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HCE-T cell line lacks cornea-specific differentiation markers compared to primary limbal epithelial cells and differentiated corneal epithelium

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Abstract

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.

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Acknowledgments

This work was supported by The Dr. Rolf M. Schwiete Foundation and HOMFOR.

The authors thank Prof. Flockerzi, Department of Experimental and Clinical Pharmacology and Toxicology, for providing the Western blot imaging system and Nanodrop. They would also like to thank Prof. Hoth, Department of Biophysics, Saarland University, for providing the qPCR system.

Funding

This study was funded by The Rolf M. Schwiete Stiftung and HOMFOR.

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Author notes

  1. Anna-Klara Rubelowski and Lorenz Latta contributed equally to this work.

Authors and Affiliations

  1. Department of Ophthalmology, Saarland University Medical Center, Homburg, Saar, Germany

    Anna-Klara Rubelowski, Lorenz Latta, Priya Katiyar, Tanja Stachon, Barbara Käsmann-Kellner, Berthold Seitz & Nóra Szentmáry

  2. Department of Ophthalmology, Semmelweis University, Budapest, Hungary

    Nóra Szentmáry

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  1. Anna-Klara Rubelowski
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Correspondence to Lorenz Latta.

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Rubelowski, AK., Latta, L., Katiyar, P. et al. HCE-T cell line lacks cornea-specific differentiation markers compared to primary limbal epithelial cells and differentiated corneal epithelium. Graefes Arch Clin Exp Ophthalmol 258, 565–575 (2020). https://doi.org/10.1007/s00417-019-04563-0

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