Name: visualization and quantification of mesenchymal cell adipogenic differentiation potential with a lineage specific marker
Uploaded: 2018-03-31T15:00:00.000+00:00
Duration: 13 min 26 s
Description: Watch this Scientific Journal Video about Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker at JoVE.com

我们感谢脂肪组织的捐赠者和研究人员, 他们收集并提供这一资源给我们进行研究使用。我们想确认 Allergan 的资金支持。我们还感谢奥克兰大学, 医学和健康科学学院以绩效为基础的研究基金, 用于资助录像和编辑的费用, 以便提交本条。

1. 为鉴别化验准备陶瓷
<ol><li>在无菌组织培养罩中制备组织培养试剂和处理活细胞。</li>
	<li>制备 A0 培养基: Dulbecco 改性鹰中、火腿 F12 培养基 (DMEM/F12)、1x glutamax、1x 青霉素/链霉素。</li>
	<li>准备完整的 ASC 培养基: DMEM/F12, 1x glutamax, 1x 青霉素/链霉素, 10% 胎牛血清。</li>
	<li>使用粘附纯化陶瓷, 在 T75 培养瓶中展开。使用荧光活化细胞分类 (陶瓷)10确认纯度。</li>
	<li>分离细胞通过从 T75 培养瓶中去除所有培养基并加入2毫升细胞离解酶。</li>
	<li>将培养瓶留在孵化器 (37 °c, 加湿, 5% CO2) 为5-10 分钟。把培养瓶从孵化器中取出, 并牢牢地敲打瓶子的侧面。用倒置显微镜检查细胞是否与培养瓶分离。</li>
	<li>在 T75 培养瓶中加入13毫升的 A0 培养基。</li>
	<li>将15毫升转化为15毫升离心管和离心机, 在 400 x g 处为5分钟。</li>
	<li>放弃上清, 并在1毫升的完全 ASC 培养基中重新悬浮细胞颗粒。</li>
	<li>使用 hemocytometer 执行单元格计数。</li>
	<li>在完整的 ASC 培养基中稀释细胞悬浮液, 使其浓度为2.5万细胞毫升-1 。</li>
	<li>将 A0 介质的200µL 添加到96井板 (平底) 的所有外围井中。这降低了含有中心细胞的井的蒸发速率。</li>
	<li>将200µL 的细胞悬浮液转移到 microwell 板上, 以达到每井 5 x 103单元的最终细胞密度。每个治疗组需要四口水井 (三项技术重复和免疫细胞化学 (ICC) 的无主抗体控制)。</li>
	<li>离开 microwell 板在孵化器 (37 °c, 加湿, 5% CO2), 直到细胞达到汇合 (3-4 天)。</li>
</ol>2. 诱导分化陶瓷
<ol><li>用1µM dexamethasome、10µM 胰岛素和200µM 吲哚美辛补充完整的 ASC 培养基, 制备脂肪分化培养基。完全分化培养基稳定在4°c 1 月, 所以只做1的化验要求。存储在4°c 和当需要加热整除到37°c 在水浴。</li>
	<li>3-4 天后, 将 microwell 板从孵化器中取出, 用脂肪分化培养基取代一半培养基。</li>
	<li>每2-3 天执行一次中等变化。 注: 最佳脂肪分化需要14天的培养在分化培养基。用传统染料染色和基因特异污渍分析分化细胞。</li>
</ol>3. 鉴别染色-传统染料基污渍
<ol><li>用 PBS 稀释16% 甲醛, 制备4% 甲醛溶液。仅稀释实验所需的用量, 并在离心管中密封牢固。 注意: 甲醛是一种潜在的致癌物质, 在吸入时会引起鼻子、喉咙和肺部的刺激。执行与油烟机内甲醛有关的所有过程。</li>
	<li>在100毫升异丙醇中溶解300毫克, 制备油红色 O 型溶液。</li>
	<li>把 microwell 盘子从孵化器里拿出来。以下步骤可在无菌环境中执行。</li>
	<li>从井中取出所有介质, 用4% 甲醛固定细胞30分钟。</li>
	<li>在孵化时间, 准备油红色 O 工作解决方案。工作解决方案包括6件油红色 O 型股, 4 份蒸馏水 (dH2o)。混合好, 让坐在室温下20分钟, 过滤前使用0.2 µm 过滤器单元。工作解决方案只稳定2小时。</li>
	<li>吹打从水井中取出所有甲醛。</li>
	<li>用60% 异丙醇洗涤细胞, 并在继续前让水井完全干燥。</li>
	<li>添加50µL 的油红 O 工作溶液, 每井和孵化在室温下10分钟。</li>
	<li>除去油红色 O 溶液, 并立即添加 dh2o 用 dh24 次清洗, 然后再在光显微镜下查看。</li>
</ol>4. 鉴别基因特异抗体的染色
<ol><li>通过溶解80克氯化钠、2克氯化钾和30克三基到 dH2O (pH 8) 的1升, 制备三缓冲盐水 (tb) 的库存溶液。使用 dH2o 在室温下存储, 以稀释1:10 来准备工作溶液。</li>
	<li>准备 immunobuffer (IB) (1% 胎牛血清 (血清) 在 TBS)。标签15毫升离心管与日期和存储在4摄氏度。IB 可用于1星期从日期。</li>
	<li>用200毫升磷酸盐缓冲盐水 (PBS) 溶解0.5 克酪蛋白和0.195 克叠氮化钠, 制备0.25% 酪蛋白阻滞剂。通过搅拌过夜, 完全溶解成分。整除15毫升离心管, 并贮存在-20 摄氏度的冷冻机中。解冻酪蛋白溶液可保持在4°c 1 月。</li>
	<li>通过稀释 DAPI 1:200 的 DAPI 溶液, 将其储存在4摄氏度, 免受光照。</li>
	<li>通过在无菌 PBS 中溶解硫柳汞, 制备库存硫柳汞溶液 (10毫克毫升-1)。存放在4°c 和稀释适当的数额到0.4 毫克 mL-1使用。</li>
	<li>固定和 permeabilize 的细胞与冰冷甲醇 (96 井板) 5 分钟洗涤细胞一次与200µL 的 tb。</li>
	<li>块与50µL 0.25% 酪蛋白在室温下10分钟, 并洗涤一次与 tb。</li>
	<li>添加50µL 的主要抗体稀释在 IB (见表 1的抗体细节) 和孵化与眼睑关闭为1小时。</li>
	<li>洗一次与200µL 的 tbs, 然后3倍与 tbs 5 分钟的温柔摇摆。</li>
	<li>添加50µL 的二级抗体稀释在 IB (参见表 2的抗体细节) 与 1:2000 DAPI 在每个, 孵化与盖子闭合 1 h. 用箔盖板以防止光漂白。</li>
	<li>用 tbs 洗一次, 然后两次与 tb 15 分钟的温柔摇摆。</li>
	<li>卸下所有的 tb 并添加200µL 的硫柳汞溶液。</li>
	<li>储存板在4摄氏度, 保护免受光, 直到成像。</li>
</ol>5. 使用高通量显微镜的成像细胞
<ol><li>利用随附软件控制的高含量筛查显微镜, 分析染有基因特异抗体和荧光共轭二次抗体 (免疫荧光) 的细胞。</li>
	<li>将 microwell 板加载到显微镜阶段。在车牌采集控制中, 选择中等幅度放大图像 (10X 物镜)。确保图像是从每个井的中心拍摄的, 每个站点之间有50µm 间距, 以确保一个单元格不会出现在两个相邻字段中。</li>
	<li>选择要图像的水井。</li>
	<li>选择适当的通道组合、曝光时间和 z 偏移参数。有关每个筛选器 (辅助表 2) 的详细信息, 请参阅表 3 。</li>
	<li>使用采集向导获取检测 (图像自动存储到数据库服务器上)。</li>
	<li>使用备用通道组合获取沾有油红色 O 的印版 (补充表 3)。</li>
</ol>6. 分析获取的图像
注意: 对数据库服务器的远程访问能够快速、轻松地评估图像分析软件所获得和使用的图像。
<ol><li>在分析软件上打开单元格评分模块。</li>
	<li>使用 DAPI 通道 (核标记) 和感兴趣的段核来检测细胞核, 其大小和信号强度高于背景。</li>
	<li>使用 FITC 通道检测 FABP4 信号。区分细胞与用户定义参数的大小和信号强度以上背景, 以选择所有 FABP4 的积极区域在每个图像。</li>
	<li>在分析软件上打开 Transfluor 模块, 分析沾有油红色 O 的板材。</li>
	<li>指定油红色 O 染色区域的大小和强度为 "坑"。</li>
</ol>

