Name: isolation, culture, and adipogenic induction of neural crest original adipose-derived stem cells from periaortic adipose tissue
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Description: Watch this Scientific Journal Video about Isolation, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue at JoVE.com

国家重点研发项目（2018YFC1312504），国家自然科学基金（81970378、81670360、81870293）和上海市科委（17411971000，17140902402）为本研究提供资金.

动物规程经上海交通大学动物护理委员会审查批准。
1. 生成 Wnt-1 Cre+/-;罗莎26RFP/+小鼠
<ol><li>交叉 Wnt-1 Cre+/-小鼠16与 Rosa26RFP/+小鼠18生成 Wnt-1 Cre+/-;罗莎26RFP/+小鼠。在25°C和45%湿度的无病原体设施中，在12小时光/暗循环下，将小鼠在4~8周大。</li>
</ol>2. PaAT的解剖
<p class="jove...

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在这项研究中，我们提出了一种可靠的方法，用于从Wnt-1 Cre+/-的PVAT中提取的NCADSCs的分离、培养和异位诱导;Rosa26RFP/+转基因小鼠，旨在生产RFP+ADSC。以前的报告显示，在NCADSCs和非NCADSCs22中，一般多能性等位干细胞（MSCs）标记的表达没有显著差异，而且NCADSCs具有很强的在体外15、22、23体细胞中分化成腺?...

全世界肥胖症的患病率正在上升，这增加了患心血管疾病和糖尿病等相关慢性病的风险。PVAT环绕血管，是血管功能中涉及的内分泌和副项的主要来源。临床研究表明，高PVAT含量是心血管疾病2、3的独立危险因素，其病理功能取决于组成脂肪衍生干细胞（ADSCs）4的表型。
虽然ADSC细胞系，如鼠3T3-L1，3T3-F442A和OP9是研究腺生成或脂肪发生5的有用细胞模型，但细胞系和原细胞之间，腺化的调控机制不同。基质血管细胞部分（SVF）中的ADSCs直接从脂肪组织分离出来，并诱导分化成腺细胞，最有可能在体内重述脂肪发生和脂肪发生6。然而，ADSCs的脆弱性、浮力以及大小和免疫表型的变化使得它们直接隔离具有挑战性。此外，不同的分离程序也会显著影响这些细胞7的表型和增生电位能力，从而强调需要一种维持ADSC完整性的协议。
脂肪组织通常被归类为形态和功能上截然不同的白色脂肪组织（WAT），或棕色脂肪组织（BAT）8，其中含有不同的ADSCs9。虽然在以前的研究中，从围肠和肠下WAT分离的ADSCs在以前的研究中具有特征9，10，11，12，较少知道从PVAT的ADSC，主要由BAT13组成。
在最近的一项研究中，我们发现，围阴弓脂肪组织（PAAT）中一部分常驻ADSC来自神经峰细胞（NcCs），这是一种来自前体14、15的迁移祖细胞的瞬态种群。Wnt1-Cre转基因小鼠用于追踪神经峰细胞发育16、17。我们越过Wnt1-Cre+小鼠与罗莎26RFP/+小鼠生成Wnt-1 Cre[/-;Rosa26RFP/+小鼠，其中NcCs及其后代被标记与红色荧光蛋白（RFP），并很容易跟踪在体内和体外15。在这里，我们描述了一种从小鼠PAAT中分离神经峰衍生ADSCs（NC衍生ADSCs，或NCADSCs）的方法，并诱导NcADSCs分化成白色脂肪细胞或棕色腺细胞。

