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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.

Involvement of mTOR-autophagy in the selection of primitive mesenchymal stem cells in chitosan film 3-dimensional culture