Mouse Meibomian Gland Progenitor Cell Organoids as an In Vitro Model of Acinar and Ductal Differentiation

Sun Woong KimKorea (Republic of) Speaker Mouse Meibomian Gland Progenitor Cell Organoids as an In Vitro Model of Acinar and Ductal DifferentiationPurpose: Recent studies have shown that two-dimensional (2D) culture of primary rabbit and immortalized human meibomian gland epithelial cells (iHMGEC) do not recapitulate normal meibocyte differentiation, but 3D-spheroid culture of iHMGEC can facilitate meibocyte differentiation. The purpose of this study was to develop mouse meibomian gland (MG) organoid which can be capable of differentiating to MG acini and duct. Methods: Mouse meibomian gland epithelial cells were isolated and were suspended in matrigel/basement membrane matrix and grown in proliferation media supplemented with Rock inhibitor (Y-27632), Noggin, A8301, FGF10, and Rspondin1 to form adult progenitor cell clusters or spheroids. Cells were then differentiated with serum-free, differentiation media with or without synthetic agonists for the nuclear lipid receptor, peroxisome proliferator activator receptor gamma (PPARγ). Cells were then evaluated for differentiation markers using western blotting, immunocytochemistry (ICC) and real-time PCR. Results: The 3D culture induced the formation of KRT5+ spheroids that were enriched with Lrig1+, Sox9+, and Lgr6+ cells at the outer layer. These MG progenitor cell organoids can be passaged more than 30 times and were still capable of developing MG-like phenotypes as indicated by lipid synthesis as well as expression of essential proteins related to meibum synthesis. When MG progenitor cell organoids were switched to differentiation media containing PPARγ agonists, accumulation of lipid droplets and cell death were observed in the center of organoids, which demonstrates that these progenitor cell organoids can differentiate and response to differentiation signals. Meibocyte differentiation marker, AWAT2+/PPARγ+ were expressed in acini-like organoid and KRT6a+ duct like organoids were also formed. Some organoids showed both duct and acini phenotypes. Conclusions: The 3D culturing of mouse MG progenitor cells can induce the formation of both acini and ductal organoids and may thus serve as a better in vitro model system for studying the regulatory mechanisms controlling meibomian gland function. Mimicking MG homeostasis, the outer layer of MG organoids is composed of proliferating cells that migrate inward, undergo terminal differentiation and generating lipid-filled meibocytes.