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作者声明没有利益冲突。

本文阐述了 FABP4 immunolabelling 对间充质基质细胞的成熟脂肪干细胞进行准确检测和定量量化的效用和优越性。FABP4 能够检测到一个沿袭特定蛋白3,4,5在成熟脂肪细胞中表达的变化, 与其他常用染料的标签大分子变化13,14如油红色 O15、尼罗河红色16和苏丹黑色17。FABP4 immunolabelling 允许可视化和分析细胞形态学的变化。这是一个独特的优势比以前描述的方法使用定量反向转录 PCR (RT-qPCR), 分析变化的成绩单水平没有任何可视化5,18,19 、20或流式细胞术, 要求单元格处于悬浮20中, 或需要细胞裂解以隔离蛋白质1920的西方印迹。FABP4 immunolabelling 是稳定的固定细胞, 不泄漏出在溶液中, 是一个胞浆蛋白当标签在一个黏附分子层的细胞, 将重叠与核允许容易的可视化和增加准确性的自动化分析。
高含量的筛选是有利的, 因为它是快速, 准确, 重现性和客观。它提供了一个成本有效使用试剂和研究员时间的平台, 因为图像采集和分析需要最少的手工操作, 并且依赖于仪器的自动处理。有几个因素被认为对高含量筛选化验的准确性和重现性至关重要31。抗原表达的量化依赖于图像质量。为了获得成功的图像分析, 每个频道的图像必须集中在一个灰度值的最佳动态范围内 (自动曝光设置是理想的)。不集中或饱和的图像导致错误的结果。
本文所述方法的一些潜在限制包括以下内容。对专门的成像设备和分析软件的要求。在本文的范围之外, 可使用替代的开源软件选项 (例如, ImageJ32、33、斐济34、CellProfiler32、35) 来执行类似的图像分割。任务;但是, 他们需要的技能, 以下载和定制的宏可用在线或编写宏/命令, 他们的特定分析管道。所有自动成像分析管道的固有特性是一个背景噪声级别。这主要归因于碎片、图像质量差或分析错误, 强调需要仔细取样准备和仔细设置成像和分析平台。发现小鼠的 FABP4 标签检测未分化的祖细胞30;虽然本研究中的控制 (包括未分化细胞) 缺乏特定的 FABP4 染色。这不足以检查在每一个生物背景下, 在使用该检测的每个生物学环境中检验未分化祖 FABP4 表达的必要性。
为了在 FABP4 标签和油红色 O 染色之间进行有效的比较, 从图像分析中得出的输出测量需要与生物学相关。因此, 测量, "% 阳性细胞" 被用于 FABP4, 和 ' 染色面积每细胞 ' 被用于油红色 O。值得注意的是, 该措施 '% 阳性细胞 ' 没有用于油红色 O 标记细胞在我们的研究, 因为它是不可能准确地将标签的脂肪滴归因于一个给定的核;脂肪滴在细胞体的大小、数量和分布上相差很大, 很少与细胞核重叠。不同组织来源的细胞之间存在油红色 O 染色变异;虽然 the% 阳性细胞测量不适合目前的研究, 另一组已成功地使用它来量化从人类腺体干细胞衍生的脂肪细胞22 , 其中油红色 O 染色出现的一个大脂肪滴, 横跨细胞质和细胞核重叠, 使数据 as% 阳性细胞呈现。
相比之下, FABP4 immunolabelling 的形态学是一致的脂肪细胞来自不同的组织来源的范围23,24,25。在能够分化的文化中, 量化细胞比例的能力有许多应用。成人间充质基质细胞对多种治疗用途具有重要的前景。临床翻译中出现的一个重要问题是, 在26之间、隔离站点27、28和隔离方法之间, 这些细胞之间的能力固有变化12和扩展单元格编号12用于治疗使用。以前已经表明, 黏附的基质细胞的文化经常是异质的, 有些细胞可以分化, 一些不是12,17 , 因为我们的 FABP4 化验显示了 ASC 文化。像这样的化验, 结合浓缩技术, 将有助于确定异种文化中的亚群体, 能够对谱系特异分化。有一个标准化的, 可量化的化验, 如这种 FABP4 化验提供了一个简单的方法来比较所有这些变量, 并有可能评估治疗结果内和之间的临床试验。
FABP4 的量化在半自动化的方式, 使客观和重现性的分析, 在许多捐助者的案例和样本。此外, 由于标签是细胞质, 它可能被用来分析肥大 (脂肪细胞大小的扩大) 除了增生 (脂肪细胞数量增加), 这是相当重要的区别, 在肥胖研究, 其中肥厚与肥胖个体的脂肪功能障碍密切相关21。肥胖症的流行已经激发了对能够控制脂质贮存的药物的大量兴趣。这种 FABP4 的检测还提供了一个简单的读数, 以筛选数以千计的化合物, 以抑制脂质积累的能力。

