Human iPS-derived-keratinocytes, a new useful model to identify and explore pathological phenotype of Epidermolysis Bullosa Simplex.. Human iPS-derived-keratinocytes, a new useful model to identify and explore pathological phenotype of Epidermolysis Bullosa Simplex.

Epidermolysis Bullosa Simplex (EBS), an autosomal dominant skin disorder, is characterized by trauma-induced skin fragility. Genetically, majority of cases are related to missense mutations in two keratin genes, KRT5 or KRT14, leading to cytolysis of basal keratinocytes and intraepidermal blistering. Only symptomatic treatment exists for EBS patients so far. Progress towards identification of new treatments have been hampered by incomplete understanding of the mechanisms underlying this disease, and availability of relevant and reliable in vitro models recapitulating the physiopathological mechanisms. Recent advances in stem cell field have fueled the prospect that these limitations could be overcome thanks to the availability of disease-specific human induced pluripotent stem cells (hiPSC). Here we generated hiPSC-derived keratinocytes from EBS patients carrying KRT5 dominant mutations and compared them to non-affected hiPSC-derived keratinocytes as well as their primary counterparts. Our results demonstrated that EBS hiPSC-derived keratinocytes displayed proliferative defects, increased capacity to migrate, alteration of ERK signaling pathway and cytoplasmic keratin filament aggregates in response to osmotic-shock treatment in a pattern similar to primary EBS keratinocytes. Of interest, EBS hiPSC-derived keratinocytes exhibited a downregulation of hemidesmosomal proteins revealing the different effects of KRT5 mutations on keratin cytoskeletal organization. Combination of culture miniaturization and treatment with the chaperone molecule 4-Phenybutiric acid (4-PBA), our results demonstrated that hiPSC-derived keratinocytes represent a suitable model for identifying novel therapies for EBS. Overall design: Comparison of gene expression profiles of 3 hiPSC-derived WT keratinocytes and 3 hiPSC-derived EBS keratinoctyes (3 different KRT5 dominant mutations)

单纯型大疱性表皮松解症（Epidermolysis Bullosa Simplex, EBS）是一种常染色体显性遗传性皮肤病，以创伤诱发性皮肤脆性为核心临床特征。遗传学角度而言，绝大多数病例与两类角蛋白基因KRT5或KRT14的错义突变相关，此类突变可诱发基底角质形成细胞溶解，进而导致表皮内水疱形成。截至目前，EBS患者仅能接受对症治疗，新型治疗方案的开发进展始终受限：一方面源于对该疾病潜在发病机制的认知尚不完整，另一方面则因缺乏能够重现其病理生理过程的相关可靠体外模型。

近年来干细胞研究领域的进展为突破上述局限提供了可能，这得益于疾病特异性人类诱导多能干细胞（hiPSC）的成功获取与应用。本研究从携带KRT5显性突变的EBS患者体内成功诱导生成了hiPSC来源的角质形成细胞，并将其与健康对照hiPSC来源的角质形成细胞以及原代对应细胞开展对比分析。

研究结果表明，EBS hiPSC来源的角质形成细胞呈现出增殖缺陷、迁移能力增强、ERK信号通路异常等特征，且在渗透压休克处理后会形成细胞质角蛋白丝聚集物，上述表型模式与原代EBS角质形成细胞高度相似。值得关注的是，EBS hiPSC来源的角质形成细胞中半桥粒蛋白表达出现下调，这提示KRT5突变对角蛋白细胞骨架的组织调控存在多维度影响。

结合微小型化培养与分子伴侣4-苯基丁酸（4-PBA）处理的联合实验策略，本研究证实hiPSC来源的角质形成细胞可作为合适的体外模型，用于筛选EBS的新型治疗手段。

总体实验设计：对3株野生型hiPSC来源的角质形成细胞与3株携带不同KRT5显性突变的EBS hiPSC来源的角质形成细胞的基因表达谱进行对比分析。