Epithelial Cell-Derived Extracellular Vesicles Trigger the Differentiation of Two Epithelial Cell Lines.

Tiago Ramos,Mohit Parekh,Stephen B Kaye,Sajjad Ahmad

doi:10.3390/ijms23031718

Abstract

Extracellular vesicles (EVs), specifically exosomes, carry a cell-type dependent cargo that is transported to the recipient cell and translated in the presence of a required machinery. Differences in the cargo carried by the corneal and conjunctival-derived EVs could be the agent that triggers the transdifferentiation of these two cell populations. Therefore, this study investigates the role of EVs in triggering the plasticity of corneal and conjunctival epithelial cells and identifies prospective miRNA and genes responsible for maintaining ocular surface homeostasis. The EVs were extracted from the conditioned media (after starving) of corneal epithelial (hTCEpi) and conjunctival (HCjE-Gi) cell lines using ultracentrifugation. HCjE-Gi cells were cultured with hTCEpi-derived EVs and vice-versa. The EVs were characterized as exosomes using Nanosight and Flow cytometry. KRT3 and KRT12 were used as associated corneal markers, whereas KRT7 and KRT13 were used as associated conjunctival markers with ΔNp63 as a differentiation marker. Shift of these markers was an indication of transdifferentiation. The cargo of the extracted exosomes from both the cell types was explored using next-generation sequencing. The hTCEpi-derived EVs induced conjunctival epithelial cells to express the corneal-associated markers KRT3 and KRT12, losing their conjunctival phenotype at both the mRNA and protein level. Simultaneously, HCjE-Gi-derived EVs induced corneal epithelial cells to express the conjunctival associated markers KRT7 and KRT13, losing their corneal phenotype. This process of differentiation was accompanied by an intermediate step of cell de-differentiation showed by up-regulation in the expression of epithelial stem cell marker ΔNp63, also shown on the ex vivo human cadaveric donor corneas. miRNA molecules (total of 11 including precursor and mature) with significant differences in their relative abundance between the two populations (p < 0.05) were found and investigated. miR-9-5p expression was higher in HCjE-Gi cells and HCjE-Gi-derived EVs when compared to hTCEpi cells and hTCEPi-derived EVs (p < 0.001). The results suggest that EVs released by the two cell types have the ability to influence the transdifferentiation of human conjunctival and corneal epithelial cells. miR-9-5p could have a role in stem cell homeostasis and cell differentiation via HES-1 gene.

Highlights

The anterior part of the eye is composed of two phenotypical and functionally distinct structures: the cornea and the conjunctiva
NanoSight® analysis confirmed the presence of Extracellular vesicle (EV) in both HCjE-Gi and hTCEpi conditioned media, at concentrations of 4.12 × 108 and
Flow cytometry showed the presence of CD63- and TSG101-positive events and the absence of GRP94-positive events in EVs extracted from both HCjE-Gi and hTCEpi conditioned media (Figure 2B) [34], suggesting that majority of the isolated EVs showed exosome-associated features

Summary

Introduction

The anterior part of the eye is composed of two phenotypical and functionally distinct structures: the cornea and the conjunctiva. The anterior sclera, which encircles the cornea, is covered by the conjunctival epithelium that extends to cover the inner surface of the eyelids. Following limbal stem cell deficiency (LSCD), conjunctival epithelial cells may migrate onto the cornea, a process called conjunctivalisation [5]. This results in loss of corneal clarity and visual impairment [5,6,7,8,9,10]. It has been shown that the process of conjunctival transdifferentiation is incomplete and the newly regenerated epithelium fails to express the corneal specific marker, keratin (KRT)

Methods

Results

Discussion

Conclusion