Abstract
Purpose :
Limbal stem cell deficiency (LSCD) is a condition caused by damage or dysfunction of Limbal stem cell niches, which leads to corneal opacity and visual impairment. Dental pulp stem cells (DPSCs) are promising candidates for corneal surface regeneration in LSCD. In this study we aimed to set up an ex-vivo model of human LSCD induced by chemical burns, and determine if the DPSC attached and proliferated on the damaged corneal surface and entered the denuded limbal niche.
Methods :
An ex vivo model of LSCD was produced by the application of NaOH-soaked filter paper disk to the limbal region of donor corneas mounted on artificial chambers. The LSCD corneas were then debrided of epithelium, as were control healthy corneas.
DPSCs were transdifferentiated into corneal epithelial-like cells by non-contact co-culture with limbal explants from healthy human corneas. These cells were seeded onto the ocular surface of Bausch and Lomb contact lenses (CLs) and labelled with Qtracker® 525. The pre-seeded CLs were combined with the experimental and healthy corneas and incubated for 5 days; control group received empty LCs. After removal of the CLs, the corneas were imaged to confirm DPSC transfer and attachment to the corneal surface, and to determine cell morphology.
Immunostaining was used to detect specific markers of corneal epithelial cells, cytokeratins 3 and 12. Cytokeratins 19, a specific marker of conjunctival epithelial was also used to confirm the cells had not become conjunctival cells and CD90, to determine if the cells had maintained their SC characteristics.
Results :
After removal of the CL from the LSCD corneas the DPSCs were located close to the limbus rather than the central cornea as seen with the healthy corneas. Immunostaining of the LSCD cornea sections showed that the differentiated DPSCs stained positive for cytokeratin 3, 12 and CD90. DPSC localized around the limbus, migrated deep to the epithelial layer. The DPSCs did not express cytokeratin 19, but were shown to restrict invasion of the cytokeratin 19 positive conjunctival tissues.
Conclusions :
Transdifferentiated DPSCs were successfully transferred to both chemically-induced LSCD and healthy corneas using CLs as carriers. The DPSCs expressed both corneal epithelial and SC markers showing the cells may have become LSC. However, they had the capacity to limit conjunctival invasion of the LSCD corneas, which itself may be of therapeutic value.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.