HCE-T cell line lacks cornea-specific differentiation markers compared to primary limbal epithelial cells and differentiated corneal epithelium
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.
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.
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.
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.
KeywordsHCE-T Limbal epithelial cells Primary corneal epithelium Cornea-specific differentiation markers Cell line
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.
This study was funded by The Rolf M. Schwiete Stiftung and HOMFOR.
Compliance with ethical standards
This article does not contain any studies with human participants or animals performed by any of the authors.
Conflict of interest
The authors declare that they have no conflict of interest.
- 5.Juretic M, Jurisic Dukovski B, Krtalic I, Reichl S, Cetina-Cizmek B, Filipovic-Grcic J, Lovric J, Pepic I (2017) HCE-T cell-based permeability model: a well-maintained or a highly variable barrier phenotype? Eur J Pharm Sci 104:23–30. https://doi.org/10.1016/j.ejps.2017.03.018 CrossRefPubMedGoogle Scholar
- 7.Yamasaki K, Kawasaki S, Young RD, Fukuoka H, Tanioka H, Nakatsukasa M, Quantock AJ, Kinoshita S (2009) Genomic aberrations and cellular heterogeneity in SV40-immortalized human corneal epithelial cells. Invest Ophthalmol Vis Sci 50(2):604–613. https://doi.org/10.1167/iovs.08-2239 CrossRefPubMedGoogle Scholar
- 14.Klinngam W, Fu R, Janga SR, Edman MC, Hamm-Alvarez SF (2018) Cathepsin S alters the expression of pro-inflammatory cytokines and MMP-9, partially through protease-activated receptor-2, in human corneal epithelial cells. Int J Mol Sci 19(11). https://doi.org/10.3390/ijms19113530 CrossRefGoogle Scholar
- 20.Tong L, Png E, Aihua H, Yong SS, Yeo HL, Riau A, Mendoz E, Chaurasia SS, Lim CT, Yiu TW, Iismaa SE (2013) Molecular mechanism of transglutaminase-2 in corneal epithelial migration and adhesion. Biochim Biophys Acta 1833(6):1304–1315. https://doi.org/10.1016/j.bbamcr.2013.02.030 CrossRefPubMedGoogle Scholar
- 23.Yamada T, Ueda T, Ugawa S, Ishida Y, Imayasu M, Koyama S, Shimada S (2010) Functional expression of transient receptor potential vanilloid 3 (TRPV3) in corneal epithelial cells: involvement in thermosensation and wound healing. Exp Eye Res 90(1):121–129. https://doi.org/10.1016/j.exer.2009.09.020 CrossRefPubMedGoogle Scholar
- 33.Li G, Xu F, Zhu J, Krawczyk M, Zhang Y, Yuan J, Patel S, Wang Y, Lin Y, Zhang M, Cai H, Chen D, Zhang M, Cao G, Yeh E, Lin D, Su Q, W-w L, Sen GL, Afshari N, Chen S, Maas RL, Fu X-D, Zhang K, Liu Y, Ouyang H (2015) Transcription factor PAX6 (paired box 6) controls limbal stem cell lineage in development and disease. J Biol Chem 290(33):20448–20454. https://doi.org/10.1074/jbc.M115.662940 CrossRefPubMedPubMedCentralGoogle Scholar
- 37.Forsdahl S, Kiselev Y, Hogseth R, Mjelle JE, Mikkola I (2014) Pax6 regulates the expression of Dkk3 in murine and human cell lines, and altered responses to Wnt signaling are shown in FlpIn-3T3 cells stably expressing either the Pax6 or the Pax6(5a) isoform. PLoS One 9(7):e102559. https://doi.org/10.1371/journal.pone.0102559 CrossRefPubMedPubMedCentralGoogle Scholar
- 43.Meyer-Hoffert U, Wu Z, Kantyka T, Fischer J, Latendorf T, Hansmann B, Bartels J, He Y, Glaser R, Schroder JM (2010) Isolation of SPINK6 in human skin: selective inhibitor of kallikrein-related peptidases. J Biol Chem 285(42):32174–32181. https://doi.org/10.1074/jbc.M109.091850 CrossRefPubMedPubMedCentralGoogle Scholar
- 51.Sasamoto Y, Hayashi R, Park S-J, Saito-Adachi M, Suzuki Y, Kawasaki S, Quantock AJ, Nakai K, Tsujikawa M, Nishida K (2016) PAX6 isoforms, along with reprogramming factors, differentially regulate the induction of cornea-specific genes. Sci Rep 6:20807. https://doi.org/10.1038/srep20807 http://www.nature.com/articles/srep20807#supplementary-information CrossRefPubMedPubMedCentralGoogle Scholar
- 52.Kiselev Y, Eriksen TE, Forsdahl S, Nguyen LH, Mikkola I (2012) 3T3 cell lines stably expressing Pax6 or Pax6(5a)—a new tool used for identification of common and isoform specific target genes. PLoS One 7(2):e31915. https://doi.org/10.1371/journal.pone.0031915 CrossRefPubMedPubMedCentralGoogle Scholar
- 54.Li W, Chen YT, Hayashida Y, Blanco G, Kheirkah A, He H, Chen SY, Liu CY, Tseng SC (2008) Down-regulation of Pax6 is associated with abnormal differentiation of corneal epithelial cells in severe ocular surface diseases. J Pathol 214(1):114–122. https://doi.org/10.1002/path.2256 CrossRefPubMedPubMedCentralGoogle Scholar
- 56.Roux IP, Romain D, Jean-Paul C, Jieqiong Q, Huiqing Z, Alain J, Olivier F, Daniel A (2018) Modeling of aniridia-related keratopathy by CRISPR/Cas9 genome editing of human limbal epithelial cells and rescue by recombinant PAX6 protein. Stem Cells 36(9). https://doi.org/10.1002/stem.2858 CrossRefGoogle Scholar