细胞过滤的细胞悬浮液。 </li><li>离心细胞悬浮液在250×g的10分钟，在20℃。吸去上清液彻底。 </li><li>洗涤细胞沉淀两次用25ml的HBSS不含Ca 2+ / Mg 2 +的并弃去上清液。 </li><li>前的最后被小时，使用一个自动细胞计数系统，可以在实验室或手动使用血细胞计数器计数细胞数。对于MF原代培养一代继续开行步3.19.1，进行分析或隶属函数的使用流式细胞仪进行步3.19.2排序。 <ol><li>分离和纯的MF培养的生长 （图1）中，丢弃从最后离心，重悬细胞沉淀上清液中的成纤维细胞分离培养基的种子高达4×10 6细胞所需要的适当的量在3ml的培养基，每孔中6孔，细胞培养处理板。继续执行步骤3.20。 </li><li>用于分析或微丝的使用流式细胞术，最多放置2×10 6在2 ml成纤维细胞分离培养基的24孔，低的结合板孵育O / N在37℃，5％CO 2的分选。这是恢复可能受影响的酶的过程中的细胞表面表位。然后收集细胞悬液，计数回收的细胞，并继续用于免疫染色和流式细胞仪分析8。 </li></ol></li><li>生长的细胞在37℃，5％的CO 2。更换介质每2 - 3天，直到形成80％汇合MF单层〜（ 图1D-E）。 </li><li>代细胞在T25烧瓶与来自6孔板中以比1:1和生长〜10 - 在成纤维细胞生长培养基中7-14天实现80 - 100％汇合。 </li><li> 2:从T25传代细胞T75细胞比1扩大文化。一旦细胞达到汇合，利用流式细胞仪如前5描述用一个锥形瓶孤立MF纯度的分析。 3:在比1冻结或通过MF文化的另一种T75烧瓶中。 注意:当通过流式细胞术分析，因此预 ​​计间充质来源的胃肠粘膜基质MF当在培养生长将具有以下表现型:EpCAM-，CD31，CD45，波形蛋白+α-SMA +，CD90 +。 </li></ol>

<ol>
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The authors have nothing to disclose.

虽然该协议是为研究中的应用开发只，在成长为一个潜在的癌症预后标志物和治疗靶点基质细胞至关重要的光，冻结"功能性"生物标本以备后用的能力提供了一个显著优势。这是一个独特的优势，为创作临床biorepository的，可作为额外的工具服务促进个性化医疗10,11的发展，类似于先前针对脂肪间充质干细胞从冷冻的基质血管级分中分离报告的结果，我们进行了未观察到在标记和增殖/免疫/?...

