Contreras和Watts实验室和William Raphael博士。这些实验由NHLBI F31 HL128035-01（组织消化方案标准化），NHLBI 5R01HL117847-02和2P01HL070687-11A1（动物）和NHLBI 5R01HL117847-02（细胞分离和培养）支持。

本文中描述的所有程序都遵循密歇根州立大学机构动物护理和使用委员会（IACUC）制定的指南。所有缓冲液和介质均应避光保护。 缓冲液，介质和仪器的制备<ol><li>制备Krebs Ringer碳酸氢盐缓冲溶液（KRBB）:135mM氯化钠，5mM氯化钾，1mM硫酸镁，0.4mM磷酸氢二钾，5.5mM葡萄糖，1％抗生素/抗真菌剂（10,000单位/ mL青霉素，10,000μg / mL链霉素，25μg/ mL两性霉素B）和10mM HEPES（pH = 7.4）。该溶液在4℃保存无菌时稳定3周。 </li><li>制备胶原酶1型溶液:在含有4％牛血清白蛋白（BSA）的KRBB中1mg / mL。该溶液应保持在37℃，稳定4小时。 </li><li>制备红细胞裂解缓冲溶液:154 mM氯化铵，10 mM碳酸氢钾，和0.1mM EDTA。保持在4°C长达一个月。 </li><li>制备MCS阻滞缓冲液:DMEM / F12培养基，10％胎牛血清（FBS），5％正常驴血清，40μL/ mL F（ab）片段驴抗鼠IgG。保持在4°C长达一个月。 </li><li>制备MCS缓冲溶液:磷酸盐缓冲盐水（PBS，pH = 7.5），0.5％BSA和2mM EDTA。保持在4°C长达一个月。通过在玻璃容器中加热至37℃，然后施加真空15秒，将脱气溶液。留下未使用的缓冲密封。 </li><li>准备基质血管分数（SVF）基础培养基:Dulbecco&#39;s-Modified Eagles培养基（DMEM）:F12,15％胎牛血清（FCS），1％抗生素/抗真菌剂（10,000单位/ mL青霉素，10,000μg/ mL链霉素，25μg / mL两性霉素B），44.05mM碳酸氢钠，100μM抗坏血酸，33μM生物素，17μM泛酸盐，2mM L-谷氨酰胺和20mM HEPES。保持无菌，在4℃下长达2周。 </li><li>准备APC＆白细胞抑制因子（10ng / mL），血小板衍生生长因子BB（10ng / mL）和碱性成纤维细胞生长因子（10ng / mL）的培养基:具有额外生长因子（包括表皮生长因子10ng / mL） 5ng / mL）。保持无菌，在4℃下长达2周。 </li><li>准备APC感应介质:具有10％FBS，2.5μg/ mL胰岛素，0.5mM 2-异丁基-1-甲基黄嘌呤（IBMX），1μM地塞米松和200pM T3（三碘甲状腺原氨酸甲状腺激素）的APC培养基。保持无菌，在4℃下长达2周。 </li></ol>脂肪细胞祖细胞分离<ol><li>根据制度指导麻醉大鼠。将大鼠置于背侧卧位。通过脚趾压缩确认麻醉深度，并对这种痛苦的刺激失去反射反应。 注意:该方案使用10周龄的Sprague Dawley大鼠和70mg / kg通过腹膜内注射递送的戊巴比妥。 </li><li> 做一个垂直的中线切口w沿着胸部胸部和会阴区域的剪刀。进入腹腔并暴露肠系膜上动脉，小肠系膜阻力血管（mPVAT）和胸主动脉（aPVAT）。 <ol><li>切断与肠系膜和主动脉的所有连接，并从动物中移除血管。通过使用解剖显微镜和填充KRBB的培养皿分离PVAT以观察血管并分离PVAT。 </li><li>在本实验中，收集性腺（GON）脂肪以代表非PVAT脂肪贮库。将含有10mM HEPES（pH = 7.4）的KRBB中的冰分离的脂肪垫放在冰上。 </li><li>在生物安全罩中，将约50mg的组织转移到具有1mL胶原酶I型溶液的1.7mL管中，并用组织剪刀（1-3mm片）切碎。 </li></ol></li><li>通过在37℃下在旋转栅栏培养箱（或用轨道振荡器的培养箱）中孵育1小时来摘取样品。在生物安全罩中，通过100和40顺序过滤消化的物质<html> μm细胞过滤器放入50mL管中。在300℃下，将滤液在4℃下离心10分钟。 注意:从这里向前的协议中的所有步骤都将在生物安全罩中进行，以保持细胞无菌。 </li><li>倒出上清液，将含有SVF细胞的小丸重新悬浮于1 mL红细胞裂解缓冲液溶液中，并转移至1.7 mL离心管中。在室温孵育细胞5分钟保护光，并在300℃下在4℃离心5分钟。 </li><li>倒出上清液，并重新悬浮SVF基础培养基中剩余的细胞沉淀。收集20μL亚样品，用台盼蓝溶液计数活细胞。 