DataSheet1_Spheroid size influences cellular senescence and angiogenic potential of mesenchymal stromal cell-derived soluble factors and extracellular vesicles.pdf

Introduction: The secretome of mesenchymal stromal cells (MSCs) serves as an innovative tool employed in the regenerative medicine approach. In this particular context, three-dimensional (3D) culture systems are widely utilized to better replicate in vivo conditions and facilitate prolonged cell maintenance during culture. The use of spheroids enables the preservation of the classical phenotypical characteristics of MSCs. However, the distinct microenvironment within the spheroid may impact the secretome, thereby enhancing the angiogenic properties of adult MSCs that typically possess a reduced angiogenic potential compared to MSCs derived from perinatal tissues due to the hypoxia created in the internal region of the spheroid.Methods: In this study, large spheroids (2,600 cells, ∼300 μm diameter) and small spheroids (1,000 cells, ∼200 μm diameter) were used to examine the role of spheroid diameter in the generation of nutrients and oxygen gradients, cellular senescence, and the angiogenic potential of secreted factors and extracellular vesicles (EVs).Results: In this study, we demonstrate that large spheroids showed increased senescence and a secretome enriched in pro-angiogenic factors, as well as pro-inflammatory and anti-angiogenic cytokines, while small spheroids exhibited decreased senescence and a secretome enriched in pro-angiogenic molecules. We also demonstrated that 3D culture led to a higher secretion of EVs with classical phenotypic characteristics. Soluble factors and EVs from small spheroids exhibited higher angiogenic potential in a human umbilical vein endothelial cell (HUVEC) angiogenic assay.Discussion: These findings highlighted the necessity of choosing the appropriate culture system for obtaining soluble factors and EVs for specific therapeutic applications.</p

Additional file 3: of Circulating healing (CH) cells expressing BST2 are functionally activated by the injury-regulated systemic factor HGFA

Table S2. Fold change values of the 87 characterizing genes derived from SAM analysis for each pairwise class comparison. (XLSX 61 kb

Additional file 5: of Circulating healing (CH) cells expressing BST2 are functionally activated by the injury-regulated systemic factor HGFA

Table S3. RT-PCR array data set for the four considered experimental groups. (XLSX 57 kb

Additional file 4: of Circulating healing (CH) cells expressing BST2 are functionally activated by the injury-regulated systemic factor HGFA

Unsupervised hierarchical clustering of considered cell populations. (TXT 41 kb

Additional file 2: of Circulating healing (CH) cells expressing BST2 are functionally activated by the injury-regulated systemic factor HGFA

Table S1. Log2 Intensity values of the 87 genes characterizing the CH cells for each considered cell population. (XLSX 69 kb

Additional file 1: of Circulating healing (CH) cells expressing BST2 are functionally activated by the injury-regulated systemic factor HGFA

Figure S1. Pairwise class comparisons detecting genes differentially expressed in CH cells. Figure S2. Identification and sorting of BST2pos CH cells. Figure S3. Quantification of BST2pos CH cells migrated toward the injury sites. Figure S4. Specificity of the used anti-RFP antibody. Figure S5. Effects of injury-related signals on BST2pos CH cells motility. (PDF 15587 kb

Standardized Method to Functionalize Plasma-Extracellular Vesicles via Copper-Free Click Chemistry for Targeted Drug Delivery Strategies

Extracellular vesicles (EVs) have emerged as potential vehicles for targeted drug delivery and diagnostic applications. However, achieving consistent and reliable functionalization of EV membranes remains a challenge. Copper-catalyzed click chemistry, commonly used for EV surface modification, poses limitations due to cytotoxicity and interference with biological systems. To overcome these limitations, we developed a standardized method for functionalizing an EV membrane via copper-free click chemistry. EVs derived from plasma hold immense potential as diagnostic and therapeutic agents. However, the isolation and functionalization of EVs from such a complex biofluid represent considerable challenges. We compared three different EV isolation methods to obtain an EV suspension with an optimal purity/yield ratio, and we identified sucrose cushion ultracentrifugation (sUC) as the ideal protocol. We then optimized the reaction conditions to successfully functionalize the plasma-EV surface through a copper-free click chemistry strategy with a fluorescently labeled azide, used as a proof-of-principle molecule. Click-EVs maintained their identity, size, and, more importantly, capacity to be efficiently taken up by responder tumor cells. Moreover, once internalized, click EVs partially followed the endosomal recycling route. The optimized reaction conditions and characterization techniques presented in this study offer a foundation for future investigations and applications of functionalized EVs in drug delivery, diagnostics, and therapeutics

Table_1_The Secretome Derived From Mesenchymal Stromal Cells Cultured in a Xeno-Free Medium Promotes Human Cartilage Recovery in vitro.DOCX

Osteoarthritis (OA) is a disabling joint disorder causing articular cartilage degeneration. Currently, the treatments are mainly aimed to pain and symptoms relief, rather than disease amelioration. Human bone marrow stromal cells (hBMSCs) have emerged as a promising paracrine mechanism-based tool for OA treatment. Here, we investigate the therapeutic potential of conditioned media (CM) and extracellular vesicles (EVs) isolated from hBMSC and grown in a xeno-free culture system (XFS) compared to the conventional fetal bovine serum-culture system (FBS) in an in vitro model of OA. First, we observed that XFS promoted growth and viability of hBMSCs compared to FBS-containing medium while preserving their typical phenotype. The biological effects of the CM derived from hBMSC cultivated in XFS- and FBS-based medium were tested on IL-1α treated human chondrocytes, to mimic the OA enviroment. Treatment with CM derived from XFS-cultured hBMSC inhibited IL-1α-induced expression of IL-6, IL-8, and COX-2 by hACs compared to FBS-based condition. Furthermore, we observed that hBMSCs grown in XFS produced a higher amount of EVs compared to FBS-culture. The hBMSC-EVs not only inhibit the adverse effects of IL-1α-induced inflammation, but play a significant in vitro chondroprotective effect. In conclusion, the XFS medium was found to be suitable for isolation and expansion of hBMSCs with increased safety profile and intended for ready-to-use clinical therapies.</p

Figure S5 from Nicotinic Acid Phosphoribosyltransferase Regulates Cancer Cell Metabolism, Susceptibility to NAMPT Inhibitors, and DNA Repair

Figure S5 shows that 2-HNA inhibits NAPRT enzymatic activity and increases glycolysis in OVCAR-5 cells.</p

Figure S6 from Nicotinic Acid Phosphoribosyltransferase Regulates Cancer Cell Metabolism, Susceptibility to NAMPT Inhibitors, and DNA Repair

Figure S6 shows that NAPRT overexpression confers resistance to FK866 to MIA PaCa2 cells, but resistance is reverted by 2-HNA.</p