Exosomes, nanoscale extracellular vesicles functioning as cell-to-cell communicators, are an emerging promising therapeutic in the field of bone tissue engineering. Here, we report the construction and evaluation of a novel cell-free tissue-engineered bone that successfully accelerated the restoration of critical-sized mouse calvarial defects through combining exosomes derived from human adipose-derived stem cells (hASCs) with poly(lactic-co-glycolic acid) (PLGA) scaffolds. The exosomes were immobilized on the polydopamine-coating PLGA (PLGA/pDA) scaffolds under mild chemical conditions. Specifically, we investigated the effects of hASC-derived exosomes on the osteogenic, proliferation, and migration capabilities of human bone marrow-derived mesenchymal stem cells in vitro and optimized their osteoinductive effects through osteogenic induction. Furthermore, an in vitro assay showed exosomes could release from PLGA/pDA scaffold slowly and consistently and in vivo results showed this cell-free system enhanced bone regeneration significantly, at least partially through its osteoinductive effects and capacities of promoting mesenchymal stem cells migration and homing in the newly formed bone tissue. Therefore, overall results demonstrated that our novel cell-free system comprised of hASC-derived exosomes and PLGA/pDA scaffold provides a new therapeutic paradigm for bone tissue engineering and showed promising potential in repairing bone defects.

Objectives:The present study aimed to investigate whether exosomes derived from miR-375-overexpressing human adipose mesenchymal stem cells (hASCs) could enhance bone regeneration. Materials and Methods:Exosomes enriched with miR-375 (Exo [miR-375]) were generated from hASCs stably overexpressing miR-375 after lentiviral transfection and identified with transmission electron microscopy, nanosight and western blotting.The construction efficiency of Exo (miR-375) was evaluated with qRT-PCR and incubated with human bone marrow mesenchymal stem cells (hBMSCs) to optimize the effective dosage. Then, the osteogenic capability of Exo (miR-375) was investigated with ALP and ARS assays. Furthermore, dual-luciferase reporter assay and western blotting were conducted to reveal the underlying mechanism of miR-375 in osteogenic regulation. Finally, Exo (miR-375) were embedded with hydrogel and applied to a rat model of calvarial defect, and μ-CT analysis and histological examination were conducted to evaluate the therapeutic effects of Exo (miR-375) in bone regeneration. Results: miR-375 could be enriched in exosomes by overexpressing in the parent cells. Administration of Exo (miR-375) at 50 μg/mL improved the osteogenic differentiation of hBMSCs. With miR-375 absorbed by hBMSCs, insulin-like growth factor binding protein 3 (IGFBP3) was inhibited by binding to its 3′UTR, and recombinant IGFBP3 protein reduced the osteogenic effects triggered by Exo (miR-375). After incorporated with hydrogel, Exo (miR-375) displayed a slow and controlled release, and further in vivo analysis demonstrated that Exo (miR-375) enhanced the bone regenerative capacity in a rat model of calvarial defect. Conclusions: Taken together, our study demonstrated that exosomes derived from miR-375-overexpressing hASCs promoted bone regeneration. S U PP O RTI N G I N FO R M ATI O N Additional supporting information may be found online in the Supporting Information section at the end of the article. How to cite this article: Chen S, Tang Y, Liu Y, et al. Exosomes derived from miR-375-overexpressing human adipose mesenchymal stem cells promote bone regeneration. Cell

Background:Cancer stem cells (CSCs) paradigm suggests that CSCs might have important clinical implications in cancer therapy. Previously, we reported that accumulation efficiency of CSCs is different post low- and high-LET irradiation in 48 h.Methods:Cancer stem cells and non-stem cancer cells (NSCCs) were sorted and functionally identified through a variety of assays such as antigen profiles and sphere formation. Inter-conversion between CSCs and NSCCs were in situ visualised. Cancer stem cells proportions were assayed over multiple generations under normal and irradiation surroundings. Supplement and inhibition of TGF-β1, as well as immunofluorescence assay of E-cadherin and Vimentin, were performed.Results:Surface antigen markers of CSCs and NSCCs exist in an intrinsic homoeostasis state with spontaneous and in situ visualisable inter-conversions, irrespective of prior radiations. Supplement with TGF-β1 accelerates the equilibrium, whereas inhibition of TGF-β signalling disturbs the equilibrium and significantly decreases CSC proportion. Epithelial mesenchymal transition (EMT) might be activated during the process.Conclusion:Our results indicate that the intrinsic inter-conversion and dynamic equilibrium between CSCs and NSCCs exist under normal and irradiation surroundings, and TGF-β might have important roles in the equilibrium through activating EMT.

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The X-linked deubiquitinase USP9X has been implicated in multiple pathological disorders including malignancies and X-linked intellectual disability. However, its biological function and substrate repertoire remain to be investigated. In this study, we utilized the tandem mass tag labeling assay to identify USP9X-regulated proteins and revealed that the expression of multiple genes is altered in USP9X-deficient cells. Interestingly, we showed that USP9X promotes stabilization of centrosome proteins PCM1 and CEP55 through its catalytic activity. Remarkably, we demonstrated that USP9X is physically associated and spatially co-localized with PCM1 and CEP55 in the centrosome, and we revealed that either PCM1 or CEP55 loss resulted in impairment of USP9X centrosome localization. Moreover, we showed that USP9X is required for centrosome duplication, and this effect is dependent on its catalytic activity and its N-terminal module, which is responsible for physical association of USP9X with PCM1 and CEP55. Collectively, our experiments identified USP9X as an integral component of the centrosome where it functions to stabilize PCM1 and CEP55 and promote centrosome biogenesis.