注意:可以隔离的SVF单元的数量因地点而异。每毫克组织收获的平均SVF平均数为:aPVAT = 5.0±2.0×10 3 ，mPVAT = 1.04±0.62×10 4 ，GON = 2.4±1.2×10 5 。 </li></ol>磁激活细胞分选_content"&gt;注意:通过在4°C下执行所有步骤，基于CD34和PDGFRα细胞表面标记从SVF分离APC。 <ol><li>旋转细胞在300×g下5分钟。倒出上清液，以1×10 6个细胞/ mL的方式将细胞沉淀重悬于MCS阻断缓冲液中并孵育20分钟。 注意:1 x 10 6 - 2 x 10 8细胞/ mL的细胞悬浮液可以有效分离。 </li><li> 在4℃下，用5μLFITC-偶联的小鼠抗CD34（1μg/ 1×10 6个细胞）孵育细胞30分钟。在300×g和4℃下旋转细胞5分钟。 <ol><li>在4℃下在黑暗中孵育4μL抗FITC微珠和96μLMCS缓冲液（总体积为100μL）5分钟以分离CD34 +和CD34-细胞。 </li></ol></li><li>将磁力分离器连接到支架上，并将MultiSort（MS）色谱柱放置在分离器的前面。放置5 mL收集管在MS柱下方的上管支架中。 </li><li>用MCS缓冲溶液冲洗准备MS柱。在柱顶上加入500μLMCS Buffer，让缓冲液通过。丢弃污水和更换收集管。 </li><li>将抗体标记的细胞悬浮液载载到制备的MS柱上。收集含有未标记细胞的流通。 </li><li>用500μL脱气的MCS Buffer 3x洗涤MS柱。收集通过的未标记细胞，并与前一步骤中的流程结合。 </li><li>从磁选机中取出MS柱，并将其放在新的收集管上。移取1 mL MCS缓冲液到MS柱上。立即用磁性标记的细胞冲洗部分，然后缓慢地施加柱子提供的柱塞，以防止多余的气体进入色谱柱。 注意:隔离的细胞编号将因现场而异。从SVF群体中分离的CD34 + APC的平均数量mg组织是:aPVAT = 2.6±0.43×10 2 ，mPVAT = 9.6±1.4×10 2 ，GON = 1.3±0.22×10 3 。 </li><li> 在300×g和4℃下旋转细胞5分钟。在4℃下将10μL的1:200溶液/ 1×10 6细胞兔抗PDGFRα中收集的CD34 +级分孵育30分钟。 <ol><li>在300×g和4℃下再次离心细胞5分钟。通过重复步骤3.3至3.7分离，用4μL抗兔IgG微珠和96μLMCS缓冲液孵育标记细胞。 注意:隔离的细胞编号将因现场而异。从每mg组织的CD34 +群体分离的PDGFRα + APC的平均数量是:aPVAT = 2.4±0.64，mPVAT = 8.4±2.4，GON = 10.4±1.9，其为先前分离群体的0.5-10％。 </li></ol></li></ol>细胞培养和脂肪生成诱导<ol><li>培养SVF和APC在6-Well组织培养板中的基础培养基中，每2天更换一次。在3次连续传代后，以1×10 2细胞/孔的黑色96孔组织培养板平板扩增测定，其在8,24,48和96小时以及50,000孔/孔在24孔板中评估，或10,000个细胞/孔在48孔组织培养板中用于脂肪形成测定，其是定性和定量的。 </li><li>补充APC基础培养基48 h后融合，诱导之前骨形态发生蛋白4（3.3 nmol / L），如分化所示10 。使用APC感应培养基在100％汇合（第0天）48小时后诱导细胞孵育细胞。 </li><li> 48小时后，更换培养基以维持APC Induction培养基中没有IBMX和地塞米松的细胞，持续14天，每48小时更换一次培养基。 </li><li> MCS隔离由FACS验证。 <ol><li>取50μL（50,000个细胞）的磁分离子样品并用FACS溶液洗涤。 </li><li>离心细胞并重悬于100μL的1,900驴抗兔IgG Dylight 405溶液中，以标记PDGFRα +细胞。孵育30分钟，保护光线和4°C。 </li><li>洗涤细胞并重悬于200μL的2％甲醛溶液中，直到用流式细胞仪分析488nm（FITC）和405nm（Dylight 405）滤膜的时间。 注意:使用亲本脂肪生成荧光测定法在微孔板读数器中测量荧光并使用前脂肪细胞作为脂质积累的校准物，定量评估培养细胞的脂质积累。脂质积累也通过脂质染料染色和在配备有照相机的倒置显微镜上进行的成像进行定量测量，使得可观察到或不含有脂质的总细胞的百分比。任何能够在485nm激发测量荧光并且在572处发射的读板器nm适用于分析以及具有能够捕获数字图像的相机的任何显微镜。 </li></ol></li></ol>

