An efficient simplified method for the generation of corneal epithelial cells from human pluripotent stem cells
An efficient simplified method for the generation of corneal epithelial cells from human pluripotent stem cells
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DOI：
10.1007/s13577-022-00713-5
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发表时间：
2022-05-12
期刊：
影响因子：
4.3
通讯作者：
Kamei, Ken-ichiro
中科院分区：
文献类型：
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作者：
Abdalkader, Rodi;Kamei, Ken-ichiro
来源于人多能干细胞(HPSCs)的角膜上皮细胞是临床前模型试验眼科药物的重要细胞来源。然而，目前的分化方案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)来从hPSCs中产生角膜上皮细胞。SSM-CI的优点是使用两种定义的培养液，在25天内最大限度地减少细胞培养时间，其中包含转化生长因子-β、WNT/β-连环蛋白途径抑制剂和碱性成纤维细胞生长因子。与传统的人角膜上皮细胞系(HCE-T)和人原代角膜上皮细胞(HPCEpC5)相比，SSM-CI培养的角膜上皮细胞分化良好，并表达相应的成熟标志物，包括PAX6和CK12。RNA-seq分析表明，hPSCs向角膜上皮细胞分化，并显著上调角膜祖细胞和成体角膜上皮细胞的表型。此外，尽管转化生长因子-β和Wnt/β-连环蛋白最初被抑制，但在后期观察到这些途径相关的转录上调，这表明它们在成熟的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的变化，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本的过度表达。因此，SSM-CI为临床前模型中使用的功能性角膜上皮细胞的产生提供了一种全面的方法。
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-beta, and Wnt/beta-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 (hPCEpC5), 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-beta and Wnt/beta-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.[GRAPHICS].