肌成纤维细胞/成纤维细胞（MFS）表示细胞在胃肠道黏膜丰富的人口。这些基质细胞就位于下方的上皮形成和在肠道内的粘膜固有层的互连网络。微丝不仅负责细胞外基质的沉积，但通过旁分泌调节可影响电解质转运，恢复原状的，屏障功能相邻上皮1,2。此外，隶属函数已被证明在炎症和组织中发挥关键作用重塑3,4。肌成纤维细胞是在肿瘤微环境，在那里他们也被称为癌相关成纤维细胞的一个关键部分，并有助于肿瘤细胞的生长，并作为一个利基癌症干细胞3。新出现的数据表明，微丝也可用作本地抗原呈递细胞。此外，隶属函数功能的固有免疫和适应性免疫反应的重要调节器，机生产线CING多种细胞因子和生长因子5。 在健康个体中，微丝细胞被认为从间充质干细胞分化和表达在其细胞表面上，所述间充质标记物，CD90 3,5，这些细胞也表达vimentin，但阴性上皮细胞和造血细胞标记物的EpCAM和CD45分别。肌成纤维细胞被建议是成纤维细胞的活化形式，并且可以从非活化的成纤维细胞用α-SMA 5的表达来区分。 在过去十年中，多种方法用于从人结肠黏膜肌成纤维细胞的分离已公布-大多是基于最初由Mahida 等人 5-8中记载的方法。虽然个别的研究报道来自各种肠粘膜，对于隶属函数的从胃肠道的多个区域（即，胃，小和升隔离没有通用协议分离程序阿尔赫肠粘膜）已经出版。本文提出的方案已经过测试，并成功地用于所有三个类型的组织上面提到的。 。此外，还没有被报道用于从冷冻胃肠粘膜分离的MF流程。 这里，我们提出一种优化方法，它是基于酶消化，并且同时允许对人体肠道粘膜的MF的培养分离和流式细胞术分析在新鲜消化，单细胞，粘膜制剂。这种技术可靠地得到原代培养与MF的表型。此外，同样的方法可以用来从胃和小肠的组织冷冻样品隔离微丝为好。从新鲜的GI组织成肌纤维细胞的分离先前已描述;然而，冷冻样品的利用带来了许多好处。即，研究人员将能够收集和存储组织免受任何数目在全世界合作者谁具有capabi的lity运送冷冻组织样本。此外，研究人员可能会发现从手术室和/或内窥镜套件和立即丢弃组织的处理的组织为隔离与他们当前的实验时间表冲突的集合。此外，由于手术的不可预测性，可能会出现组织采购非常晚了一天，这将限制左为加工组织的时间。冷冻组织进行后续处理会减轻这些挑战。 最后，这些方法已被成功地用于在结肠，胃和小肠肌纤维母细胞在疾病状态如大肠癌和炎症性肠疾病的隔离。

<table border="0" cellpadding="0" cellspacing="0" width="719">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">MEM, 1X</td>
			<td style="width:111px;">Corning</td>
			<td style="width:119px;">MT-10-010-CV</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Hanks&#39; Balanced Salt Solution</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:119px;">H6648</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Sodium pyruvate</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:119px;">S8636</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Antibiotic-Antimycotic, 100X</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:119px;">15240-062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Ciprofloxacin HCl</td>
			<td style="width:111px;">Corning</td>
			<td style="width:119px;">61-277-RF</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">L-Glutamine, 100x</td>
			<td style="width:111px;">Corning</td>
			<td style="width:119px;">25-005-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">MEM Nonessential Amino Acids</td>
			<td style="width:111px;">Corning</td>
			<td style="width:119px;">25-025-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Dimethyl Sulfoxide (DMSO) Hybri-Max</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:119px;">D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">DL-Dithiothreitol solution (DTT)</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:119px;">646563</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">0.5M EDTA, pH 8.0</td>
			<td style="width:111px;">Cellgro</td>
			<td style="width:119px;">46-034-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">DNAse</td>
			<td style="width:111px;">Worthington</td>
			<td style="width:119px;">LS002139</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">Collagenase from&#160;Clostridium histolyticum, Type I</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:119px;">C1639</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">Collagenase from&#160;Clostridium histolyticum, Type II</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:119px;">C1764</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">Collagenase from Clostridium histolyticum, Type IV</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:119px;">C5138</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Accumax cell dissociation solution</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:119px;">A7089</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">gentleMACS C Tubes</td>
			<td style="width:111px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-093-237</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">gentleMACS Dissociator</td>
			<td style="width:111px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-093-235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Countess Automated Cell Counter</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:119px;">C10227</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Cell Strainer (70&#181;m)</td>
			<td style="width:111px;">Corning</td>
			<td style="width:119px;">352350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">PE Mouse Anti-Human Vimentin</td>
			<td style="width:111px;">BD Biosciences</td>
			<td style="width:119px;">562337</td>
			<td>Clone RV202</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">FITC Monoclonal Anti-Actin, &#945;-Smooth Muscle</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:119px;">F3777</td>
			<td>Clone 1A4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">APC Mouse Anti-Human CD90</td>
			<td style="width:111px;">BD Biosciences</td>
			<td style="width:119px;">559869</td>
			<td>Clone 5E10</td>
		</tr>
	</tbody>
</table>

isolation of cd 90+ fibroblast/myofibroblasts from human frozen gastrointestinal specimens