国际细胞治疗学会 (ISCT) 确定多能间充质基质细胞的关键要求之一是细胞必须具有分化成脂肪、成骨和软骨血统的能力。1. 传统的方法测量分化成这三血统依赖于检测大分子产品使用化学染料1。染料, 如油红色 O (在细胞中染色脂肪滴在已经历成脂), 是廉价和易于使用;然而, 当间充质细胞分化为各自的谱系2时, 它们无法检测出基因表达的具体变化。在这里, 我们已经开发了一种分化检测, 量化的蛋白质表达脂肪谱系特定标记, 脂肪酸结合 protein-4 (FABP4)3,4,5。FABP4 最初在小鼠 3T3-L1 脂肪细胞3中发现, 后来被发现在人皮下脂肪组织6中表达。它是一种胞浆蛋白, 它作为一个伴侣来指导细胞的脂肪酸摄取, 并参与了脂肪4的过程。
用于分化检测的前体细胞为脂肪源性间质基质细胞 (陶瓷)7,8。陶瓷与骨髓间充质干细胞 (BM-MSCs) 有许多性质, 这是成人的主要骨髓间充质干细胞,8,9。在临床应用中, 陶瓷提供了一些优于 BM MSCs 的优势, 因为更大的细胞产量可以从更容易接近的组织来源中分离出来8,9。一个孤立的细胞群需要满足一定的标准来定义为陶瓷。首先, 它们必须在标准培养条件下表现出对塑料组织培养容器的粘附1。它们还必须显示特定的表面抗原表达式1。落后陶瓷的特征是 CD34 的阳性表面抗原表达, CD73, CD90, CD105 的表达低和 CD45 和 HLA-DR10的阴性表达。陶瓷在塑料上培养28天 (黏附纯化陶瓷) 显示 CD73、CD90 和 CD105 的阳性表达, CD34、CD45 和 HLA-DR10的阴性表达。最后, 单元格必须保留区分为各种沿袭的能力1,7,8。
脂肪分化协议诱导上调的 FABP4 表达在其他脂肪细胞谱系基因, 所以我们使用免疫化学可视化 FABP4 蛋白在单元格中, 然后量化 FABP4 表达在单细胞水平使用全自动荧光高含量筛查显微镜。这种方法优于传统染料, 因为它能够高度明确地确认脂肪细胞谱系分化。这种基因特异谱系检测结合高含量筛选方法也可以量化的细胞比例在异构细胞的准备, 能够分化下来的特定血统。在我们的研究中, 我们使用 FABP4 检测来确认细胞培养后新鲜分离陶瓷脂肪分化潜能的丧失。

