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Hui Lin, Hong He, Samuel C Yiu; Isolation and Characterization of Lacrimal Gland Progenitor Cells Induced by Duct Ligation. Invest. Ophthalmol. Vis. Sci. 2014;55(13):58.
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© ARVO (1962-2015); The Authors (2016-present)
Lacrimal gland (LG) progenitor cells are believed to play important roles in regeneration of damaged lacrimal glands and therefore may have therapeutic role in aqueous deficient dry eye. Here we report the regenerative potential of LG progenitor cells after duct ligation.
New Zealand White rabbits (aged 6-8 weeks) were randomized into ligated (day 7 & day 10 subgroups) and control groups. In the ligated groups, the main secretory duct of LGs was ligated for 3 days and then reopened. The LGs were harvested for frozen slides, RNA extractionand cell isolation. The cells were expanded in serum free medium. Immuno-staining was performed on tissues and on P0-P4 cells. Flow cytometry was performed on fresh isolated cells. Real-time polymerase chain reaction (PCR) was performed on tissues and P0-P2 cells. BrdU staining was examined on P0-P2 cells. Single cell clonal assay was performed. The P1 cells were seeded onto decellularized LG scaffold. Beta-hexosaminidase (β-HEX) secretion assay was performed for assessing acinar cell function.
In contrast to the control group, ligated tissues demonstrated more△Np63, K14 and nestin-positive cells on immuno-staining, and significantly higher mRNA level of K14, △Np63, nestin, ABCG2, BMP-1 and SNAI2 on real-time PCR, especially in day 7 samples. Flow cytometry demonstrated 8 folds PCK-positive cells, 3 folds △Np63-positive cells and 2 folds nestin-positive cells in the fresh isolated cells from day 7 samples over control. LG epithelial cells isolated from day 7 samples expanded successfully and passaged to P9 and expressed △Np63 and nestin. Although the expanded cells from the control group also expressed △Np63 and nestin, they could be passaged to P5 only. Most expanded cells in both groups were PCK positive and BrdU positive. The expanded cells of day 7 group also showed higher mRNA levels of nestin, △Np63, ABCG2, BMP-1 and SNAI2 than control. Single clonal assay showed that 1% of the expanded cells in P1 of day 7 group could form confluent monolayer and then be continuously passaged to P8. Finally, β-HEX activity was higher in constructs seeded with cells from ligated group.
Regeneration and epithelial-mesenchymal transitions occur in ligated rabbit LGs. Progenitor cells, isolated and expanded from ligated tissue, can also be cultured in decellularizedLG tissue and have secretory function.
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