成纤维细胞/肌成纤维细胞（MFS）已获得越来越多的关注他们在发病中的作用及其对肿瘤微环境的两个伤口愈合和推广贡献。虽然目前有很多技术的MF的胃肠道（GI）组织中分离，该协议引入了一种新的从冰冻组织这些间质细胞的分离的元素。冷冻胃肠组织标本不仅允许研究者获得来自世界各地的合作者，生物库，和商业供应商的样品，它也允许新鲜样品的延迟处理。所描述的协议将始终如一地产生特征梭形细胞表达标记CD90，α-SMA和波形对MF型。因为这些细胞来自患者样品衍生，使用原代细胞也赋予密切模仿隶属函数的受益于疾病状态，即癌和炎症性肠疾病。这种技术具有蜂ñ证实胃，小肠和结肠MF原代培养一代。主培养物的MF可以使用在实验繁多在若干通路和它们的纯度评估由两个免疫细胞化学和流式细胞术分析。

使用此酶消化协议中，我们一直能够隔离并从胃肠手术标本和活检（图1）生长的MF人口肠道CD90 +粘膜基质细胞。 5播种单个核细胞悬液到6孔板（ 图1A-B）之后-可见MF菌落数量激增可能在第2天进行观察。对MF原代培养达到〜50 - 70％汇合由7天- 11（图1C-D）。 <img alt="图1" src...

Watch this Scientific Journal Video about Isolation of CD 90+ Fibroblast/Myofibroblasts from Human Frozen Gastrointestinal Specimens at JoVE.com

Isolation of CD 90+ Fibroblast/Myofibroblasts from Human Frozen Gastrointestinal Specimens

Here, a protocol to isolate and establish primary fibroblast/myofibroblast (MF) cultures from frozen gastric, small intestinal, and colonic tissue-yielding cells with a MF phenotype-is presented. These cells express CD90, &#945;-SMA and vimentin. MFs can be used for a variety of functional assays including enzymatic activity and cytokine production.

从人类冷冻胃肠标本分离CD 90+成纤维细胞/肌成纤维细胞


Fibroblasts/myofibroblasts (MFs) have been gaining increasing attention for their role in pathogenesis and their contributions to both wound healing and ...

isolation-cd-90-fibroblastmyofibroblasts-from-human-frozen

Research

JoVE Journal

Developmental Biology

9.5K 次观看.  University of Texas Medical Branch. Here, a protocol to isolate and establish primary fibroblast/myofibroblast (MF) cultures from frozen gastric, small intestinal, and colonic tissue-yielding cells with a MF phenotype-is presented. These cells express CD90, α-SMA and vimentin. MFs can be used for a variety of functional assays including enzymatic activity and cytokine production. 

视频: Isolation of CD 90+ Fibroblast/Myofibroblasts from Human Frozen Gastrointestinal Specimens