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		<tr>
			<td>4% PFA</td>
			<td>BBI life sciences</td>
			<td>E672002-0500</td>
			<td>Lot #: EC11FA0001</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>ABCONE (China)</td>
			<td>A47902</td>
			<td>1% working concentration</td>
		</tr>
		<tr>
			<td>Anti-cebp/&#945;</td>
			<td>ABclonal</td>
			<td>A0904</td>
			<td>1:1000 working concentration</td>
		</tr>
		<tr>
			<td>Anti-mouse IgG, HRP-linked</td>
			<td>CST</td>
			<td>7076</td>
			<td>1:5000 working concentration</td>
		</tr>
		<tr>
			<td>Anti-perilipin</td>
			<td>Abcam</td>
			<td>AB61682</td>
			<td>1 &#956;g/mL working concentration; lot #: GR66486-54</td>
		</tr>
		<tr>
			<td>Anti-PPARy</td>
			<td>SANTA CRUZ</td>
			<td>sc-7273</td>
			<td>0.2 &#956;g/mL working concentration</td>
		</tr>
		<tr>
			<td>Anti-rabbit IgG, HRP-linked</td>
			<td>CST</td>
			<td>7074</td>
			<td>1:5000 working concentration</td>
		</tr>
		<tr>
			<td>Anti-&#946;-Tubulin</td>
			<td>CST</td>
			<td>2146</td>
			<td>1:1000 working concentration</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>VWR life sciences</td>
			<td>0332-100G</td>
			<td>50 mg/mL working concentration; lot #: 0536C008</td>
		</tr>
		<tr>
			<td>Collagenase, Type I</td>
			<td>Gibco</td>
			<td>17018029</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma-Aldrich</td>
			<td>D4902</td>
			<td>0.1 &#181;M working concentration</td>
		</tr>
		<tr>
			<td>Erythrocyte Lysis Buffer</td>
			<td>Invitrogen</td>
			<td>00-4333</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Corning</td>
			<td>R35-076-CV</td>
			<td>50 mg/mL working concentration; lot #: R2040212FBS</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Gibco</td>
			<td>14025092</td>
			<td></td>
		</tr>
		<tr>
			<td>HDMEM</td>
			<td>Gelifesciences</td>
			<td>SH30243.01</td>
			<td>Lot #: AD20813268</td>
		</tr>
		<tr>
			<td>IBMX</td>
			<td>Sigma-Aldrich</td>
			<td>I7018</td>
			<td>0.5 mM working concentration</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>I3536</td>
			<td>1 &#956;g/mL working concentration</td>
		</tr>
		<tr>
			<td>Microsurgical forceps</td>
			<td>Suzhou Mingren Medical Equipment Co.,Ltd. (China)</td>
			<td>MR-F201A-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsurgical scissor</td>
			<td>Suzhou Mingren Medical Equipment Co.,Ltd. (China)</td>
			<td>MR-H121A</td>
			<td></td>
		</tr>
		<tr>
			<td>Oil Red O solution</td>
			<td>Sigma-Aldrich</td>
			<td>O1516</td>
			<td>0.3% working concentration</td>
		</tr>
		<tr>
			<td>PBS (Phosphate buffered saline)</td>
			<td>ABCONE (China)</td>
			<td>P41970</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>PrimeScript RT reagent Kit</td>
			<td>TAKARA</td>
			<td>RR047A</td>
			<td>Lot #: AK4802</td>
		</tr>
		<tr>
			<td>RNeasy kit</td>
			<td>TAKARA</td>
			<td>9767</td>
			<td>Lot #: AHF1991D</td>
		</tr>
		<tr>
			<td>Rosa26RFP/+ mice</td>
			<td>JAX</td>
			<td>No.007909</td>
			<td>C57BL/6 backgroud; male and female</td>
		</tr>
		<tr>
			<td>Rosiglitazone</td>
			<td>Sigma-Aldrich</td>
			<td>R2408</td>
			<td>1 &#956;M working concentration</td>
		</tr>
		<tr>
			<td>Standard forceps</td>
			<td>Suzhou Mingren Medical Equipment Co.,Ltd. (China)</td>
			<td>MR-F424</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissor</td>
			<td>Suzhou Mingren Medical Equipment Co.,Ltd. (China)</td>
			<td>MR-S231</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Premix Ex Taq</td>
			<td>TAKARA</td>
			<td>RR420A</td>
			<td>Lot #: AK9003</td>
		</tr>
		<tr>
			<td>Triiodothyronine</td>
			<td>Sigma-Aldrich</td>
			<td>T2877</td>
			<td>10 nM working concentration</td>
		</tr>
		<tr>
			<td>Wnt1-Cre+;PPAR&#947;flox/flox mice</td>
			<td>JAX</td>
			<td>No.009107</td>
			<td>C57BL/6 backgroud; male and female</td>
		</tr>
	</tbody>
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isolation, culture, and adipogenic induction of neural crest original adipose-derived stem cells from periaortic adipose tissue

