In-Vitro Effects of Secreted Frizzled-Related Protein 1 (SFRP1) On Human Corneal Epithelial Cells

Abstract

Purpose: Limbal corneal epithelial cells (LCECs) are responsible for corneal epithelial cell regeneration. However, corneal central epithelial cells (CCECs) are also suggested to display potential for self-renewal. Additionally, a better understanding of molecules that regulate corneal epithelial cell regeneration is important for studying conditions affecting the cornea, for example, keratoconus. Given our previous findings of reduced levels of secreted frizzled-related protein 1 (SFRP1) in tears from keratoconus patients compared to controls, we investigated the effects of SFRP1 on the proliferation and survival of cultured central and limbal human corneal epithelial cells.

Material and methods: Limbal and central corneal explants were established from postmortem human corneas, and cultured in CnT-PR, an epithelial-specific tissue culture media. Subcultured cells from explants were immunostained for the cytokeratins CK3, 12, 19, and the proliferative/oligopotent markers Ki67 and p63. BrdU flow cytometry, Alamar Blue and LDH assays were used to assess effects of SFRP1 treatment on central and LCECs.

Results: Primary limbal and central corneal epithelial cells were successfully cultured in vitro to confluence (P6 and P4, respectively). They all expressed varying levels of cytokeratins CK3, CK12 and CK19, and Ki67 and p63. Additionally, they showed significantly increased metabolic activity after SFRP1 treatment (p < 0.05), with a maximum response at 1 μg/mL of SPRF1. No difference in proliferation was detected in SFRP1 treated LCECs; however, a reduction in cell death was noted (p < 0.05).

Conclusion: Similar to the LCECs, primary human CCECs can be cultured in vitro, and expressed epithelial markers. SFRP1 demonstrated an improvement on the metabolic activity of both CCECs and LCECs, which in LCECs could be resulted from reduced cell death. This may have implications in degenerative corneal disorders, such as keratoconus.

Keywords: Primary human corneal epithelial cells; SFRP1; cell death; corneal explants; keratoconus; tissue culture.