PURPOSE Human corneal epithelial cell-transformed (HCE-T) cell line is used as a widely accepted barrier model for pharmacological investigations in the context of eye application. The differentiation of (limbal) corneal epithelial into mature corneal epithelium coincides with the expression of established differentiation markers. If these differentiation mechanisms are disturbed, it will lead to ocular surface disease. In this study, we want to compare the expression of differentiation markers in the HCE-T cell line to differentiated primary epithelial cells (pCECs) and primary limbal epithelial cell (LEC) culture. This is necessary in order to decide whether HCE-T cells could be a tool to study the differentiation process and its regulatory networks in corneal epithelium. METHODS Primary limbal epithelial cells (LECs) for cell culture and primary corneal epithelial cells (pCECs) as differentiated tissue samples were obtained from the limbus or central cornea region of corneal donors. HCE-T cell line was purchased from RIKEN Institute RCB-2280.Expression levels of conjunctival- and corneal-specific keratin and adhesion markers (KRT3, KRT12, KRT13, KRT19, DSG1), stem cell and differentiation markers (PAX6, ABCG2, ADH7, TP63, ALDH1A1), and additional (unvalidated) putative differentiation and stem cell markers (CTSV, SPINK7, DKK1) were analyzed with qPCR. Additionally, KRT3, KRT12, DSG1, and PAX6 protein levels were analyzed with Western blot. RESULTS KRT3, KRT12, DSG1, PAX6, ADH7, and ALDH1A1 mRNA expressions were higher in LECs and magnitudes higher in pCECs compared to HCE-T cells. KRT3, KRT12, PAX6, ALDH1A1, ADH7, TP63, and CTSV mRNAs have shown increasing mRNA expression from HCE-T < HCE-T cultured in keratinocyte serum-free medium (KSFM) < LEC < to pCEC.KRT3 and KRT12 protein expressions were only slightly increased in LEC compared to HCE-T samples, and the strongest signals were seen in pCEC samples. DSG1 protein expression was only detected in pCECs. PAX6 protein expression was hardly detected in HCE-T cells, and no difference could be seen between LECs and pCECs. CONCLUSIONS The HCE-T cell line is even less differentiated than LECs regarding the investigated markers and therefore might also lack the ability to express differentiation markers at protein level. Hence, this cell line is not suitable to study corneal differentiation processes. Primary LECs in the way cultured here are not an ideal system compared to differentiated epithelium in organ culture but should be preferred to HCE-T cells if corneal differentiation markers are investigated. Other cell models or differentiation protocols should be developed in the future to gain new tools for research on ocular surface diseases.

中文翻译：

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与原代角膜缘上皮细胞和分化的角膜上皮细胞相比，HCE-T 细胞系缺乏角膜特异性分化标记。

目的人角膜上皮细胞转化 (HCE-T) 细胞系被用作广泛接受的屏障模型，用于眼部应用背景下的药理学研究。（角膜缘）角膜上皮分化为成熟角膜上皮与已建立的分化标志物的表达一致。如果这些分化机制受到干扰，就会导致眼表疾病。在这项研究中，我们想要比较分化标志物在 HCE-T 细胞系与分化原代上皮细胞 (pCEC) 和原代角膜缘上皮细胞 (LEC) 培养物中的表达。这是必要的，以便决定 HCE-T 细胞是否可以成为研究角膜上皮分化过程及其调节网络的工具。方法 用于细胞培养的原代角膜缘上皮细胞 (LEC) 和作为分化组织样本的原代角膜上皮细胞 (pCEC) 从角膜供体的角膜缘或中央角膜区域获得。HCE-T 细胞系购自 RIKEN Institute RCB-2280。结膜和角膜特异性角蛋白和粘附标记物（KRT3、KRT12、KRT13、KRT19、DSG1）、干细胞和分化标记物（PAX6、ABCG2、ADH7）的表达水平、TP63、ALDH1A1）和其他（未经验证的）推定分化和干细胞标记物（CTSV、SPINK7、DKK1）用 qPCR 进行了分析。此外，还使用蛋白质印迹分析了 KRT3、KRT12、DSG1 和 PAX6 蛋白水平。结果 与 HCE-T 细胞相比，LEC 中 KRT3、KRT12、DSG1、PAX6、ADH7 和 ALDH1A1 mRNA 表达更高，pCEC 中的表达量级更高。KRT3、KRT12、PAX6、ALDH1A1、ADH7、TP63 和 CTSV mRNA 显示从角质形成细胞无血清培养基 (KSFM) < LEC < 到 pCEC 中培养的 HCE-T < HCE-T 的 mRNA 表达增加。与 HCE 相比，LEC 中的 KRT3 和 KRT12 蛋白表达仅略微增加-T 个样本，并且在 pCEC 样本中看到了最强的信号。仅在 pCEC 中检测到 DSG1 蛋白表达。在 HCE-T 细胞中几乎检测不到 PAX6 蛋白表达，并且 LEC 和 pCEC 之间没有差异。结论 HCE-T 细胞系在所研究的标志物方面甚至比 LEC 的分化程度更低，因此可能也缺乏在蛋白质水平表达分化标志物的能力。因此，该细胞系不适合研究角膜分化过程。与器官培养中的分化上皮细胞相比，此处培养的初级 LEC 不是理想的系统，但如果研究角膜分化标记，则应优先于 HCE-T 细胞。未来应开发其他细胞模型或分化方案，以获得研究眼表疾病的新工具。