Abstract
The barrier function of the corneal epithelium is crucial for protection of the eye, and when the corneal epithelium is wounded, it can pose significant challenges to proper healing, leading to chronic conditions, vision impairment and potentially high healthcare costs. Limbal epithelial stem cells (LESCs) are key for cornea wound healing and regeneration but are diminished in the cornea when wounded or when genetic disorders induce LESC deficiencies. Prior studies show that electric field stimulation (ES) can enhance wound healing by promoting cell migration. Electrically stimulating LESCs may enhance wound healing by promoting the migration and proliferation of cells into damaged regions of the cornea, thereby restoring LESC populations back to healthy levels. The hypothesis was that ES enhances LESC proliferation and differentiation. In vitro experiments included evaluating the effects of direct current (DC) ES (100 mV/mm or 200 mV/mm for 3 hours) on LESC behavior. For the video microscopy results, the effects on proliferation were more ambiguous in the LESCs. However, directionality and speed increased in LESCs stimulated at 200 mv/mm, while results at 100 mV/mm were more variable. Proliferative and migratory behaviors of dividing cells (parent and daughter cells) were quantified and analyzed in videos of electrically stimulated LESCs. Video analysis showed increased migratory behavior following cell division, suggesting ES may stimulate post-mitotic migration. Quantitative Reverse Transcription PCR (RT-qPCR) results in LESCs electrically stimulated at 100 mV/mm showed no expression of Krt12, indicating ES did not enhance differentiation. Resazurin assays, trypan blue cell count, and immunofluorescence results all showed trends of slightly increased proliferation at 100 mV/mm (p > 0.05) and no differentiation.
These findings suggest that electrical stimulation, particularly at 200 mV/mm, may enhance migration and potentially proliferation of LESCs, while having little to no effect on differentiation. The effects appear to be voltage-dependent, as 100 mV/mm stimulation yielded weaker and more variable outcomes. Further studies are needed to determine the optimal ES parameters and to understand the mechanisms behind these responses. This work provides early evidence supporting the therapeutic potential of ES in promoting wound healing in LESC-deficient or damaged corneas. The thesis was conducted over 8 months.