Ex Vivo Expansion And Differentiation Of Rat Meibomian Gland Progenitor Cells

Purpose:There is no effective treatment for Meibomian gland dysfunction(MGD),in this study we modified the expansion of meibomian gland peogenitor cells and investigated the molecular signatures of duct and acinus of Meibomian gland(MG)in vivo and Meibomian gland progenitor cells in vitro,besides,offered a new choice for screening drugs for MGD ex vivo.Methods:Fresh MG tissues were isolated from eyelids of 8 weeks old SD rats and enzymatically digested into single cells,and clonal cultured with supplemental hormonal epithelial medium(SHEM)with mitomycin C-treated 3T3 cells as feeder layers,and added the Y27632 to increase the Cloning efficiency.MG tissues and it's progenitor cells were harvested to examine the specific markers by quantitative real-time PCR(qRT-PCR)and immunofluorescence(IF)staining.Oil Red O staining was performed to detect the lipid deposition of MG progenitor cells.Results:Clones of MG progenitor cells emerged after 3 to 4 days' culture and reached confluent around day 7-8.Adding the Y26632 can increase the cloning efficiency of MG progenitor cells in vitro.qRT-PCR results showed that p63,stem cell markers K15,sox9 and Krt5,Krtl4,K10 were detected in MG tissues and progenitor cells after 7 days'culture.The lipid metabolism and differentiation related gene expression of SCD1,Acat1,Awatl,Hmgcs1,Far,Elovl4 and PPAR-? decreased evidently during ex vivo culture.The IF result shows that K14 was expressed in MG tissue and all MG progenitor cells after cultivation.p63 positive cells were located in the basal of acinus and duct in vivo while were all positive after in vitro culture,Sox9 emerged randomly in basal layer of acinus and in vitro culture,Tenascin-C(TNC)were positive surrounding the basal cells of acinus.K10 emerged in cells that stratified in clones.Oil Red O staining shows there is nearly no lipid droplets can be observed in cultured cells.Conclusions:The progenitor cells of MG have some characteristic of stem cells.The Meibomian gland acini cells cultured in vitro can maintain the phenotype as the basal cell of acinus and duct.There may have one or two specifically cell lineages can differentiate into duct or acinus cells.With this culture method we can expand MG progenitor cells which be used to investigate the biology of Meibomian gland and screen new drugs for MGD.