Corneal epithelial cells derived from human pluripotent stem cells (hPSCs) are an important cell source for preclinical models to test ophthalmic drugs. However, current differentiation protocols lack instructions regarding optimal culturing conditions, which hinders the quality of cells and limits scale-up. Here, we introduce a simplified small molecule-based corneal induction method (SSM-CI) to generate corneal epithelial cells from hPSCs. SSM-CI provides the advantage of minimizing cell culturing time using two defined culturing media containing TGF-β, and Wnt/β-catenin pathway inhibitors, and bFGF growth factor over 25 days. Compared to the conventional human corneal epithelial cell line (HCE-T) and human primary corneal epithelial cells (hPCEpCs), corneal epithelial cells generated by SSM-CI are well-differentiated and express relevant maturation markers, including PAX6 and CK12. RNA-seq analysis indicated the faithful differentiation of hPSCs into corneal epithelia, with significant upregulation of corneal progenitor and adult corneal epithelial phenotypes. Furthermore, despite the initial inhibition of TGF-β and Wnt/β-catenin, upregulation of these pathway-related transcripts was observed in the later stages, indicating their necessity in the generation of mature corneal epithelial cells. Moreover, we observed a shift in gene signatures associated with the metabolic characteristics of mature corneal epithelial cells, involving a decrease in glycolysis- and an increase in fatty acid oxidation. This was also attributed to the overexpression of metabolic enzymes and transporter-related transcripts responsible for fatty acid metabolism. Thus, SSM-CI provides a comprehensive method for the generation of functional corneal epithelial cells for use in preclinical models.

中文翻译：

---

一种从人多能干细胞中产生角膜上皮细胞的有效简化方法

源自人类多能干细胞 (hPSC) 的角膜上皮细胞是临床前模型测试眼科药物的重要细胞来源。然而，目前的分化协议缺乏关于最佳培养条件的说明，这阻碍了细胞的质量并限制了放大。在这里，我们介绍了一种简化的基于小分子的角膜诱导方法 (SSM-CI)，以从 hPSC 生成角膜上皮细胞。SSM-CI 的优势在于，使用两种含有 TGF-β、Wnt/β-连环蛋白通路抑制剂和 bFGF 生长因子的确定培养基，可在 25 天内最大限度地缩短细胞培养时间。与传统的人角膜上皮细胞系（HCE-T）和人原代角膜上皮细胞（hPCEpCs）相比，SSM-CI 产生的角膜上皮细胞分化良好，并表达相关的成熟标志物，包括 PAX6 和 CK12。RNA-seq 分析表明 hPSCs 忠实地分化为角膜上皮细胞，角膜祖细胞和成人角膜上皮表型显着上调。此外，尽管最初抑制了 TGF-β 和 Wnt/β-catenin，但在后期观察到这些通路相关转录物的上调，表明它们在成熟角膜上皮细胞的产生中是必要的。此外，我们观察到与成熟角膜上皮细胞代谢特征相关的基因特征发生变化，包括糖酵解减少和脂肪酸氧化增加。这也归因于负责脂肪酸代谢的代谢酶和转运蛋白相关转录物的过度表达。因此，SSM-CI 为生成用于临床前模型的功能性角膜上皮细胞提供了一种综合方法。