title: Activation of canonical Wnt pathway promotes proliferation of retinal stem cells derived from adult mouse ciliary margin.
abstract: Adult retinal stem cells represent a possible cell source for the treatment of retinal degeneration. However, only a small number of stem cells reside in the ciliary margin. The present study aimed to promote the proliferation of adult retinal stem cells via the Wnt signaling pathway. Ciliary margin cells from 8-week-old mice were dissociated and cultured to allow sphere colony formation. Wnt3a, a glycogen synthase kinase (GSK) 3 inhibitor, fibroblast growth factor (FGF) 2, and a FGF receptor inhibitor were then applied in the culture media. The primary spheres were dissociated to prepare either monolayer or secondary sphere cultures. Wnt3a increased the size of the primary spheres and the number of Ki-67-positive proliferating cells in monolayer culture. The Wnt3a-treated primary sphere cells were capable of self-renewal and gave rise to fourfold the number of secondary spheres compared with nontreated sphere cells. These cells also retained their multilineage potential to express several retinal markers under differentiating culture conditions. The Wnt3a-treated cells showed nuclear accumulation of beta-catenin, and a GSK3 inhibitor, SB216763, mimicked the mitogenic activity of Wnt3a. The proliferative effect of SB216763 was attenuated by an FGF receptor inhibitor but was enhanced by FGF2, with Ki-67-positive cells reaching over 70% of the total cells. Wnt3a and SB216763 promoted the proliferation of retinal stem cells, and this was partly dependent on FGF2 signaling. A combination of Wnt and FGF signaling may provide a therapeutic strategy for in vitro expansion or in vivo activation of adult retinal stem cells.
authors.affiliation: Department of Cell Fate Modulation, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Kumamoto-city, Japan.
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Inoue
authors.forename: Tetsushi
authors.initials: T
authors.lastname: Kagawa
authors.forename: Mikiko
authors.initials: M
authors.lastname: Fukushima
authors.forename: Takeshi
authors.initials: T
authors.lastname: Shimizu
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Yoshinaga
authors.forename: Shinji
authors.initials: S
authors.lastname: Takada
authors.forename: Hidenobu
authors.initials: H
authors.lastname: Tanihara
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Taga
mesh_terms: D000818:Animals; D002455:Cell Division; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D002924:Ciliary Body / Q000166:cytology* / Q000378:metabolism / Q000502:physiology; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors; D019394:Ki-67 Antigen / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D012171:Retinal Vessels / Q000166:cytology / Q000378:metabolism*; D015398:Signal Transduction; D013234:Stem Cells / Q000502:physiology*; D051153:Wnt Proteins / Q000378:metabolism / Q000502:physiology*; D060508:Wnt3 Protein; D060509:Wnt3A Protein; D051176:beta Catenin / Q000378:metabolism
chemical_list: D019394:Ki-67 Antigen; D051153:Wnt Proteins; D060508:Wnt3 Protein; D060509:Wnt3A Protein; C555511:Wnt3a protein, mouse; D051176:beta Catenin; D016222:Fibroblast Growth Factor 2; D038362:Glycogen Synthase Kinase 3