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
Summary
该方案允许从诱导多能干细胞（iPSC）产生纯脂肪细胞群。维甲酸用于将iPSCs分化为间充质干细胞（MSC），用于产生脂肪细胞。然后，采用基于尼罗河红染色的分选方法获得纯脂肪细胞。
Abstract
诱导多能干细胞（iPSC）技术的最新进展允许产生不同的细胞类型，包括脂肪细胞。然而，目前的分化方法效率低，不能产生均匀的脂肪细胞群。在这里，我们通过使用基于全反式视黄体的方法以高产量生产间充质干细胞（MSCs）来规避这个问题。通过调节控制细胞增殖、存活和粘附的途径，我们的分化策略允许有效生成分化成纯多能MSC群体的胚胎体（EB）。该方法产生的大量MSC为产生脂肪细胞提供了理想的来源。然而，脂肪细胞分化导致的样品异质性仍然是一个挑战。因此，我们使用基于尼罗河红的方法使用FACS纯化含脂成熟脂肪细胞。这种分选策略使我们能够建立一种可靠的方法来使用具有降低样品异质性和增强细胞功能的脂肪细胞池来模拟脂肪细胞相关的代谢紊乱。
Introduction
间充质干细胞（MSCs）是产生中胚层起源细胞（如脂肪细胞，骨细胞和软骨细胞）的有效短暂资源，可以进一步用于模拟其各自的遗传疾病。然而，以前的方法依赖于从成人组织1获得这些MSC，这带来了从供体获得大量MSC的挑战，并且限制了它们在次优体外培养条件下保持功能存活1，2。这些障碍产生了对体外产生MSCs的方案的巨大需求。人诱导多能干细胞（iPSCs）可用作MSC的宝贵来源，表现出MSC特征3，4，5。iPSCs衍生的间充质干细胞可用作多种疾病的治疗选择。此外，iPSCs衍生的MSCs产生脂肪细胞的能力使其成为研究人类脂肪生成，肥胖和脂肪细胞相关疾病的有价值的体外人类模型。
目前脂肪细胞的分化方案可分为两组，一组涉及使用化学或基于蛋白质的混合物分化脂肪细胞，结果产量为30%-60%6，7，8，9，而另一组涉及遗传操作以稳健地诱导控制脂肪细胞发育的关键转录因子，以产生80%-90%10的产量，11.然而，基因操作并不能概括脂肪细胞分化的自然过程，并且经常掩盖脂肪生成过程中到达的微妙范式，使其对疾病建模目的无效12，13。因此，我们提出了一种通过使用尼罗河红荧光标记含脂脂肪细胞来将化学衍生的成熟脂肪细胞与未成熟脂肪细胞进行分类的方法。
在这里，我们提出了一种协议，涉及将iPSC衍生的拟胚体（EB）与全反式 维甲酸瞬时孵育以产生大量快速增殖的MSC，这可以进一步用于产生脂肪细胞14。我们还提出了一种通过使用亲脂性染料荧光标记其脂滴来从异质分化池中分选化学衍生的成熟脂肪细胞的方法;尼罗河红。这将允许产生具有增强功能的成熟脂肪细胞的纯群体，以准确模拟脂肪细胞相关的代谢紊乱。
Protocol
该研究已获得适当的机构研究伦理委员会的批准，并按照1964年《赫尔辛基宣言》及其后来的修正案或类似的伦理标准中规定的伦理标准进行。该协议已获得HMC（编号16260/16）和QBRI（编号：2016-003）的机构审查委员会（IRB）的批准。这项工作还针对H1和H9等hESC进行了优化。血液样本是在完全知情同意的情况下从健康个体获得的。iPSCs由健康个体的外周血单核细胞（PBMC）产生。 <stro…
Representative Results
间充质分化过程中细胞的示意图和形态：iPSC 分化为 MSC 涉及跨越 EB 形成、MSC 分化和 MSC 扩增的不同发育阶段（图 1）。在这些发育阶段，细胞由于受到不同的刺激化学物质而获得各种形态。在开始分化时，细胞被铺在悬浮液中，并且预期是圆形的，具有明确的细胞边界，同时直径为小到中等尺寸（图2）。在分化的初始阶段选择悬浮培养细胞使其与自?…
Discussion
该协议至关重要，因为它能够提供高产量和高效率的MSC。这种大规模生产MSCs是通过将iPSCs衍生的EB与10μM的RA14，15瞬时孵育来实现的。用 10 μM RA 瞬时处理将 MSC 产量提高了 11.2 至 1542 倍14，15，该方案适用于 iPSC 和 hPSC。在此剂量和治疗持续时间下，RA通过直接或间接调节参与细胞增殖，凋亡以及细胞间和ECM细?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作由卡塔尔国家研究基金（QNRF）资助（资助号NPRP10-1221-160041）。Maryam Aghadi得到了卡塔尔国家研究基金（QNRF）的GSRA奖学金的支持。
Materials
| Adiponectin | Abcam | ab22554 | Adipocyte maturation marker |
| anti-CD105 | BD Pharmingen | 560839 | MSC differentiation marker |
| anti-CD14 | BD Pharmingen | 561712 | MSC differentiation marker |
| anti-CD19 | BD Pharmingen | 555415 | MSC differentiation marker |
| anti-CD34 | BD Pharmingen | 555824 | MSC differentiation marker |
| anti-CD44 | abcam | ab93758 | MSC differentiation marker |