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

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

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

从人类骨骼肌成肌原细胞和成纤维细胞的分离和定量免疫细胞化学表征

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

科研

发育生物学

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

从脑室周围地区的成年大鼠和人类脊髓神经分离干/祖细胞

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

使用非整合附加型质粒从冷冻血沉棕黄层诱导多能干细胞的产生

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

树突棘从锥体神经元的三维定量从人类诱导多能干细胞衍生

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human development and investigate the pathophysiology of disease. By employing site-specific nucleases (SSNs) for genome editing, the rapid derivation of new hPSC lines harboring specific genetic alterations in an otherwise isogenic setting becomes possible. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 are the most commonly used SSNs. All of these nucleases function by introducing a double stranded DNA break at a specified site, thereby promoting precise gene editing at a genomic locus. SSN-meditated genome editing exploits two of the cell&#39;s endogenous DNA repair mechanisms, non-homologous end joining (NHEJ) and homology directed repair (HDR), to either introduce insertion/deletion mutations or alter the genome using a homologous repair template at the site of the double stranded break. Electroporation of hPSCs is an efficient means of transfecting SSNs and repair templates that incorporate transgenes such as fluorescent reporters and antibiotic resistance cassettes. After electroporation, it is possible to isolate only those hPSCs that incorporated the repair construct by selecting for antibiotic resistance. Mechanically separating hPSC colonies and confirming proper integration at the target site through genotyping allows for the isolation of correctly targeted and genetically homogeneous cell lines. The validity of this protocol is demonstrated here by using all three SSN platforms to incorporate EGFP and a puromycin resistance construct into the AAVS1 safe harbor locus in human pluripotent&#160;stem cells.

Genome editing of human pluripotent stem cells (hPSCs) can be done quickly and efficiently. Presented here is a robust experimental procedure to genetically engineer hPSCs as exemplified by editing the AAVS1 safe harbor locus to express EGFP and introduce antibiotic resistance.

建立基因组编辑的人多能干细胞系:从定位到隔离

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

从人胎盘组织源分离和间充质干扩张/基质细胞

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34- pericytes and vascular CD34+CD146- adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium.

从人类心脏组织血管周围多能前体细胞群的分离

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

从人类脐带和胎盘胎儿分离和鉴定间充质干细胞

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and colleagues1. Later, others documented the existence of similar types of EPCs originating from bone marrow2,3. More recently, Yoder and Ingram showed that EPCs derived from umbilical cord blood had a higher proliferative potential compared to ones isolated from adult peripheral blood4,5,6. Apart from being involved in postnatal vasculogenesis, EPCs have also shown promise as a cell source for creating tissue-engineered vascular and heart valve constructs7,8. Various isolation protocols exist, some of which involve the cell sorting of mononuclear cells (MNCs) derived from the sources mentioned earlier with the help of endothelial and hematopoietic markers, or culturing these MNCs with specialized endothelial growth medium, or a combination of these techniques9. Here, we present a protocol for the isolation and culture of EPCs using specialized endothelial medium supplemented with growth factors, without the use of immunosorting, followed by the characterization of the isolated cells using Western blotting and immunostaining.

The goal of this protocol is to isolate endothelial progenitor cells from umbilical cord blood. Some of the applications include using these cells as a biomarker for identifying patients with cardiovascular risk, treating ischemic diseases, and creating tissue-engineered vascular and heart valve constructs.

人脐血内皮祖细胞的分离

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of the heart after ischemic injury. When activated, epicardial cells undergo a process known as epithelial to mesenchymal transition (EMT) to provide cells to the regenerating myocardium. Furthermore, the epicardium contributes via secretion of essential paracrine factors. To fully appreciate the regenerative potential of the epicardium, a human cell model is required. Here we outline a novel cell culture model to derive primary epicardial derived cells (EPDCs) from human adult and fetal cardiac tissue. To isolate EPDCs, the epicardium is dissected from the outside of the heart specimen and processed into a single cell suspension. Next, EPDCs are plated and cultured in EPDC medium containing the ALK 5-kinase inhibitor SB431542 to maintain their epithelial phenotype. EMT is induced by stimulation with TGF&#946;. This method enables, for the first time, the study of the process of human epicardial EMT in a controlled setting, and facilitates gaining more insight in the secretome of EPDCs that may aid heart regeneration. Furthermore, this uniform approach allows for direct comparison of human adult and fetal epicardial behavior.

The epicardium plays a crucial role in the development and repair of the heart by providing cells and growth factors to the myocardial wall. Here, we describe a method to culture human primary epicardial cells that enables the study and comparison of their developmental and adult characteristics.

人成体和胎心标本中原发性心外膜细胞的分离培养

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...