Department of Medical Research, Taipei Veterans General Hospital, Taipei, Taiwan.
Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan.
Sci Rep. 2017 Aug 31;7(1):10113. doi: 10.1038/s41598-017-10708-0.
Mesenchymal stem cells (MSCs) in conventional monolayer culture are heterogeneous and contain a significant portion of senescent cells. MSCs cultured on chitosan film form 3-dimenional spheres, increase in stemness and differentiation capability; however, the underlying mechanisms remain elusive. We first demonstrate chitosan film culture induces apoptosis at 2 days, with specificity in late senescent cells. Especially in senescent cells, chitosan film culture activates mTOR, which activates S6K/S6/4E-BP1 to enhance fibronection synthesis and peripheral dead cell attachment, and phosphorylates ULK1 at S757 to further inactivate ULK1, LC3A and autophagy, thereby inducing apoptosis. Combination of chitosan film culture with mTOR inhibition prevents peripheral dead cell attachment, thereby further increasing pluripotent gene expression, in vitro osteogenesis and in vivo bone formation. These data successfully figure out the role of mTOR signaling in chitosan film culture and develop a method by combination of rapamycin treatment for promoting stemness and differentiation capability in MSCs.
常规单层培养的间充质干细胞（MSCs）是异质的，其中包含很大一部分衰老细胞。在壳聚糖膜上培养的 MSCs 形成三维球体，增加了干细胞特性和分化能力；然而，其潜在机制仍不清楚。我们首先证明壳聚糖膜培养在第 2 天诱导细胞凋亡，并且对晚期衰老细胞具有特异性。特别是在衰老细胞中，壳聚糖膜培养激活 mTOR，mTOR 激活 S6K/S6/4E-BP1 以增强纤维连接蛋白的合成和外周死亡细胞的附着，并使 ULK1 在 S757 磷酸化，从而进一步失活 ULK1、LC3A 和自噬，从而诱导细胞凋亡。壳聚糖膜培养与 mTOR 抑制的联合使用可防止外周死亡细胞附着，从而进一步增加多能基因表达、体外成骨和体内骨形成。这些数据成功地阐明了 mTOR 信号在壳聚糖膜培养中的作用，并开发了一种通过雷帕霉素治疗联合使用的方法，以促进 MSCs 的干细胞特性和分化能力。