| anti-CD45 | BD Pharmingen | 560975 |
MSC differentiation marker |
| anti-CD73 | BD Pharmingen | 550256 | MSC differentiation marker |
| anti-CD90 | BD Pharmingen | 555596 | MSC differentiation marker |
| bFGF | R&D | 233-FP | MSC culture media supplement |
| C/EBPA | Abcam | ab40761 | Adipocyte maturation marker |
| Dexamethasone | Torics | 1126 | Adipocyte differentiation media supplement |
| FABP4 | Abcam | ab93945 | Adipocyte maturation marker |
| Fetal bovine serum | ThermoFisher | 10082147 | MSC culture media supplement |
| Glutamax | ThermoFisher | 35050-061 | MSC culture media supplement |
| IBMX | Sigma Aldrich | I5879 | Adipocyte differentiation media supplement |
| Indomethacin | Sigma Aldrich | I7378 | Adipocyte differentiation media supplement |
| Insulin | Sigma Aldrich | 91077C | Adipocyte differentiation media supplement |
| Knockout DMEM | ThermoFisher | 12660012 | Basal media for preparing matrigel |
| Low glucose DMEM | ThermoFisher | 11885084 | MSC culturing media |
| Matrigel | Corning | 354230 | Coating matrix |
| MEM-alpha | ThermoFisher | 12561056 | Adipocyte differentiation media |
| Nilered | Sigma Aldrich | 19123 | Sorting marker for adipocyte |
| Penicillin | ThermoFisher | 15140122 | MSC/Adipocyte media supplement |
| Phosphate-buffered saline | ThermoFisher | 14190144 | wash buffer |
| Pierce™ 20X TBS Buffer | Thermo Fisher | 28358 | wash buffer |
| PPARG | Cell Signaling Technology | 2443 | Adipocyte maturation marker |
| ReLeSR | Stem Cell Technologies | 5872 | Dissociation reagent |
| Retinoic acid | Sigma Aldrich | R2625 | MSC differentiation media supplement |
| Rock inhibitor | Tocris | 1254/10 | hPSC culture media supplement |
| Roziglitazone | Sigma Aldrich | R2408 | Adipocyte differentiation media supplement |
| StemFlex | ThermoFisher | A334901 | hPSC culture media |
| Triton | Thermo Fisher | 28314 | Permebealization reagent |
| Trypsin | ThermoFisher | 25200072 | Dissociation reagent |
| Tween 20 | Sigma Aldrich | P7942 | Wash buffer |
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Cite This Article
Aghadi, M., Karam, M., Abdelalim, E. M. Robust Differentiation of Human iPSCs into a Pure Population of Adipocytes to Study Adipocyte-Associated Disorders. J. Vis. Exp. (180), e63311, doi:10.3791/63311 (2022).