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作者没有什么可以披露的。

本实验的中心焦点是PVAT库的APC的分离，扩增和成脂诱导。在这里，我们提出了基于表达表面标志物CD34和PDGFRα的细胞的鉴定来分离APC的方案。这些表面蛋白先前在具有高增殖率的APC上鉴定，并且在各种脂肪贮积14,15中分化成白色或棕色脂肪细胞的潜力。通过选择基于这些特异性标记的细胞，我们能够从与所选择的PVAT中观察到的脂肪细胞表型匹配的多个脂肪库中分离相似的APC群体?...

血管周围脂肪组织（PVAT）由于其紧邻血管，是脉管系统功能1中的主要旁分泌信号成分。这种脂肪组织的扩张取决于存在于2,3的脂肪细胞祖细胞（APC）的表型。脂肪组织中细胞的分离是困难的，因为原发性脂肪细胞是脆弱的，浮力的和尺寸范围的。某些分离技术还可以通过增加炎症蛋白质合成和减少脂肪形成基因表达来改变细胞表型和形态，强调维持细胞完整性的协议的重要性。 原代细胞和特异性前脂肪细胞亚群的培养给出体内生长的还原性方法，并保持相当的细胞遗传组成5 ，尽管工作ti我与这些细胞由于老化或老化的恶化而受到限制6 。来自不同脂肪库（包括皮下和网膜库）的前脂肪细胞也表现出增殖7的差异，这强调了从特定解剖部位收集细胞的重要性。来自非PVAT白色脂肪贮库的前体细胞已经在先前的研究7,8,9中进行了表征，但是对于PVAT APC表型已知较少。 这里描述的技术允许对特定和定义的APC群体进行分析，对其形态，生存力以及增殖和分化潜力的影响最小。磁激活细胞分选（MCS）适用于下游应用，如培养，因为珠粒溶解而不改变细胞。 MCS也是经济的，一旦抗体结合浓度已经被标准化，流式细胞术检测的需要很小。使用PVAT前体的体外研究也可以看出这些原代细胞可能具有的潜力。