<table><tbody><tr><td>Tris Base</td><td>Invitrogen</td><td>15504-020</td><td>Tris buffered saline</td></tr><tr><td>Sodium Chloride</td><td>Merck</td><td>1064041000</td><td>Tris buffered saline</td></tr><tr><td>Potassium Chloride</td><td>Merck</td><td>1049360500</td><td>Tris buffered saline</td></tr><tr><td>Sodium Azide</td><td>Scharlau</td><td>SO0091</td><td>Casein blocker solution</td></tr><tr><td>Casein</td><td>Sigma</td><td>C7078-500G</td><td>Casein blocker solution</td></tr><tr><td>PBS tablet</td><td>Sigma</td><td>P4417-100TAB</td><td>Phosphate buffered saline</td></tr><tr><td>DMEM/F12</td><td>Gibco</td><td>11330-032</td><td>Cell culture</td></tr><tr><td>Penicillin/streptomycin</td><td>Invitrogen</td><td>15140-122</td><td>Cell culture</td></tr><tr><td>Glutamax</td><td>Gibco</td><td>35050-061</td><td>Cell culture</td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td>10091-148</td><td>Cell culture</td></tr><tr><td>TrypLE Select Enzyme (1X), no phenol red</td><td>Gibco</td><td>12563-029</td><td>Cell dissociation enzyme</td></tr><tr><td>96 well plate (flat bottom)</td><td>Falcon</td><td>FAL353072</td><td>Cell culture equipment</td></tr><tr><td>T75 culture flask, with filter cap</td><td>Greiner</td><td>658175</td><td>Cell culture equipment</td></tr><tr><td>Insulin</td><td>Sigma&amp;nbsp;</td><td>19278-5ML</td><td>Adipogenic differentiation media</td></tr><tr><td>dexamethasone</td><td>Sigma</td><td>D2915-100MG</td><td>Adipogenic differentiation media</td></tr><tr><td>indomethacin</td><td>sigma</td><td>I7378-5G</td><td>Adipogenic differentiation media</td></tr><tr><td>Oil-Red O</td><td>Sigma</td><td>O-0625</td><td>Classic stain for adipogenesis</td></tr><tr><td>Isopropanol</td><td>Merck</td><td>1.09634</td><td>Classic stain for adipogenesis</td></tr><tr><td>16% formaldehyde</td><td>Pierce</td><td>28908</td><td>Cell fixation</td></tr><tr><td>Acetone</td><td>Merck</td><td>100983</td><td>Cell fixation</td></tr><tr><td>DAPI</td><td>Sigma</td><td>D9542</td><td>Immunocytochemistry</td></tr><tr><td>Anti rabbit IgG alexa 488</td><td>Molecular Probes</td><td>A11008</td><td>Immunocytochemistry</td></tr><tr><td>Thimerosal</td><td>Sigma</td><td>T5125-10G</td><td>Immunocytochemistry</td></tr><tr><td>Rabbit anti-human fatty acid binding protein 4 (FABP4) antibody</td><td>Cayman Chemicals</td><td>10004944</td><td>Marker of adipogenesis</td></tr><tr><td>Centrifuge tubes (15mL)</td><td>Greiner</td><td>GR188271</td><td>General</td></tr><tr><td>Centrifuge tubes (50mL)</td><td>Greiner</td><td>GR227261-P</td><td>General</td></tr><tr><td>ImageXpress Micro XLS</td><td>Molecular Devices</td><td></td><td>high content screening machine</td></tr><tr><td>MetaXpress</td><td>Molecular Devices</td><td></td><td>high content screening software, version 5.4.01</td></tr></tbody></table>

visualization and quantification of mesenchymal cell adipogenic differentiation potential with a lineage specific marker

几种染料目前可用于检测骨髓间充质细胞分化为脂肪脂。染料, 如油红色 O, 是廉价的, 易于使用和广泛利用的实验室分析的脂肪潜力的间充质细胞。然而, 它们并不特定于基因转录的变化。我们已经开发了一种基因特异的分化分析, 以研究何时间充质细胞将其命运转变为脂肪血统。免疫标签对脂肪酸结合 protein-4 (FABP4), 一个血统特定的标记脂肪分化, 使可视化和量化的分化细胞。用96井微板块格式量化脂肪分化潜能的能力, 对许多应用有很好的影响。数以百计的临床试验涉及使用成人间充质基质细胞, 目前很难将治疗结果与此类临床试验之间的关系, 特别是在这方面。这种简单的高通量 FABP4 检测为评估患者源性细胞的分化潜能提供了定量的检测方法, 是比较不同分离和扩张方式的有力工具。这一点尤其重要, 因为越来越多的人认识到在间充质细胞产品中对患者进行的细胞异质性。该方法在高通量药物筛选方面也有潜在效用, 特别是在肥胖和糖尿病前期研究中。

