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This unit describes protocols for isolating subpopulations of extracellular vesicles (EVs) purified from human adipose tissue–derived mesenchymal stromal cells by density gradient centrifugation and for characterizing them by flow cytometry (FCM). Determining the optimal strategy for isolating EVs is a critical step toward retrieving the maximal amount while ensuring the recovery of different vesicular subtypes. The first protocol details density gradient centrifugation to isolate both exosomes and microvesicles. In the second protocol, characterization of EV subpopulations by FCM is depicted, taking advantage of non‐conventional modalities, in accordance with the latest technical indications. The procedures described here can be easily reproduced and can be employed regardless of the cell type used to obtain EVs. © 2019 by John Wiley & Sons, Inc.

Cardiovascular side effects are major shortcomings of cancer treatments causing cardiotoxicity and late-onset cardiomyopathy. While doxorubicin (Dox) has been reported as an effective chemotherapy agent, unspecific impairment in cardiomyocyte mitochondria activity has been documented. We demonstrated that the human fetal amniotic fluid-stem cell (hAFS) secretome, namely the secreted paracrine factors within the hAFS-conditioned medium (hAFS-CM), exerts pro-survival effects on Dox-exposed cardiomyocytes. Here, we provide a detailed comparison of the cardioprotective potential of hAFS-CM over the secretome of mesenchymal stromal cells from adipose tissue (hMSC-CM). hAFS and hMSC were preconditioned under hypoxia to enrich their secretome. The cardioprotective effects of hAFS/hMSC-CM were evaluated on murine neonatal ventricular cardiomyocytes (mNVCM) and on their fibroblast counterpart (mNVFib), and their long-term paracrine effects were investigated in a mouse model of Dox-induced cardiomyopathy. Both secretomes significantly contributed to preserving mitochondrial metabolism within Dox-injured cardiac cells. hAFS-CM and hMSC-CM inhibited body weight loss, improved myocardial function, reduced lipid peroxidation and counteracted the impairment of mitochondrial complex I activity, oxygen consumption, and ATP synthesis induced by Dox. The hAFS and hMSC secretomes can be exploited for inhibiting cardiotoxic detrimental side effects of Dox during cancer therapy, thus ensuring cardioprotection via combinatorial paracrine therapy in association with standard oncological treatments.

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The secretome of mesenchymal stromal cells (MSCs) derived from different tissue sources is considered an innovative therapeutic tool for regenerative medicine.Although adipose tissue-and bone marrow-derived MSCs (ADSCs and BMSCs, respectively) share many biological features, the different tissue origins can be mirrored by variations in their secretory profile, and in particular in the secreted extracellular vesicles (EVs). In this study, we carried out a detailed and comparative characterization of middle-and small-sized EVs (mEVs and sEVs, respectively) released by either ADSCs or BMSCs. Their involvement in an endochondral ossification setting was investigated using ex vivo metatarsal culture models that allowed to explore both blood vessel sprouting and bone growth plate dynamics. Although EVs separated from both cell sources presented similar characteristics in terms of size, concentration, and marker expression, they exhibited different characteristics in terms of protein content and functional effects. ADSC-EVs overexpressed proangiogenic factors in comparison to the BMSC-counterpart, and, consequently, they were able to induce a significant increase in endothelial cord outgrowth. On the other hand, BMSC-EVs contained a higher amount of pro-differentiation and chemotactic proteins, and they were able to prompt growth plate organization. The present study highlights the importance of selecting the appropriate cell source of EVs for targeted therapeutic applications. K E Y W O R D S cartilage and bone development, ex vivo metatarsal culture model, extracellular vesicles, mesenchymal stromal cells, regenerative medicine Significance statement Mesenchymal stromal cells are a great source for regenerative medicine applications. Cells derived from different tissues might have distinctive characteristics. Many

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Three‐dimensional (3D) cancer tumor models are becoming vital approaches for high‐throughput drug screening, drug targeting, development of novel theranostic systems, and personalized medicine. Yet, it is becoming more evident that the tumor progression and metastasis is fueled by a subpopulation of stem‐like cells within the tumor that are also called cancer stem cells (CSCs). This study aimed to develop a tumoroid model using CSCs. For this purpose CD133+ cells were isolated from SaOS‐2 osteosarcoma cell line with magnetic‐activated cell sorting. To evaluate tumoroid formation ability, the cells were incubated in different cell numbers in agar gels produced by 3D Petri Dish® method. Subsequently, CD133+ cells and CD133− cells were co‐cultured to investigate CD133+ cell localization in tumoroids. The characterization of tumoroids was performed using Live&Dead staining, immunohistochemistry, and quantitative polymerase chain reaction analysis. The results showed that, CD133+, CD133− and SaOS‐2 cells were all able to form 3D tumoroids regardless of the initial cell number, but, while 72 hr were needed for CD133+ cells to self‐assemble, 24 hr were enough for CD133− and SaOS‐2 cells. CD133+ cells were located within tumoroids randomly with high cell viability. Finally, when compared to two‐dimensional (2D) cultures, there were 5.88, 4.14, 6.95, and 1.68‐fold higher messenger RNA expressions for Sox2, OCT3/4, Nanog, and Nestin, respectively, in CD133+ cells that were cultured within 3D tumoroids, showing longer maintenance of stem cell phenotype in 3D, that can allow more relevant screening and targeting efficiency in pharmaceutical testing. It was concluded that CSC‐based tumoroids are propitious as 3D tumor models to fill the gap between conventional 2D in vitro culture and in vivo animal experiments for cancer research.