<table><tbody><tr><td>&lt;strong&gt;Tissue Dissection&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dissecting Dishes</td><td>Handmade with Silicone</td><td></td><td></td></tr><tr><td>Culture Petri Dish</td><td>Pyrex</td><td></td><td>7740 Glass</td></tr><tr><td>Silicone Elastomer</td><td>Dow Corning</td><td>Sylgard 170</td><td>Kit</td></tr><tr><td>Braided Silk Suture</td><td>Harvard Apparatus</td><td>51-7615</td><td>SP104</td></tr><tr><td>Stereomicroscope MZ6</td><td>Leica</td><td></td><td>10447254</td></tr><tr><td>Stereomaster Microscope Fiber-Optic Light Source</td><td>Fisher Scientific</td><td>12562-36</td><td></td></tr><tr><td>Vannas Scissors</td><td>George Tiemann &amp;amp; Co</td><td>160-150</td><td></td></tr><tr><td>Splinter Forceps</td><td>George Tiemann &amp;amp; Co</td><td>160-55</td><td></td></tr><tr><td>Tissue Scissors</td><td>George Tiemann &amp;amp; Co</td><td>105-400</td><td></td></tr><tr><td>&lt;strong&gt;KRBB Solution&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Sodium Chloride</td><td>Sigma-Aldrich</td><td>7647-14-5</td><td></td></tr><tr><td>Potassium Chloride</td><td>Sigma-Aldrich</td><td>7447-40-7</td><td></td></tr><tr><td>Magnesium Sulfate</td><td>Sigma-Aldrich</td><td>7487-88-9</td><td></td></tr><tr><td>Potassium Phosphate Dibasic</td><td>Sigma-Aldrich</td><td>7758-114</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>50-99-7</td><td></td></tr><tr><td>Antibiotic/Antimicotic</td><td>Corning</td><td>30-004-CI</td><td></td></tr><tr><td>HEPES</td><td>Corning</td><td>25-060-CI</td><td></td></tr><tr><td>&lt;strong&gt;Tissue Digestion&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Collagenase Type 1</td><td>Worthington Biochemical</td><td>LS004196</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Fisher Scientific</td><td>9048-46-8</td><td></td></tr><tr><td>Red Blood Cell Lysis Buffer</td><td>BioLegend</td><td>420301</td><td>1X Working Solution</td></tr><tr><td>Water Bath</td><td>Thermo-Fisher Scientific</td><td>2876</td><td>Reciprocal Shaking Bath</td></tr><tr><td>Biosafety Cabinet</td><td>Thermo-Fisher Scientific</td><td>1385</td><td></td></tr><tr><td>Rotisserie Incubator</td><td>Daigger</td><td>EF4894C</td><td></td></tr><tr><td>100 &amp;micro;m Cell Strainer</td><td>Thermo-Fisher Scientific</td><td>22-363-549</td><td>Yellow</td></tr><tr><td>40 &amp;micro;m Cell Strainer</td><td>Thermo-Fisher Scientific</td><td>22-363-547</td><td>Blue</td></tr><tr><td>Hemocytometer</td><td>Cole-Parmer</td><td>UX-79001-00</td><td></td></tr><tr><td>Trypan Blue</td><td>Sigma-Aldrich</td><td>93595</td><td></td></tr><tr><td>&lt;strong&gt;Cell Isolation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>OctoMACS Kit</td><td>Miltenyi Biotech</td><td>130-042-108</td><td></td></tr><tr><td>(DMEM):F12 Medium</td><td>Corning</td><td>90-090</td><td>Medium Base</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Corning</td><td>35016CV</td><td>USA