通过RNA-Seq分析，人类原主角细胞的体外分化特征
Summary
这里介绍的是一个逐步的过程，通过接触抑制来分离人类原性角蛋白细胞的体外分化，然后通过RNA-seq分析在分子水平上进行表征。
Abstract
人类原发性角蛋白细胞经常用作体外模型，用于表皮分化和相关疾病的研究。报告了利用各种诱导条件以二维（2D）水下方式培养的角蛋白细胞体外分化的方法。这里描述的是2D体外角细胞分化方法通过接触抑制和随后的分子表征RNA-seq的过程。简言之，角蛋白细胞生长在定义的角蛋白细胞介质中，辅以生长因子，直到它们完全汇合。分化是由角蛋白细胞之间的密切接触引起的，并因排除介质中的生长因子而进一步刺激。利用RNA-seq分析，发现1）分化的角蛋白细胞在分化过程中表现出不同的分子特征，2）动态基因表达模式在表皮分层期间与细胞大体相似。至于与正常角蛋白细胞分化的比较，携带转录因子p63突变的角蛋白细胞表现出改变的形态和分子特征，符合其分化缺陷。最后，本协议详细介绍了2D体外角细胞分化的步骤及其分子特征，重点是RNA-seq数据的生物信息学分析。由于RNA提取和RNA-seq程序有据可查，因此它不是本协议的重点。体外角蛋白细胞分化的实验程序及生物信息分析管道可用于研究健康与患病角蛋白细胞表皮分化期间的分子事件。
Introduction
人类原发性角蛋白细胞来源于人类皮肤，常被用作细胞模型，研究表皮1、2、3、4的生物学。表皮的分层可以通过角蛋白细胞分化进行建模，无论是2D水下单层方式，还是3D空气提升器官模型2、3、5、6、7。虽然 3D 模型在评估表皮结构和功能方面变得越来越重要，但由于 2D 分化模型的便利性和生成大量用于分析的细胞的可能性，因此仍然广泛使用。
各种条件已应用于诱导角蛋白细胞分化的2D，包括血清的添加，高浓度的钙，较低的温度和抑制表皮生长因子受体2，3。这些方法中的每一个方法都经过了许多角蛋白细胞分化标记基因的验证，并证明在评估角蛋白细胞分化（包括在病理条件下）是有效的。然而，当对标记基因的特定面板进行2、3的检查时，这些诱导条件也显示了其分化效率和动力学的差异。
其中一种方法涉及角蛋白细胞接触抑制和消耗生长因子在培养媒介8。研究表明，当细胞达到全密度时，角蛋白细胞可以自发分化。 将文化媒介中的增长因素排除在外，可以进一步增强差异化。将接触抑制和减损生长因子相结合的方法已被证明在使用多个表皮标记3时，产生具有与正常分层表皮相似的基因表达模式的分化角蛋白细胞，表明该模型适合研究正常的角蛋白细胞分化。最近，利用该模型对角蛋白细胞分化进行了两次综合基因表达分析，结果报告了9、10。研究人员在分子水平上验证了该模型，并表明它可用于研究正常和患病的角蛋白细胞分化。
本协议描述了使用RNA-seq进行体外分化方法和分化细胞分子分析的过程。它还说明了分化日（增殖阶段）、第2天、第4天和第7天（分别早期、中间和晚期分化）细胞转录组的特点。表明，分化的角蛋白细胞显示基因表达模式，在表皮分层期间，它们与细胞大体相似。为了研究这种方法是否可用于研究皮肤病理学，我们应用了相同的实验和分析管道，以研究携带转录因子p63突变的角蛋白细胞，这些突变来自结膜、异体发育不良和唇裂（EEC）综合征11，12的患者。该协议侧重于角蛋白细胞的体外分化以及随后对RNA-seq的生物信息学分析。完整程序的其他步骤，如RNA提取、RNA-seq样品制备和库结构，都记录得很好，很容易遵循，尤其是在使用许多常用商业试剂盒时。因此，这些步骤仅在协议中简要说明。数据表明，该管道适用于研究健康与患病角细胞表皮分化期间的分子事件。
Protocol
Representative Results
Discussion
本工作描述了一种利用RNA-seq分析诱导人类角蛋白细胞分化和后续表征的方法。在目前的文献中，许多关于人类角蛋白细胞分化的研究使用另外两种方法，高钙浓度或以血清作为诱导分化的方法2，3，23。前一份报告仔细比较了这三种不同的方法3，表明这些方法可以代表角蛋白细胞分化的不同生物学。在同一份…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项研究得到了荷兰科学研究组织的支持（NWO/ALW/MEERVOUD/836.12.010，H.Z.）（NWO/ALW/公开竞争/ALWOP 376，H.Z.，J.G.A.S.）;拉德布德大学奖学金（H.Z.）;和中国奖学金委员会助学金201406330059（J.Q.）。
Materials
| Bioanalyzer 2100 | Agilent | G2929BA | |
| Bovine pituitary extract (BPE) | Lonza | Part of the bulletKit | |
| CFX96 Real-Time system | Bio-Rad | qPCR machine | |
| Dulbecco's Phosphate-Buffered Saline (DPBS) | Sigma-Aldrich | D8537 | |
| Epidermal Growth Factor (EGF) | Lonza | Part of the bulletKit | |
| Ethanolamine >= 98% | Sigma-Aldrich | E9508 | |
| High Sensitivity DNA chips | Agilent | 5067-4626 | |
| Hydrocortison | Lonza | Part of the bulletKit | |
| Insulin | Lonza | Part of the bulletKit | |
| iQ SYBR Green Kit | BioRad | 170-8886 | |
| iScript cDNA synthesis | Bio rad | 1708890 | |
| KAPA Library Quant Kit | Roche | 07960255001 | Low concentration measure kit |
| KAPA RNA HyperPrep Kit with RiboErase | Roche | KK8540 | RNAseq kit |
| KGM Gold Keratinocyte Growth Medium BulletKit | Lonza | 192060 | |
| Nanodrop | deNovix | DS-11 FX (model) | Nanodrop and Qbit for DNA and RNA measurements |
| NEXTflex DNA barcodes -24 | Illumnia | NOVA-514103 | 6 bp long primers |
| Penicillin-Streptomycin | Gibco | 15140122 | |
| RNA Pico Chip | Agilent | 5067-1513 |
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