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
1Department of Cardiology, Shanghai Chest Hospital,Shanghai Jiao Tong University, 2Shanghai Institute of Nutrition and Health, Shanghai Institute for Biological Sciences,Chinese Academy of Sciences, 3Shanghai Key Laboratory of Hypertension, Shanghai Institute of Hypertension,Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, 4Department of Cardiology, Shanghai General Hospital,Shanghai Jiao Tong University School of Medicine, 5Department of Cardiology, Shanghai Key Laboratory of Clinical Geriatric Medicine,Huadong Hospital Affiliated to Fudan University
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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 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。 2. PaAT的解剖 <p class="jove…
Representative Results
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…
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| 4% PFA | BBI life sciences | E672002-0500 | Lot #: EC11FA0001 |
| Agarose | ABCONE (China) | A47902 | 1% working concentration |
| Anti-cebp/α | ABclonal | A0904 | 1:1000 working concentration |
| Anti-mouse IgG, HRP-linked | CST | 7076 | 1:5000 working concentration |
| Anti-perilipin | Abcam | AB61682 | 1 μg/mL working concentration; lot #: GR66486-54 |
| Anti-PPARy | SANTA CRUZ | sc-7273 | 0.2 μg/mL working concentration |
| Anti-rabbit IgG, HRP-linked | CST | 7074 | 1:5000 working concentration |
| Anti-β-Tubulin | CST | 2146 | 1:1000 working concentration |
| BSA | VWR life sciences | 0332-100G | 50 mg/mL working concentration; lot #: 0536C008 |
| Collagenase, Type I | Gibco | 17018029 | |
| Dexamethasone | Sigma-Aldrich | D4902 | 0.1 µM working concentration |
| Erythrocyte Lysis Buffer | Invitrogen | 00-4333 | |
| FBS | Corning | R35-076-CV | 50 mg/mL working concentration; lot #: R2040212FBS |
| HBSS | Gibco | 14025092 | |
| HDMEM | Gelifesciences | SH30243.01 | Lot #: AD20813268 |
| IBMX | Sigma-Aldrich | I7018 | 0.5 mM working concentration |
| Insulin | Sigma-Aldrich | I3536 | 1 μg/mL working concentration |
| Microsurgical forceps | Suzhou Mingren Medical Equipment Co.,Ltd. (China) | MR-F201A-1 | |
| Microsurgical scissor | Suzhou Mingren Medical Equipment Co.,Ltd. (China) | MR-H121A | |
| Oil Red O solution | Sigma-Aldrich | O1516 | 0.3% working concentration |
| PBS (Phosphate buffered saline) | ABCONE (China) | P41970 | |
| Penicillin-Streptomycin | Gibco | 15140122 | |
| PrimeScript RT reagent Kit | TAKARA | RR047A | Lot #: AK4802 |
| RNeasy kit | TAKARA | 9767 | Lot #: AHF1991D |
| Rosa26RFP/+ mice | JAX | No.007909 | C57BL/6 backgroud; male and female |
| Rosiglitazone | Sigma-Aldrich | R2408 | 1 μM working concentration |
| Standard forceps | Suzhou Mingren Medical Equipment Co.,Ltd. (China) | MR-F424 | |
| Surgical scissor | Suzhou Mingren Medical Equipment Co.,Ltd. (China) | MR-S231 | |
| SYBR Premix Ex Taq | TAKARA | RR420A | Lot #: AK9003 |
| Triiodothyronine | Sigma-Aldrich | T2877 | 10 nM working concentration |
| Wnt1-Cre+;PPARγflox/flox mice | JAX | No.009107 | C57BL/6 backgroud; male and female |
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Cite This Article
Qi, Y., Miao, X., Xu, L., Fu, M., Peng, S., Shi, K., Li, J., Ye, M., Li, R. Isolation, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue. J. Vis. Exp. (157), e60691, doi:10.3791/60691 (2020).