Origins</td></tr><tr><td>Normal Donkey Serum</td><td>AbCam</td><td>AB7475</td><td></td></tr><tr><td>Anti-CD34</td><td>Santa Cruz</td><td>SC-7324</td><td>FITC-conjugated</td></tr><tr><td>Anti-PDGFR&amp;alpha;</td><td>Thermo-Fisher Scientific</td><td>PA5-17623</td><td></td></tr><tr><td>Donkey Anti-Rabbit IgG</td><td>Jackson ImmunoResearch</td><td>712-007-003</td><td></td></tr><tr><td>Phosphate-Buffered Saline (PBS) 10X</td><td>Corning</td><td>46-013-CM</td><td>1X Working Solution</td></tr><tr><td>EDTA</td><td>Fisher Scientific</td><td>15575020</td><td></td></tr><tr><td>&lt;strong&gt;Cell Culture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Cell Incubator</td><td>Thermo-Fisher Scientific</td><td>51030285</td><td>Heracell 160i&amp;nbsp;</td></tr><tr><td>6-Well Plates</td><td>Corning</td><td>3516</td><td>TC-Treated</td></tr><tr><td>48-Well Plates</td><td>Corning</td><td>3548</td><td>TC-Treated</td></tr><tr><td>96-Well Plates, Black Wall</td><td>Corning</td><td>353376</td><td>TC-Treated</td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma-Aldrich</td><td>144-55-8</td><td>TC-Treated</td></tr><tr><td>Fetal Calf Serum (FCS)</td><td>Corning</td><td>35011CV</td><td>USA Origins</td></tr><tr><td>Ascorbic Acid</td><td>Sigma-Aldrich</td><td>50-81-7</td><td></td></tr><tr><td>Biotin</td><td>Sigma-Aldrich</td><td>58-85-5</td><td></td></tr><tr><td>Pantothenate</td><td>Sigma-Aldrich</td><td>137-08-6</td><td></td></tr><tr><td>L-Glutamine</td><td>Corning</td><td>61-030</td><td></td></tr><tr><td>Bone Morphogenic Protein 4 (BMP4)</td><td>Prospec Bio</td><td>CYT-081</td><td></td></tr><tr><td>Epidermal Growth Factor (EGF)</td><td>PeproTech</td><td>400-25</td><td></td></tr><tr><td>Leukemia Inhibitory Factor</td><td>PeproTech</td><td>250-02</td><td></td></tr><tr><td>Platelet-derived Growth Factor BB</td><td>Prospec Bio</td><td>CYT-740</td><td></td></tr><tr><td>Basic Fibroblast Growth Factor (bFGF)</td><td>PeproTech</td><td>450-33</td><td></td></tr><tr><td>Insulin</td><td>Corning</td><td>25-800-CR</td><td>ITS Solution</td></tr><tr><td>IBMX</td><td>Sigma-Aldrich</td><td>28822-58-4</td><td></td></tr><tr><td>Dexamethasone</td><td>Sigma-Aldrich</td><td>50-02-2</td><td></td></tr><tr><td>T3 (Triiodothyronine)</td><td>Sigma-Aldrich</td><td>6893-023</td><td></td></tr><tr><td>&lt;strong&gt;Cell Analysis&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CyQUANT Proliferation Assay</td><td>Thermo-Fisher Scientific</td><td>C7026</td><td></td></tr><tr><td>AdipoRed Fluorescence Assay Reagent</td><td>Lonza</td><td>PT-7009</td><td></td></tr><tr><td>Oil Red O Lipid Dye Reagent</td><td>Sigma-Aldrich</td><td>O1391</td><td>In Solution</td></tr><tr><td>M1000 Microplate Reader</td><td>Tecan</td><td></td><td></td></tr><tr><td>Eclipse Inverted Microscope</td><td>Nikon</td><td></td><td></td></tr><tr><td>Digital Sight DS-Qil Camera</td><td>Nikon</td><td></td><td></td></tr></tbody></table>

