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Osteosarcoma is a bone tumor with a high incidence in children and adolescents. Chemotherapy for osteosarcoma is limited, and effective targeted drugs are urgently needed to treat osteosarcoma. Exosomes as a natural nano drug delivery platform have been widely studied and proven to have good drug delivery performance. However, the low production of exosomes hinders its development as a carrier. Exosome mimetics (EMs) as an alternative product of exosomes solve the problem of low production of exosomes and maintain the good performance of exosomes as carriers. In this study, bone marrow mesenchymal stem cells (BMSCs) were sequentially extruded to generate EMs to encapsulate doxorubicin (EM-Dox) to treat osteosarcoma. The results showed that we successfully prepared EMs of BMSC, and EM-Dox was prepared using an active-loading approach. Our engineered EM-Dox demonstrated significantly more potent tumor inhibition activity and fewer side effects than free doxorubicin. This novel biological nanomedicine system provides a promising opportunity to develop novel precision medicine for osteosarcoma.

To develop exosome-mimetics derived from bone marrow mesenchymal stem cells (EM) as a novel nanoscale drug delivery system(nanoDDS) with improved tumor targeting activity, therapeutic effect, and biosafety, and to evaluate the therapeutic effect of doxorubicin loaded EM (EM-Dox) on neuroblastoma (NB) in vitro and in vivo.EM was prepared by serial extrusion of bone marrow mesenchymal stem cells (BMSCs), ammonium sulfate gradient method was used to promote the active loading of doxorubicin, and EM-Dox was obtained after removal of free doxorubicin by dialysis. The obtained EM and EM-Dox were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), Western Blot assay(WB), and the yield of exosomes and EM was further compared. Confocal fluorescent microscopy was used to verify the uptake of EM-Dox and free doxorubicin (Free-Dox) by NB cells. CCK-8 assay, cell cycle assay, and cell apoptosis assay were used to evaluate the antitumor effect of EM-Dox on NB cells in vitro. In addition, the targeted therapeutic effect and biosafety of EM-Dox against NB were evaluated in tumor-bearing nude mice.TEM, NTA, and WB verified that both EM and EM-Dox feature highly similar morphology, size and marker protein expression in comparison with naturally occurred exosomes, but the particle size of EM-Dox increased slightly after loading doxorubicin. The protein yield and particle yield of EM-Dox were 16.8 and 26.3-folds higher than those of exosomes, respectively. Confocal fluorescent microscopy showed that EM and doxorubicin had a definite co-localization. EM-Dox was readily internalized in two well-established human NB cell lines. The intracellular content of doxorubicin in cells treated with EM-Dox was significantly higher than that treated with Free-Dox. CCK-8 assay and flow cytometry confirmed that EM-Dox could inhibit NB cell proliferation, induce G2/M phase cell cycle arrest, and promote NB cell apoptosis in vitro. In vivo bioluminescence imaging results demonstrated that EM-Dox effectively targets NB tumors in vivo. Compared with Free-Dox, EM-Dox had a significantly increased inhibitory effect against NB tumor proliferation and progression in vivo, without inducing any myocardial injury.EM-Dox showed significantly increased anti-tumor activity in comparison with free doxorubicin in vitro and in vivo, and scalable EMs may represent a new class of NanoDDS that can potentially replace naturally occurred exosomes in preclinical or clinical translations.

Renal fibrosis occurs largely through epithelial-mesenchymal transition (EMT). This study explored the beneficial effects of a human umbilical cord mesenchymal stem cell-loaded decellularized kidney scaffold (ucMSC-DKS) on renal fibrosis in a rodent model of post-transplantation renal failure, and the underlying mechanism.Rat-derived DKSs were examined after preparation, and then recellularized with human ucMSCs to prepare cell-loaded patches. A rat model of renal failure was established after subtotal nephrectomy (STN). The cell patches were transplanted to remnant kidneys. Changes in renal function, histology, EMT, and proteins related to the transforming growth factor-β (TGF-β)/Smad signaling pathway in the remnant kidneys were examined 8 weeks after surgery, compared with non-cell patch controls.The DKSs were acellular and porous, with rich cytokine and major extracellular matrix components. The ucMSCs were distributed uniformly in the DKSs. Renal function was improved, renal fibrosis and EMT were reduced, and the TGF-β/Smad signaling pathway was inhibited compared with controls at 8 weeks after ucMSC-DKS patch transplantation.The ucMSC-DKS restores renal function and reduces fibrosis by reducing EMT via the TGF-β/Smad signaling pathway in rats that have undergone STN. It provides an alternative for renal fibrosis treatment.

Non-obstructive azoospermia is the most challenging type of male infertility. Stem cell based therapy provides the potential to enhance the recovery of spermatogenesis following cancer therapy. Bone marrow-derived mesenchymal stem cells (BMSCs) possess the potential to differentiate or trans-differentiate into multi-lineage cells, secrete paracrine factors to recruit the resident stem cells to participate in tissue regeneration, or fuse with the local cells in the affected region. In this study, we tested whether spermatogenically-induced BMSCs can restore spermatogenesis after administration of an anticancer drug. Allogeneic BMSCs were co-cultured in conditioned media derived from cultured testicular Sertoli cells in vitro, and then induced stem cells were transplanted into the seminiferous tubules of a busulfan-induced azoospermatic rat model for 8 weeks. The in vitro induced BMSCs exhibited specific spermatogonic gene and protein markers, and after implantation the donor cells survived and located at the basement membranes of the recipient seminiferous tubules, in accordance with what are considered the unique biological characteristics of spermatogenic stem cells. Molecular markers of spermatogonial stem cells and spermatogonia (Vasa, Stella, SMAD1, Dazl, GCNF, HSP90α, integrinβ1, and c-kit) were expressed in the recipient testis tissue. No tumor mass, immune response, or inflammatory reaction developed. In conclusion, BMSCs might provide the potential to trans-differentiate into spermatogenic-like-cells, enhancing endogenous fertility recovery. The present study indicates that BMSCs might offer alternative treatment for the patients with azoospermatic infertility after cancer chemotherapy.