本研究采用3种不同方法测定陶瓷的脂肪分化电位。免疫荧光标记 FABP4, 表明该标记局部化的细胞质的分化细胞, 和标签重叠的 DAPI 标签核 (图 1A)。自动图像分析显示, 早期通道细胞分化组中 FABP4 阳性细胞% 的24.6 倍增加, 较晚期通道细胞增加6.9 倍 (图 1B)。油红色 O 染色是局部的脂肪滴分散在整个细胞质的分化细胞, 和标签很少重叠的 DAPI 标签核;然而从目视检查中, 与早期通道细胞相比, 晚期通道细胞中与油红色 O 脂滴有关的细胞核较少 (图 2A)。自动图像分析显示, 早期通道细胞中每个细胞的油红色 O 染色面积增加了11.1 倍, 后期通道细胞中每个细胞的染色面积增加了53.9 倍 (图 2B)。对两个污点进行了成像, 然后使用该软件中的单元评分 (FABP4) 或 Transfluor (油红色 O) 模块进行量化 (补充图 1, 补充表 2-5)。FABP4 表达式的上调被西方印迹分析证实 (图 3,补充图 5)。所有3种分析方法均显示, 早期通道陶瓷的脂肪分化电位高于晚期通道细胞。脂肪差异不影响每个井的总细胞数 (补充图 2, 3)。然而, FABP4-positive 分化细胞的细胞核大小明显小于晚期通道陶瓷的对照细胞 (补充图 3).
我们证明, 随着细胞在文化中的扩张, 或传代, 在脂肪分化的文化中, 细胞的百分比下降, 与以前发布的数据11一致。必须指出的是, 在本研究中无法控制年龄、体重指数或既往病史的因素, 例如,12 , 可能导致不同捐献者样品之间的变异 (补充表 1)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57153/57153fig1.jpg"> 图 1: FABP4 immunolabelling 表明, 早期通道细胞具有更大的分化潜能, 而非晚期通道细胞.A) 以 DAPI (核染色) 和 FABP4 为标记的早期和晚期通道、控制和分化细胞的代表性图像与合并和分割图像一起显示。分割图像显示有差异的细胞与绿色覆盖和未分化的细胞与红色覆盖。B) FABP4 表达细胞在早期和晚期细胞脂肪分化显著增加, 但早期通道细胞的分化明显高于晚期通道细胞 (曼惠特尼测试 * 表示 p < 0.05 和 *** 表示 p < 0.001)。所显示的数据是平均跨早期通道 (p4; n=8) 或后期通道 (p10; n=4) 捐赠样品, 电子扫描电镜.<a href="/files/ftp_upload/57153/57153fig1large.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/57153/57153fig2.jpg"> 图 2: 油红色 O 染色表明, 早期通道细胞具有更大的分化潜能比后期细胞.A) DAPI (核染色) 和油红 O (灰、脂滴染色) 的代表性图像与合并和分割图像一起显示。分割图像描述所有细胞核与绿色覆盖和油红色 O 染色的脂滴与红色覆盖物。B) 在脂肪分化的情况下, 发现油红色 O 染色区显著增加。早期通道细胞与晚期通道细胞相比, 每个细胞的油红色 O 染色面积更大。每个细胞的油红色 O 染色面积 (微米2) 在这里用作脂肪分化电位的量度。所显示的数据是平均跨早期通道 (p4; n=8) 或后期通道 (p10; n=4) 捐赠样品, 电子扫描电镜 (曼惠特尼测试 * 表示 p = < 0.05 和 *** 表示 p = < 0.001)。<a href="/files/ftp_upload/57153/57153fig2large.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/57153/57153fig3.jpg"> 图 3: FABP4 蛋白表达水平的西方印迹分析区分的早期和晚期陶瓷.FABP4 带的光密度的半定量分析作为总蛋白质装载控制的比率, β肌动蛋白 (ACTB), 表明 FABP4 immunolabelling 在早期的段落明显地增加了, 并且到较少程度晚段落区分细胞.所显示的数据是平均跨早期通道 (p4; n=8) 或后期通道 (p10; n=4) 捐赠样品, 电子扫描电镜 (曼惠特尼测试 * 表示 p = < 0.05 和 *** 表示 p = < 0.001)。<a href="/files/ftp_upload/57153/57153fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>靶抗原</td>
			<td>同种 (克隆)</td>
			<td>物种</td>
			<td>稀释</td>
		</tr>
<tr>
<td>FABP4</td>
			<td>多</td>
			<td>兔</td>
			<td>1:100</td>
		</tr>
</tbody></table>表 1: 原发性抗体
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>靶向抗体</td>
			<td>荧光标签</td>
			<td>物种</td>
			<td>稀释</td>
		</tr>
<tr>
<td>兔 IgG</td>
			<td>Alexa 氟488</td>
			<td>山羊</td>
			<td>1:200</td>
		</tr>
</tbody></table>表 2: 二级抗体
辅助图 1: 图像分析虚拟环境概述.这些染色的细胞使用自动的高通量荧光显微镜成像, 以避免任何偏见的来源。使用 ImageXpress 微 XLS 获取的图像被直接保存到 MDCStore 数据管理器数据库中, 并可通过虚拟桌面连接进行查看, 用户可以使用它们来优化和测试其特定的分析参数。使用专用的 "自动运行" 实例分析图像, 使多个不同的用户可以将 "作业" 发送到自动运行队列。用户可以在 "作业" 完成后通过虚拟实例检索其数据。<a href="/files/ftp_upload/57153/Supplementary_Figure_1.jpg" target="_blank">请单击此处下载此图.</a>
补充图 2: 使用 FABP4 染色板, 比较控制井的总细胞数和用于早期和晚期通道陶瓷的区分井.所显示的数据是平均跨早期通道 (p4; n=8) 或后期通道 (p10; n=4) 捐赠样本, 电子扫描电镜。早期和晚期陶瓷的对照和分化细胞无统计学意义差异。与晚期通道细胞相比, 早期通道细胞每井的细胞都多。<a href="/files/ftp_upload/57153/Supplementary_Figure_2.tif" target="_blank">请单击此处下载此图.</a>
补充图 3: 使用油红色 O 色板, 比较控制井和有区别的井的总细胞计数在及早和晚段落陶瓷.所显示的数据是平均跨早期通道 (p4; n=8) 或后期通道 (p10; n=4) 捐赠样本, 电子扫描电镜。早期和晚期陶瓷的对照和分化细胞无统计学意义差异。<a href="/files/ftp_upload/57153/Supplementary_Figure_3.tif" target="_blank">请单击此处下载此图.</a>
补充图 4: 在以后的段落中, FABP4 阳性的分化细胞的细胞核面积明显小于控制井中的细胞.早期对照和分化细胞间细胞核面积无统计学意义差异。所显示的数据是平均跨早期通道 (p4; n=8) 或后期通道 (p10; n=4) 捐赠样本, 电子扫描电镜. (不配对 t 测试. ** 表示 P = < 0.001)。显示的是。<a href="/files/ftp_upload/57153/Supplementary_Figure_4.tif" target="_blank">请单击此处下载此图.</a>
补充图 5: 西方污点凝胶的图像。红色通道描绘的是β肌动蛋白。绿色通道描述 FABP4 immunolabelling.<a href="/files/ftp_upload/57153/Supplementary_Figure_5.tiff" target="_blank">请单击此处下载此图.</a>
补充表 1: 捐助者的临床病理资料<a href="/files/ftp_upload/57153/Supplementary_table_1.xlsx" target="_blank">请单击此处下载此表.</a>
辅助表 2: 图像设置 (FABP4)<a href="/files/ftp_upload/57153/Supplementary_table_2.xlsx" target="_blank">请单击此处下载此表.</a>
辅助表 3: 成像设置 (油红色 O)<a href="/files/ftp_upload/57153/Supplementary_table_3.xlsx" target="_blank">请单击此处下载此表.</a>
补充表 4: 使用的分析参数表 (FABP4)。单元评分模块。<a href="/files/ftp_upload/57153/Supplementary_table_4.xlsx" target="_blank">请单击此处下载此表.</a>
补充表 5: 使用的分析参数表 (油红色 O)。反流体模块。<a href="/files/ftp_upload/57153/Supplementary_table_5.xlsx" target="_blank">请单击此处下载此表.</a>

