Abstract
Purpose :
Dry eye disease affects up to 250 million people worldwide and causes symptoms like corneal itching, damage and in severe cases blindness. Nevertheless, research on the human lacrimal gland (LG) is limited due to low availability of material and the absence of an epithelial lacrimal gland cell line. To enable a better mechanistic understanding and translational research on dry eye disease, we set out to establish a human lacrimal gland epithelial cell line (hLGEC).
Methods :
Primary human LG cells of a female donor were seeded on a 3T3-fibroblast feeder layer. We used electroporation to introduce a SV40 antigen carrying plasmid into the outgrown cell pool prior to clonal selection. The successful transfection was validated using RT-PCR. After verifying the expression of selected hLGEC genes, three single cell clones (C1,C2,C3) with most distinctive epithelial character were selected for further experiments. RNA sequencing and RT-PCR (n=3) was performed to verify the expression of different epithelial marker genes of the human LG. Utilizing scanning electron microscopy and a FITC/Dextran transwell diffusion assay (n=3) confirmed the epithelial character of the selected cells. The presence and localization of characteristic membrane proteins and transcription factors of hLGEC were detected by immunofluorescence.
Results :
We successfully selected human single cell clones with features of hLGEC. Immunostaining showed the presence of typical transcription factors such as PAX6 and FOXC1 in the nucleus. In addition, the markers AQP5 (C2,C3: p<0,0001), CSTB (C1,C2,C3: p<0,0001), CST6 (C2,C3: p<0,0001), FOXC1 (C1,C2,C3: p<0,0001), MYL9 (C1: p=0,0009, C3: p<0,0001) and PAX6 (C1,C2,C3: p<0,0001) show significantly higher expression in the clones than in a human fibroblast cell line. As shown by the transwell assay, a functional barrier formed after 2 (p<0,0001) and 4 days (p<0,0001), respectively, and the epithelial character of the clones was morphologically validated for all three clones. RT-PCR showed expression of the tear proteins lactoferrin and lysozyme.
Conclusions :
This project establishes a cell line with the characteristics of human lacrimal gland epithelial cells that will contribute to improved research opportunities. This cell line will be functionally validated by studying soluble factors in the supernatant after stimulation. In addition, potential treatment options can be tested with a compound library.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.