Activation of canonical Wnt pathway promotes proliferation of retinal stem cells derived from adult mouse ciliary margin
Activation of canonical Wnt pathway promotes proliferation of retinal stem cells derived from adult mouse ciliary margin
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DOI：
10.1634/stemcells.2005-0124
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发表时间：
2006-01-01
期刊：
影响因子：
5.2
通讯作者：
Taga, T
中科院分区：
文献类型：
--
作者：
Inoue, T;Kagawa, T;Taga, T
成人视网膜干细胞是治疗视网膜变性的一种可能的细胞来源。然而，只有少量的干细胞驻留在睫状缘。本研究旨在通过Wnt信号通路促进成体视网膜干细胞的增殖。将来自8周龄小鼠的睫状缘细胞分离并培养以允许球形集落形成。然后在培养基中应用Wnt 3a、糖原合成酶激酶（GSK）3抑制剂、成纤维细胞生长因子（FGF）2和FGF受体抑制剂。解离原代球体以制备单层或二次球体培养物。Wnt 3a增加了原代球的大小和单层培养中Ki-67阳性增殖细胞的数量。Wnt 3a处理的初级球细胞能够自我更新，并产生了四倍的次级球的数量相比，未经处理的球细胞。这些细胞还保留了其多谱系潜力，在分化培养条件下表达几种视网膜标记物。Wnt 3a处理的细胞显示β-连环蛋白的核积累，并且GSK 3抑制剂SB 216763模拟Wnt 3a的促有丝分裂活性。SB 216763的增殖作用被FGF受体抑制剂减弱，但被FGF 2增强，Ki-67阳性细胞达到总细胞的70%以上。Wnt 3a和SB 216763促进视网膜干细胞的增殖，并且这部分依赖于FGF 2信号传导。Writ和FGF信号传导的组合可以为成人视网膜干细胞的体外扩增或体内激活提供治疗策略。
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 Writ and FGF signaling may provide a therapeutic strategy for in vitro expansion or in vivo activation of adult retinal stem cells.