Watch this Scientific Journal Video about Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker at JoVE.com

Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

传统的评价脂肪分化的方法既便宜又容易使用, 但并不特定于基因表达的变化。我们已经开发出一种用谱系特异标记将间充质细胞分化为成熟脂肪的实验。这种检测方法在基础研究和临床医学中有不同的应用。

带谱系特异标记的间充质细胞脂肪分化电位的可视化与量化

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy ...

visualization-quantification-mesenchymal-cell-adipogenic

Research

JoVE Journal

Biology

9.7K 次观看.  The University of Auckland. 传统的评价脂肪分化的方法既便宜又容易使用, 但并不特定于基因表达的变化。我们已经开发出一种用谱系特异标记将间充质细胞分化为成熟脂肪的实验。这种检测方法在基础研究和临床医学中有不同的应用。

视频: Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel cell population specifically selected for growth in human serum supplemented medium. These cells are characterized by morphological, phenotypic, and molecular features distinct from MSCs and we named them Mesodermal Progenitor Cells (MPCs). MPCs are round, with a thick highly refringent core region; they show strong, trypsin resistant adherence to plastic. Failure to expand MPCs directly revealed that they are slow in cycling. This is as also suggested by Ki-67 negativity. On the other hand, culturing MPCs in standard medium designed for MSC expansion, gave rise to a population of exponentially growing MSC-like cells. Besides showing mesenchymal differentiation capacity MPCs retained angiogenic potential, confirming their multiple lineage progenitor nature. Here we describe an optimized highly reproducible protocol to isolate and characterize hBM-MPCs by flow cytometry (CD73, CD90, CD31, and CD45), nestin expression, and F-actin organization. Protocols for mesengenic and angiogenic differentiation of MPCs are also provided. Here we also suggest a more appropriate nomenclature for these cells, which has been re-named as "Mesangiogenic Progenitor Cells".

Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow

Here we describe an optimized, highly reproducible protocol to isolate Mesodermal Progenitor Cells (MPCs) from human bone marrow (hBM). MPCs were characterized by flow cytometry and nestin expression. They showed the ability to give rise to exponentially growing MSC-like cell cultures while retaining their angiogenic potential.

从人骨髓分离Mesangiogenic祖细胞（多用途储值卡）

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel ...

科研

发育生物学

Expansion and Adipogenesis Induction of Adipocyte Progenitors from Perivascular Adipose Tissue Isolated by Magnetic Activated Cell Sorting

Expansion of Perivascular Adipose Tissue (PVAT), a major regulator of vascular function through paracrine signaling, is directly related to the development of hypertension during obesity. The extent of hypertrophy and hyperplasia depends on depot location, sex, and the type of Adipocyte Progenitor Cell (APC) phenotypes present. Techniques used for APC and preadipocytes isolation in the last 10 years have drastically improved the accuracy at which individual cells can be identified based on specific cell surface markers. However, isolation of APC and adipocytes can be a challenge due to the fragility of the cell, especially if the intact cell must be retained for cell culture applications.
Magnetic-activated Cell Sorting (MCS) provides a method of isolating greater number of viable APC per weight unit of adipose tissue. APC harvested by MCS can be used for in vitro protocols to expand preadipocytes and differentiate them into adipocytes through use of growth factor cocktails allowing for analysis of the prolific and adipogenic potential retained by the cells. This experiment focused on the aortic and mesenteric PVAT depots, which play key roles in the development of cardiovascular disease during expansion. These protocols describe methods to isolate, expand, and differentiate a defined population of APC. This MCS protocol allows isolation to be used in any experiment where cell sorting is needed with minimal equipment or training. These techniques can aid further experiments to determine the functionality of specific cell populations based on the presence of cell surface markers.

Here we report a method for isolation of Adipocyte Progenitor Cell (APC) populations from Perivascular Adipose Tissue (PVAT) using Magnetic-activated Cell Sorting (MCS). This method allows for an increased isolation of APC per gram of adipose tissue when compared to Fluorescence-Activated Cell Sorting (FACS).

通过磁性活化细胞分选分离的血管周围脂肪组织的脂肪细胞祖细胞的扩增和脂肪生成诱导

Expansion of Perivascular Adipose Tissue (PVAT), a major regulator of vascular function through paracrine signaling, is directly related to the ...

Isolation, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of cardiovascular disease. ADSCs derived from different adipose tissues show distinct features, and those from the PVAT have not been well characterized. In a recent study, we reported that some ADSCs in the periaortic arch adipose tissue (PAAT) descend from the neural crest cells (NCCs), a transient population of migratory cells originating from the ectoderm.
In this paper, we describe a protocol for isolating red fluorescent protein (RFP)-labeled NCCs from the PAAT of Wnt-1 Cre+/-;Rosa26RFP/+ mice and inducing their adipogenic differentiation in vitro. Briefly, the stromal vascular fraction (SVF) is enzymatically dissociated from the PAAT, and the RFP+ neural crest derived ADSCs (NCADSCs) are isolated by fluorescence activated cell sorting (FACS). The NCADSCs differentiate into both brown and white adipocytes, can be cryopreserved, and retain their adipogenic potential for ~3&#8211;5 passages. Our protocol can generate abundant ADSCs from the PVAT for modeling PVAT adipogenesis or lipogenesis in vitro. Thus, these NCADSCs can provide a valuable system for studying the molecular switches involved in PVAT differentiation.

We present a protocol for the isolation, culture, and adipogenic induction of neural crest derived adipose-derived stem cells (NCADSCs) from the periaortic adipose tissue of Wnt-1 Cre+/-;Rosa26RFP/+ mice. The NCADSCs can be an easily accessible source of ADSCs for modeling adipogenesis or lipogenesis in vitro.