血管周围过多的脂肪组织（血管内脂肪组织，也称为PVAT）与心血管疾病的高风险有关。从不同的脂肪组织衍生的ADSCs表现出明显的特征，而来自PVAT的ADSC的特征并不明显。在最近的一项研究中，我们报告说，围阴弓脂肪组织（PAAT）中的一些ADSCs从神经峰细胞（NcCs）下降，这是一种来自外端的迁移细胞的瞬态群体。
在本文中，我们描述了从Wnt-1 Cre+/-的PAAT中分离红色荧光蛋白（RFP）标记的NCC的协议;Rosa26RFP/+小鼠在体外诱导其异体分化。简单地说，基质血管分数（SVF）与PAAT酶分离，RFP+神经波峰衍生的ADSCs（NCADSCs）通过荧光活化细胞分选（FACS）分离。NCADSC 分化成棕色和白色的脂肪细胞，可以冷冻保存，并保留其可吸波潜力的 +3⁄5 段落。我们的协议可以从PVAT生成大量的ADSCs，用于在体外模拟PVAT腺化或脂肪发生。因此，这些NCADSCs可以为研究PVAT分化所涉及的分子开关提供有价值的系统。

使用上述协议，我们从 5⁄6 Wnt-1 Cre+/-获得 ±0.5±1.0 x 106 ADSCs;Rosa26RFP/+小鼠（48周大，雄性或雌性）。
图1显示了从小鼠中收集PAAT的流程图。NCADSCs的形态与其他小鼠脂肪组织的ADSC相似。培养的NCADSC在培养7~8天后达到80-90%的汇合，而NCADSC具有扩大的成纤维细胞样形态（图2B，C）。
<p class="jove_cont...

Watch this Scientific Journal Video about Isolation, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue at JoVE.com

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

我们提出了从Wnt-1 Cre+/-的围阴脂肪组织中提取神经峰衍生的干细胞（NCADSCs）的分离、培养和异位诱导方案;罗莎26RFP/+小鼠。NCADSCs 是 ADSC 的一个容易获取的来源，用于在体外模拟脂肪发生或脂肪发生。

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 ...