expansion and adipogenesis induction of adipocyte progenitors from perivascular adipose tissue isolated by magnetic activated cell sorting

通过旁分泌信号传导血管功能的主要调节剂血管周围脂肪组织（PVAT）的扩张与肥胖症中高血压的发展直接相关。肥大和增生的程度取决于储存位置，性别和存在的脂肪细胞祖细胞（APC）表型的类型。在过去十年中用于APC和前脂肪细胞分离的技术大大提高了基于特定细胞表面标志物识别个体细胞的准确性。然而，由于细胞的脆弱性，APC和脂肪细胞的分离可能是一个挑战，特别是如果必须保留完整细胞用于细胞培养应用。 磁性细胞分选（ MCS）提供了一种分离更多数量的活力APC每重量单位的脂肪组织的方法。通过MCS收获的APC可以用于体外方案扩大prea通过使用生长因子鸡尾酒将其分化成脂肪细胞，从而分析由细胞保留的多产和成脂潜能。该实验主要集中在主动脉和肠系膜PVAT库，其在扩张期间对心血管疾病的发展起关键作用。这些协议描述了分离，扩展和区分定义的APC群体的方法。该MCS协议允许隔离用于需要最少设备或培训的细胞分选的任何实验。这些技术可以帮助进一步的实验以基于细胞表面标志物的存在来确定特定细胞群的功能。

脂肪细胞前脂肪细胞的增殖能力和脂肪细胞前体的脂肪形成潜力是体外维持的特征11 。 在使用定量DNA测定法在8,24,48和96小时后评估雄性大鼠的aVVAT，mPVAT和GON的分离的SVF和APC的体外增殖。在与来自同一位点的SVF细胞相比，与来自aPVAT的APC相比，在96小时时增殖较少的APC，在任何时间点均未观察到SVF扩增率的位置差异（ 图...

Watch this Scientific Journal Video about Expansion and Adipogenesis Induction of Adipocyte Progenitors from Perivascular Adipose Tissue Isolated by Magnetic Activated Cell Sorting at JoVE.com

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

在这里，我们报告了使用磁激活细胞分选（MCS）从血管周围脂肪组织（PVAT）分离脂肪细胞祖细胞（APC）种群的方法。当与荧光活化细胞分选（FACS）相比时，该方法允许APC每克脂肪组织的APC分离增加。

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 ...

expansion-adipogenesis-induction-adipocyte-progenitors-from

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

9.4K 次观看.  Michigan State University. 在这里，我们报告了使用磁激活细胞分选（MCS）从血管周围脂肪组织（PVAT）分离脂肪细胞祖细胞（APC）种群的方法。当与荧光活化细胞分选（FACS）相比时，该方法允许APC每克脂肪组织的APC分离增加。 

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

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells (ASCs)

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix (ECM) remodeling. In vivo, ECM-rich environment consisting of fibrillar collagens provides a structural support to adipose tissues during the progression and regression of obesity. Physiological ECM remodeling mediated by matrix metalloproteinases (MMPs) plays a major role in regulating adipose tissue size and function1,2. The loss of physiological collagenolytic ECM remodeling may lead to excessive collagen accumulation (tissue fibrosis), macrophage infiltration, and ultimately, a loss of metabolic homeostasis including insulin resistance3,4. When a phenotypic change of the adipose tissue is observed in gene-targeted mouse models, isolating primary ASCs from fat depots for in vitro studies is an effective approach to define the role of the specific gene in regulating the function of ASCs. In the following, we define an immunomagnetic separation of Sca1high ASCs.

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs

We present the techniques required to isolate the stromal vascular fraction (SVF) from mouse inguinal (subcutaneous) and perigonadal (visceral) adipose tissue depots to assess their gene expression and collagenolytic activity. This method includes the enrichment of Sca1high adipose-derived stem cells (ASCs) using immunomagnetic cell separation.

脂肪库专用SCA1的免疫磁性分离<sup&gt;高</sup&gt;脂肪干细胞（ASCs）

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix ...

科研

发育生物学

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 ...