神经冠状原脂肪衍生干细胞的分离、培养和异位诱导，来自围阴脂肪组织

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of ...

Robust Differentiation of Human iPSCs into a Pure Population of Adipocytes to Study Adipocyte-Associated Disorders

Recent advances in induced pluripotent stem cell (iPSC) technology have allowed the generation of different cell types, including adipocytes. However, the current differentiation methods have low efficiency and do not produce a homogenous population of adipocytes. Here, we circumvent this problem by using an all-trans retinoic-based method to produce mesenchymal stem cells (MSCs) in high yield. By regulating pathways governing cell proliferation, survival, and adhesion, our differentiation strategy allows the efficient generation of embryonic bodies (EBs) that differentiate into a pure population of multipotent MSCs. The high number of MSCs generated by this method provides an ideal source for generating adipocytes. However, sample heterogeneity resulting from adipocyte differentiation remains a challenge. Therefore, we used a Nile red-based method for purifying lipid-bearing mature adipocytes using FACS. This sorting strategy allowed us to establish a reliable way to model adipocyte-associated metabolic disorders using a pool of adipocytes with reduced sample heterogeneity and enhanced cell functionality.

The protocol allows the generation of a pure adipocyte population from induced pluripotent stem cells (iPSCs). Retinoic acid is used to differentiate iPSCs into mesenchymal stem cells (MSCs) which are used for producing adipocytes. Then, a sorting approach based on Nile red staining is used to obtain pure adipocytes.

将人 iPSC 稳健分化为纯脂肪细胞群以研究脂肪细胞相关疾病

Recent advances in induced pluripotent stem cell (iPSC) technology have allowed the generation of different cell types, including adipocytes. However, the ...

Generation of Mesenchymal Stem Cells from Human Umbilical Cord Tissue and their Differentiation into the Skeletal Muscle Lineage

Exploring the therapeutic potential of mesenchymal stem cells is contingent upon the ease of isolation, potency toward differentiation, and the reliability and robustness of the source. We describe here a stepwise protocol for the isolation of mesenchymal stem cells from human umbilical cord tissue (uMSCs), their immunophenotyping, and the propagation of such cultures over several passages. In this procedure, the viability of the uMSCs is high because there is no enzymatic digestion. Further, the removal of blood vessels, including the umbilical cord arteries and the vein, ensures that there is no contamination of cells of endothelial origin. Using flow cytometry, uMSCs upon isolation are CD45&#8722;CD34&#8722;, indicating an absence of cells from the hematopoietic lineage. Importantly, they express key surface markers, CD105, CD90, and CD73. Upon establishment of cultures, this paper describes an efficient method to induce differentiation in these uMSCs into the skeletal muscle lineage. A detailed analysis of myogenic progression in differentiated uMSCs reveals that uMSCs express Pax7, a marker for myogenic progenitors in the initial stages of differentiation, followed by the expression of MyoD and Myf5, and, finally, a terminal differentiation marker, myosin heavy chain (MyHC).

We describe a protocol for the isolation of mesenchymal stem cells from human umbilical cord tissue and their differentiation into the skeletal muscle lineage.

从人脐带组织中生成间充质干细胞及其分化为骨骼肌谱系

Exploring the therapeutic potential of mesenchymal stem cells is contingent upon the ease of isolation, potency toward differentiation, and the ...

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor response to insulin, local hypoxia, and inflammation. By stimulating the differentiation of functional white adipocytes from progenitors, radical hypertrophy of the adipocyte population can be prevented and, consequently, the metabolic health of adipose tissue can be improved along with a reduction of inflammation. Also, by stimulating a differentiation of beige/brown adipocytes, the total body energy expenditure can be increased, resulting in weight loss. This approach could prevent the development of obesity co-morbidities such as type 2 diabetes and cardiovascular disease.
This paper describes the isolation, expansion, and differentiation of white and beige adipocytes from a subset of human adipose tissue endothelial cells that co-express the CD31 and CD34 markers. The method is relatively cheap and is not labor-intensive. It requires access to human adipose tissue and the subcutaneous depot is suitable for sampling. For this protocol, fresh adipose tissue samples from morbidly obese subjects [body mass index (BMI) &#62;35] are collected during bariatric surgery procedures. Using a sequential immunoseparation from the stromal vascular fraction, enough cells are produced from as little as 2&#8211;3 g of fat. These cells can be expanded in culture over 10&#8211;14 days, can be cryopreserved, and retain their adipogenic properties with passaging up to passage 5&#8211;6. The cells are treated for 14 days with an adipogenic cocktail using a combination of human insulin and the PPAR&#947; agonist-rosiglitazone.
This methodology can be used for obtaining proof of concept experiments on molecular mechanisms that drive adipogenic responses in adipose endothelial cells, or for screening new drugs that can enhance the adipogenic response directed either towards white or beige/brown adipocyte differentiation. Using small subcutaneous biopsies, this methodology can be used to screen out non-responder subjects for clinical trials aimed to stimulate beige/brown and white adipocytes for the treatment of obesity and co-morbidities.

The differentiation of white and beige adipocytes from adipose tissue vascular progenitors bears potential for metabolic improvement in obesity. We describe protocols for a CD34+CD31+ endothelial cell isolation from human fat and for a subsequent in vitro expansion and differentiation into white and beige adipocytes. Several downstream applications are discussed.

人网膜和皮下脂肪组织 CD34+CD31+ 内皮细胞的分离、扩增和脂肪诱导

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor ...

