title: Pluripotin enhances the expansion of rabbit limbal epithelial stem/progenitor cells in vitro.
abstract: This study was designed to demonstrate the effects of pluripotin on the proliferation, senescence and colony formation efficiency of rabbit limbal epithelial cells (RLECs) in vitro. Rabbit primary limbal epithelial cells were harvested and cultured in the presence of pluripotin. The cell proliferation was measured using the 3-(4,5)-dimethylthiahiazo(-z-y1)-3 5-di-phenytetrazoliumromide (MTT) assay and was also observed by confocal microscopy with Ki67 staining, whereas cell senescence was detected by senescence-associated β-galactosidase (SA-β-gal) staining. The colony morphology, colony-forming efficiency and colony size were observed and compared. The characteristics of the proliferating cells were examined by immunofluorescent staining using antibodies against deltaNP63, ABCG2 and Keratin 3/12. The results showed that pluripotin significantly promoted the proliferation of RLECs and increased the dividing cells with positive Ki67 staining at the concentrations lower than 400 nM. The colony-forming efficiency increased from 13.5% in the control cells to 26.4% in the 200 or 400 nM pluripotin-treated cells. The number of colonies of moderate size (600-900 μm) increased significantly in the presence of pluripotin (above 60.0% at 200 nM or 400 nM) compared with the untreated normal cells (18.6%), whereas the number of small-sized colonies (<600 μm) decreased from 79.5% for the control cells to lower than 35.0% at 200 nM or 400 nM pluripotin. Moreover, the cells treated with pluripotin stained negative with SA-β-gal, while the untreated cells showed visible positive staining. Immunofluorescent staining suggested that the pluripotin treatment resulted in higher positive staining for the limbal stem cell markers (deltaNP63 and ABCG2) and down-regulated of differentiated corneal epithelial cell marker (Keratin 3/12). This study confirmed that the small molecular compound pluripotin promoted the proliferation of rabbit limbal epithelial cells by improving the expansion of limbal stem/progenitor cells in vitro.
authors.affiliation: State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Shandong Eye Institute, Shandong Academy of Medical Sciences, Qingdao, China.
authors.forename: Haoyun
authors.initials: H
authors.lastname: Duan
authors.forename: Yao
authors.initials: Y
authors.lastname: Wang
authors.forename: Lingling
authors.initials: L
authors.lastname: Yang
authors.forename: Mingli
authors.initials: M
authors.lastname: Qu
authors.forename: Qian
authors.initials: Q
authors.lastname: Wang
authors.forename: Weiyun
authors.initials: W
authors.lastname: Shi
authors.forename: Qingjun
authors.initials: Q
authors.lastname: Zhou
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects*; D002470:Cell Survival; D016922:Cellular Senescence / Q000187:drug effects; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins; D053551:Keratin-12 / Q000378:metabolism; D053558:Keratin-3 / Q000378:metabolism; D019394:Ki-67 Antigen / Q000378:metabolism; D016850:Limbus Corneae / Q000166:cytology* / Q000378:metabolism; D018613:Microscopy, Confocal; D010455:Peptides / Q000494:pharmacology*; D011817:Rabbits; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D018528:ATP-Binding Cassette Transporters; D015415:Biomarkers; D036341:Intercellular Signaling Peptides and Proteins; D053551:Keratin-12; D053558:Keratin-3; D019394:Ki-67 Antigen; D010455:Peptides; D015534:Trans-Activators; C076687:CS1 peptide; D001616:beta-Galactosidase