我们简单实用的方法可用于获得丰富的ADSCS研究PVAT体外脂肪发生，并测试正常药物对抗肥胖和相关心血管疾病。此方法的优点是生物素的 NCADSC 的固有荧光，您需要为细胞贴上用于 FACS 的 flou-铬结合抗体的标签，或磁激活细胞分选。该方法可用于从外皮细胞中获得丰富的ADSC，用于研究PVAT脂肪生成，或体外光生成。使用年轻的小鼠和有烧灼和诱导性致病的经验意味着3T3细胞是成功的关键，该协议的成功。演示这个程序的将是研究生齐一丁和我实验室的技术员徐连。对于PAAT解剖，在用75%乙醇对小鼠尸体进行消毒5分钟后，使用剪刀剪断腹部的皮肤并分离。沿着从骨盆到颈部的腹中线切开，然后打开腹部。移动肝脏以暴露膜片，并切割中线两侧的隔膜和肋骨。剥回肋骨，露出心肺。取出肺和胸腺。将 PAAT 与大菜和心脏一起提取到培养皿中。切断大音管的母管根切掉心脏。然后，在大音和下降大音之间做一个切口。小心地将两个结构周围的脂肪组织以及左右常见的角状动脉与后胸壁分开。重要的是要将PAAT与根部分离，并且作为一个更大的血管血管，血管壁细胞的存在会影响FACS的效率。将组织放入含有冰冷HSS缓冲液的培养皿中。并使用无菌钳子去除尽可能多的血管和筋膜。然后，将脂肪组织转移到两毫升Eppendorf管中，在冰上含有5毫升的冰冷HSS缓冲液。当所有PAAT被收集后，将收获的脂肪组织转移到一个新的两毫升微离心管中，其中包含一毫升新鲜准备的消化介质。用手术剪刀把组织切碎。将组织浆料转移到含有9毫升新鲜消化介质的50毫米管中。并使用一毫升移液器对组织进行10次三角化。获得均匀溶液后，在37摄氏度下孵育管30至45分钟，每分钟100转，每5至10分钟检查一次，以防止过度消化不良。当一个浅黄色同质脂肪组织溶液可以观察到在轻轻的旋转管，停止消化与5毫升HDMEM辅以10%胎儿牛血清。经过彻底混合后，将脂肪组织样品离心。频闪血管细胞分数将可见为棕色颗粒。将颗粒重新悬浮在培养培养的10毫升中，并通过70微米细胞滤株过滤细胞悬浮液。通过另一个离心收集细胞。轻轻将颗粒重新悬浮在五毫升红细胞解液缓冲液中。将悬浮液转移到 15 毫升管中。10分钟后，在10毫升PBS中用两个离心停止反应，并辅以1%的胎儿牛血清。第二次离心后，将颗粒重新悬浮在五毫升培养培养剂的冰上。并收集细胞与最后的离心。然后，将细胞重新挂起在五毫升的 FACS 缓冲液中进行计数。为了建立一个NCADSC培养物，在按照标准协议被FACS隔离后，将细胞放在每厘米平方浓度的5倍至第三细胞，一个12个井培养板。不完全的培养基在37摄氏度和5%的二氧化碳20至24小时。第二天，用37摄氏度的PBS清洗细胞，用新鲜的培养媒介喂养细胞。当培养达到80至90%的汇合时，用每井0.25%的两毫升二毫升处理细胞，在37摄氏度下治疗3至5分钟。当细胞从板底分离时，用两毫升培养基中和酶，通过离心收集收获的细胞。将颗粒重新悬浮在一毫升新鲜培养介质中进行计数。并在新的12井培养板中播种细胞，每厘米浓度为5倍至3个细胞。然后，重新悬浮培养基中剩余的细胞，并辅以10%二甲基硫化物，冷冻储存在液氮中。为了诱导致原性分化，当细胞达到80-90%的汇合时，酌情用棕色或白色致原诱导介质治疗细胞两天。在孵化结束时，用PBS轻轻清洗细胞两次，然后向每个井中加入适当的新鲜致生诱导培养基，将细胞返回到细胞培养箱中再进行三到五天的孵化。养殖的 NCADSC 在培养七到八天后达到 80% 到 90% 的汇合率。并演示一种膨胀的玻璃纤维，如形态学。为了进一步确认其致病性潜力，可以对分化的NCADSC进行油红色染色，以检测成熟的脂肪细胞。通常，在8天的白色或棕色致原诱导后观察到成熟的脂肪细胞，超过60%的NCADSC表现出致发性分化。请注意，NCADSC 在传传后表现出大大降低的致生潜力。免疫印迹和定量实时PCR显示，在8天的白色脂肪诱导后，脂肪细胞特异性相关蛋白质和基因的表达在脂肪分化的NCADSC中显著增加。同样，在八天棕色脂肪诱导后，脂肪细胞特异性基因和棕色脂肪细胞特异性基因的表达也显著增加。由于大多数NCADSC分离协议简单、经济，不需要使用抗体，因此，IFP的强流体原因强度可以提高FACS的营养效率。

Research

JoVE Journal

Developmental Biology

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

隔离和人力富集脂肪基质细胞的成骨增强

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the ...

科研

发育生物学

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

人类神经干细胞从外周血造血祖细胞直接诱导

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

培养人多能性和神经干细胞的基本和临床前研究一个封闭的细胞培养系统

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

隔离和脂肪干细胞分化的猪从皮下脂肪组织

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

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

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

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 ...

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

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 ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

从人多能干细胞使用高度可重复的小分子为基础的分化协议心肌细胞规模化生产

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Chondrogenic Differentiation Induction of Adipose-derived Stem Cells by Centrifugal Gravity

脂肪干细胞的软骨细胞分化诱导离心重力

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes ...

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

BMPR-IB +快速分离脂肪来源的使用基质细胞在颅骨缺损愈合模型

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own ...

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

在体内荧光亲脂膜染料对大鼠膝骨关节炎模型中人脂肪间充质干细胞的追踪

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the ...