Abstract
Purpose :
The loss or deficiency of limbal stem cells results in the imbalance of corneal-conjunctival homeostasis frequently leading to corneal surface re-epithelisation by the conjunctival epithelium, a process called cornea conjunctivalisation. The migrating conjunctival epithelia can differentiate towards a corneal-like epithelium in response to the corneal microenvironment. We hypothesize that exosomes, a class of extracellular vesicles carriers of selectively uptaken RNA molecules, may play a role as functional agents of cell differentiation to revert the process of cornea conjunctivalisation.
Methods :
Two phenotypical and functionally different epithelial cells (corneal and conjunctival) were used and the exosomes extracted from their conditioned media using differential ultracentrifugation. Exosome populations (N=3) were characterized regarding their size, concentration, membrane and cargo composition. Conjunctival epithelial cells (N=3) were cultured with corneal-derived exosomes and vice-versa. The extent of cell plasticity was characterized in terms of corneal (keratin 3 and 12) and conjunctival (keratin 7 and 13) specific markers expression shift towards the exosome population of origin. Kruskal-Wallis Test followed by Dunn’s Multiple Comparison Test were used to assess the statistically significance of results.
Results :
Corneal-derived exosomes induced conjunctival cells to express higher levels of both corneal epithelial associated markers and to lose their conjunctival phenotype compared to control samples, verified at both mRNA and protein levels (p<0.05). On the opposite direction, conjunctival-derived exosomes induced corneal cells to express both conjunctival epithelial associated markers losing their corneal phenotype (p<0.05). The keratin shift was preceded by an intermediate step of cell dedifferentiation as an up-regulation in the expression of epithelial stem cell markers ΔNp63α and ABCB5 was appreciated (p<0.05). The cargo from both exosome populations showed to be positive for the presence of keratin 3, keratin 7, ΔNp63α, and ABCB5 mRNA and for 1600 microRNAs molecules. Only 11 microRNAs, all with roles in cell differentiation, showed to be significantly different expressed between the two exosome populations.
Conclusions :
Our results are consistent with our hypothesis that exosomes might be used as agents to induce cell differentiation with potential therapeutic purposes.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.