免疫与感染

Differentiation Capacity of Human Aortic Perivascular Adipose Progenitor Cells

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic lineages. Adipogenic differentiation of progenitor cells is a major mechanism driving adipose tissue expansion and dysfunction in response to obesity. Understanding changes to perivascular adipose tissue (PVAT) is thus clinically relevant in metabolic disease. However, previous studies have been predominately performed in the mouse and other animal models. This protocol uses human thoracic PVAT samples collected from patients undergoing coronary artery bypass graft surgery. Adipose tissue from the ascending aorta was collected and used for explantation of the stromal vascular fraction. We previously confirmed the presence of adipose progenitor cells in human PVAT with the capacity to differentiate into lipid-containing adipocytes. In this study, we further analyzed the differentiation potential of cells from the stromal vascular fraction, presumably containing multi-potent progenitor cells. We compared PVAT-derived cells to human bone marrow MSC for differentiation into adipogenic, osteogenic, and chondrogenic lineages. Following 14 days of differentiation, specific stains were utilized to detect lipid accumulation in adipocytes (Oil red O), calcific deposits in osteogenic cells (Alizarin Red), or glycosaminoglycans and collagen in chondrogenic cells (Masson&#8217;s Trichrome). While bone marrow MSC efficiently differentiated into all three lineages, PVAT-derived cells had adipogenic and chondrogenic potential, but lacked robust osteogenic potential.

The goal of this protocol is to test the ability of progenitor cells derived from human perivascular adipose tissue to differentiate into multiple cell lineages. Differentiation was compared to mesenchymal stem cells derived from human bone marrow, which is known to differentiate into adipocyte, osteocyte, and chondrocyte lineages.

人主动脉周脂肪祖细胞的分化能力

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic ...

生物学

Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident adipose progenitor cells (APCs) play a key role in adipose tissue homeostasis and can contribute to the tissue pathology. The growing use of single cell analysis technologies &#8211; including single-cell RNA-sequencing and single-cell proteomics &#8211; is transforming the stem/progenitor cell field by permitting unprecedented resolution of individual cell expression changes within the context of population- or tissue-wide changes. In this article, we provide detailed protocols to dissect mouse epididymal adipose tissue, isolate single adipose tissue-derived cells, and perform fluorescence activated cell sorting (FACS) to enrich for viable Sca1+/CD31-/CD45-/Ter119- APCs. These protocols will allow investigators to prepare high quality APCs suitable for downstream analyses such as single cell RNA sequencing.

We present a simple method to isolate highly viable adipose progenitor cells from mouse epididymal fat pads using fluorescence activated cell sorting.

从小鼠表皮脂肪脂肪组织中制备脂肪祖细胞

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident ...

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors (FAPs) and Myogenic Progenitors (MPs) in Skeletal Muscle in the Rat

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in muscle homeostasis, injury, and repair. Current protocols for FAPs identification and isolation use flow cytometry/fluorescence-activated cell sorting (FACS) and studies evaluating their function in vivo to date have been undertaken exclusively in mice. The larger inherent size of the rat allows for a more comprehensive analysis of FAPs in skeletal muscle injury models, especially in severely atrophic muscle or when investigators require substantial tissue mass to conduct multiple downstream assays. The rat additionally provides a larger selection of muscle functional assays that do not require animal sedation or sacrifice, thus minimizing morbidity and animal use by enabling serial assessments. The flow cytometry/FACS protocols optimized for mice are species specific, notably restricted by the characteristics of commercially available antibodies. They have not been optimized for separating FAPs from rat or highly fibrotic muscle. A flow cytometry/FACS protocol for the identification and isolation of FAPs and MPs from both healthy and denervated rat skeletal muscle was developed, relying on the differential expression of surface markers CD31, CD45, Sca-1, and VCAM-1. As rat-specific, flow cytometry-validated primary antibodies are severely limited, in-house conjugation of the antibody targeting Sca-1 was performed. Using this protocol, successful Sca-1 conjugation was confirmed, and flow cytometric identification of FAPs and MPs was validated by cell culture and immunostaining of FACS-isolated FAPs and MPs. Finally, we report a novel FAPs time-course in a prolonged (14 week) rat denervation model. This method provides the investigators the ability to study FAPs in a novel animal model.

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors FAPs and Myogenic Progenitors MPs in Skeletal Muscle in the Rat

This protocol outlines a method to isolate Fibro-adipogenic progenitors (FAPs) and myogenic progenitors (MPs) from rat skeletal muscle. Utilization of the rat in muscle injury models provides increased tissue availability from atrophic muscle for the analysis and a larger repertoire of validated methods to assess muscle strength and gait in free-moving animals.

大鼠骨骼肌中纤维-脂肪源性祖细胞（FAPs）和肌源性祖细胞（MPs）的鉴定、分离和表征

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in ...

Differentiation and Imaging of Brown Adipocytes from the Stromal Vascular Fraction of Interscapular Adipose Tissue from Newborn Mice

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with multiple lipid droplets, a central nucleus, a high mitochondrial content, and the expression of uncoupling protein 1 (UCP1). BAT has been proposed as a potential therapeutic target for obesity and its associated metabolic disorders due to its ability to dissipate metabolic energy as heat. To investigate BAT function and regulation, brown adipocyte culturing is indispensable. The present protocol optimizes tissue processing and cell differentiation for culturing brown adipocytes from newborn mice. Additionally, procedures for the imaging of differentiated adipocytes with both confocal immunofluorescence and transmission electron microscopy are shown. In the brown adipocytes differentiated with the techniques described herein, the major defining features of classical BAT are preserved, including high UCP1 levels, increased mitochondrial mass, and very close physical contact between the lipid droplets and mitochondria, making this method a valuable tool for BAT studies.

Preadipocytes are isolated from the stromal vascular fraction of interscapular brown adipose tissue from newborn mice and differentiated into cells that accumulate lipid droplets, express molecular markers, and show the mitochondrial morphology of mature brown adipocytes. These cells are further analyzed by immunofluorescence and transmission electron microscopy.

新生小鼠肩胛间脂肪组织基质血管部分棕色脂肪细胞的分化和成像

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with ...