<meta charset="utf8"></head><body>用于纤维成脂祖细胞培养的小鼠心脏和骨骼肌消化

Fibro-Adipogenic Progenitor Isolation, Expansion, and Differentiation from the Spiny Mouse Model

Due to its exceptional repair program, the spiny mouse is an emerging research model for regenerative medicine. Fibro-adipogenic progenitors are tissue-resident cells that are able to differentiate into adipocytes, fibroblasts, and chondrocytes. Fibro-adipogenic progenitors are fundamental for orchestrating tissue regeneration as they are responsible for extracellular matrix remodeling after injury. This study focuses on investigating the specific role of fibro-adipogenic progenitors in spiny mouse cardiac repair and skeletal muscle regeneration. To this end, a protocol has been optimized for the purification of spiny mouse fibro-adipogenic progenitors by flow cytometry from enzymatically dissociated skeletal and cardiac muscle. The population obtained from this protocol is capable of expanding in vitro, and can be differentiated to myofibroblasts and adipocytes. This protocol offers a valuable tool for researchers to examine the distinctive properties of spiny mouse, and to compare them to the Mus musculus. This will provide insights that could advance the understanding of regenerative mechanisms in this intriguing model.

<meta charset="utf8"></head><body>作者聚焦：使用研究棘状小鼠模型的创新方案简化细胞分析

This protocol outlines the isolation of skeletal and cardiac muscle fibro-adipogenic progenitors (FAPs) from spiny mouse (Acomys) via enzymatic dissociation and fluorescence-activated cell sorting. The FAPs obtained from this protocol can be effectively expanded and differentiated to myofibroblasts and adipocytes.

来自 Spiny 小鼠模型的 Fibro-Adipogenic Progenitor 分离、扩增和分化

Due to its exceptional repair program, the spiny mouse is an emerging research model for regenerative medicine. Fibro-adipogenic progenitors are ...

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

Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute functionally to the expansion and remodeling of WAT in adulthood. A tremendous barrier to studying the implications of this cellular heterogeneity is the inability to readily isolate functionally distinct cell subpopulations from WAT SVF for in vitro and in vivo analyses. Single-cell sequencing technology has recently identified functionally distinct fibro-inflammatory and adipogenic PDGFR&#946;+ perivascular cell subpopulations in intra-abdominal WAT depots of adult mice. Fibro-inflammatory progenitors (termed, &#8220;FIPs&#8221;) are non-adipogenic collagen producing cells that can exert a pro-inflammatory phenotype. PDGFR&#946;+ adipocyte precursor cells (APCs) are highly adipogenic both in vitro and in vivo upon cell transplantation. Here, we describe multiple methods for the isolation of these stromal cell subpopulations from murine intra-abdominal WAT depots. FIPs and APCs can be isolated by fluorescence-activated cell sorting (FACS) or by taking advantage of biotinylated antibody-based immunomagnetic bead technology. Isolated cells can be used for molecular and functional analysis. Studying the functional properties of stromal cell subpopulation in isolation will expand our current knowledge of adipose tissue remodeling under physiological or pathological conditions on the cellular level.

This protocol describes the technical approach to isolate adipogenic and fibro-inflammatory stromal cell subpopulations from murine intra-abdominal white adipose tissue (WAT) depots by fluorescence-activated cell sorting or immunomagnetic bead separation.

从小鼠腹内脂肪库分离成脂肪和纤维炎症基质细胞亚群

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute ...

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 ...

小鼠白色脂肪组织的半自动分离

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their mechanisms. Immortalized white preadipocyte cell lines are publicly available and widely used. However, the emergence of beige adipocytes in white adipose tissue in response to external cues is difficult to recapitulate to the full extent using publicly available white adipocyte cell lines. Isolation of the stromal vascular fraction (SVF) from murine adipose tissue is commonly executed to obtain primary preadipocytes and perform adipocyte differentiation. However, mincing and collagenase digestion of adipose tissue by hand can result in experimental variation and is prone to contamination. Here, we present a modified semi-automated protocol that utilizes a tissue dissociator for collagenase digestion to achieve easier isolation of the SVF, with the aim of reducing experimental variation, reducing contamination, and increasing reproducibility. The obtained preadipocytes and differentiated adipocytes can be used for functional and mechanistic analyses.

作者聚焦：使用组织解离剂从小鼠白色脂肪组织中半自动分离基质血管组分  

This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases reproducibility.

使用组织解离器从小鼠白色脂肪组织中半自动分离基质血管部分

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their ...

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

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