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Tiago Ramos, Stewart Rosalind, Stephen Kaye, Sajjad Ahmad; The role of the extracellular matrix on human corneal and conjunctival epithelial plasticity. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4344.
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© ARVO (1962-2015); The Authors (2016-present)
To determine if epithelial cells (ECs) from the human cornea and conjunctiva can be modulated in response to extracellular matrix (ECM) cues in vitro. This was investigated in both established human EC lines and primary ECs. These studies test the hypothesis that ocular surface ECs have a degree of plasticity. This is of importance due to its clinical significance.
Human corneal and conjunctival EC lines (hTCE-Pi and HCjE-Gi respectively) and primary corneal and conjunctival ECs from fresh human cadaveric tissue (Liverpool Research Eye Bank) were used for the purposes of these studies. All experiments were performed in triplicate. ECs were seeded on plastic for 9 days. Using an established protocol, cells were removed leaving the ECM attached to the plastic. Fresh corneal ECs (CoEC) were then cultured on ECM from conjunctival ECs (CjEC) for 5 days and vice versa. Marker expression for corneal epithelium, keratin (K) 3 and K12; conjunctival epithelium, K7 and K13, and MUC5AC) and stem cell (SC) markers (p63 and ABCB5) was analysed using real-time PCR, Western blots (WB) and flow cytometry (FC).
The following statistically significant results (p value at least <0.05; n=3) are from experiments performed for the human corneal and conjunctival EC lines and these were verified on primary ECs from fresh human cadaveric cornea and conjunctiva. CoECs cultured on ECM from CjECs (compared to those cultured on CoEC ECM) showed down-regulation of CoEC markers K3 (WB and FC) and K12 (FC); up-regulation of CjEC markers K7 and K13 (PCR and FC) and goblet cell marker MUC5AC (PCR); and up-regulation of SC markers p63 (FC) and ABCB5 (PCR and FC). CjECs plated on ECM from CoECs (compared to those cultured on CjEC ECM) showed down-regulation of CjEC markers K7 and K13 (PCR, WB and FC); down-regulation of CoEC markers K3 and K12 (WB and FC); and up-regulation of SC markers p63 (WB and FC) and ABCB5 (PCR, WB and FC).
These results are consistent with our hypothesis that ECM cues modulate the plasticity of ECs of the human ocular surface. Moreover, there is some evidence from these results that this plasticity involves a process of de-differentiation (increased expression of SC markers). Further work is being conducted to understand the mechanisms underlying this plasticity and the composition of the ECM deposited by the cells that enables this.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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