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Benjamin Botsford, Hui Lin, Helen P Makarenkova, Samuel C Yiu; Comparison of lacrimal gland progenitor cells isolated from duct ligation-induced tissues and intact tissues. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5655. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Lacrimal gland (LG) progenitor cells are believed to play an important role in the regeneration of damaged LG and therefore may have a therapeutic role in aqueous deficient dry eye disease. In this study, we compare the proliferation and differentiation potential of LG progenitor cells isolated from duct ligation-induced samples and intact tissues.
LG regeneration was induced by ligation of the main secretory duct for 3 days and then reopening the duct for 7 days prior to cell isolation. LG progenitor cells were obtained from regenerating and control (unoperated) LGs for preparation of cell culture. Gene and protein expression of stem cell markers Delta Np63, ABCG2 and nestin were evaluated by qRT PCR and immunostaining in every passage. LG progenitor cells were also prepared to form spheroids and then transferred into laminin gel for preparation of the 3D organoid cultures. Organoids were stained with differentiation markers lactoferrin and Rab3D and were evaluated by immunofluorescent microscopy.
LG progenitor cells isolated from injured samples expanded successfully, could be passaged up to passage 12 (P12), and expressed a high level of Delta Np63 and nestin. Although control cells from the intact tissues also expressed Delta Np63, ABCG2 and nestin, they could not be expanded beyond P6. The cell doubling time was shorter in cultures obtained from regenerating LGs. The expanded cells from ligated samples also continuously had higher mRNA levels of Delta Np63, nestin, ABCG2 than the cells from intact tissues. LG progenitor cells obtained from both groups were able to form LG organoids containing differentiated LG epithelial acinar cells, with a higher success rate in cultures prepared from operated LGs.
LG progenitor cells from both intact and regenerating tissues can be grown in culture and form 3D organoids. The results provide further insights into LG stem/progenitor cell physiology for engineering of a LG construct to treat severe cases of tear deficiency in the future.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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