Shin Hyewon, Kim Se-Eun, Kim C-Yoon, Lee Suemin, Lee Ji-Heon, Baek Jieun, Chung Gujin, Kim Min Woo, Oh Jeong-Seop, Park Shinhye, Lee Yun Hyeong, Jeong Youngin, Park Jeong Hwan, Kim Yoonseo, Lee Myeonghee, Lee Seul-Gi, Chung Hyung Min
College of Veterinary Medicine, Konkuk University, Gwangjin, Seoul, Republic of Korea.
R&D Center, CLECELL Inc., Beobwon-ro, Songpa-gu, Seoul, Korea.
PLoS One. 2025 Aug 18;20(8):e0330306. doi: 10.1371/journal.pone.0330306. eCollection 2025.
Artificial skin models have emerged as valuable tools for evaluating cosmetic ingredients and developing treatments for skin regeneration. Among them, 3D skin equivalent models (SKEs) using human primary skin cells are widely utilized and supported by standardized testing guidelines. However, primary cells face limitations such as restricted donor availability and challenges in conducting genotype-specific studies. To overcome these issues, recent approaches have focused on differentiating skin cells from human-induced pluripotent stem cells (hiPSCs). In this study, we developed a protocol to differentiate high-purity skin cells, such as fibroblasts (hFIBROs) and keratinocytes (hKERAs), from hiPSCs. To construct the hiPSC-derived SKE (hiPSC-SKE), a dermis was first formed by culturing a collagen and hFIBROs mixture within an insert. Subsequently, hKERAs were seeded onto the dermis, and keratinization was induced under air-liquid culture conditions to establish an epidermis. Histological analysis with hematoxylin and eosin staining confirmed that the hiPSC-SKE recapitulated the layered architecture of native human skin and expressed appropriate epidermal and dermal markers. Moreover, exposure to Triton X-100, a known skin irritant, led to marked epidermal damage and significantly reduced cell viability, validating the model's functional responsiveness. These findings indicate that the hiPSC-SKE model represents a promising alternative for various skin-related applications, including the replacement of animal testing.
人工皮肤模型已成为评估化妆品成分和开发皮肤再生治疗方法的重要工具。其中,使用人类原代皮肤细胞的三维皮肤等效模型(SKEs)得到广泛应用,并得到标准化测试指南的支持。然而,原代细胞面临一些局限性,如供体来源受限以及进行基因型特异性研究时存在挑战。为克服这些问题,最近的方法聚焦于将皮肤细胞从人类诱导多能干细胞(hiPSCs)中分化出来。在本研究中,我们开发了一种从hiPSCs中分化出高纯度皮肤细胞的方案,如成纤维细胞(hFIBROs)和角质形成细胞(hKERAs)。为构建hiPSC来源的SKE(hiPSC-SKE),首先通过在插入物中培养胶原蛋白和hFIBROs混合物形成真皮。随后,将hKERAs接种到真皮上,并在气液培养条件下诱导角质化以建立表皮。苏木精和伊红染色的组织学分析证实,hiPSC-SKE重现了天然人类皮肤的分层结构,并表达了适当的表皮和真皮标记物。此外,暴露于已知的皮肤刺激物 Triton X-100会导致明显的表皮损伤并显著降低细胞活力,验证了该模型的功能反应性。这些发现表明,hiPSC-SKE模型是各种皮肤相关应用的一个有前景的替代方案,包括替代动物试验。