Extracellular vesicles (EVs) from mesenchymal stromal cells (MSC) are emerging as valuable therapeutic agents for tissue regeneration and immunomodulation, but their clinical applications have so far been limited by the technical restraints of current isolation and characterisation procedures. This study shows for the first time the successful application of Raman spectroscopy as label-free, sensitive and reproducible means of carrying out the routine bulk characterisation of MSC-derived vesicles before their use in vitro or in vivo, thus promoting the translation of EV research to clinical practice. The Raman spectra of the EVs of bone marrow and adipose tissue-derived MSCs were compared with human dermal fibroblast EVs in order to demonstrate the ability of the method to distinguish the vesicles of the three cytotypes automatically with an accuracy of 93.7%. Our data attribute a Raman fingerprint to EVs from undifferentiated and differentiated cells of diverse tissue origin, and provide insights into the biochemical characteristics of EVs from different sources and into the differential contribution of sphingomyelin, gangliosides and phosphatidilcholine to the Raman spectra themselves.

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Background In the last years, several clinical trials have proved the safety and efficacy of adipose-derived stem/stromal cells (ASC) in contrasting osteoarthritis (OA). Since ASC act mainly through paracrine mechanisms, their secretome (conditioned medium, CM) represents a promising therapeutic alternative. ASC-CM is a complex cocktail of proteins, nucleic acids, and lipids released as soluble factors and/or conveyed into extracellular vesicles (EV). Here, we investigate its therapeutic potential in an in vitro model of OA. Methods Human articular chondrocytes (CH) were induced towards an OA phenotype by 10 ng/ml TNFα in the presence of either ASC-CM or EV, both deriving from 5 × 105 cells, to evaluate the effect on hypertrophic, catabolic, and inflammatory markers. Results Given the same number of donor cells, our data reveal a higher therapeutic potential of ASC-CM compared to EV alone that was confirmed by its enrichment in chondroprotective factors among which TIMP-1 and -2 stand out. In details, only ASC-CM significantly decreased MMP activity (22% and 29% after 3 and 6 days) and PGE2 expression (up to 40% at day 6) boosted by the inflammatory cytokine. Conversely, both treatments down-modulated of ~ 30% the hypertrophic marker COL10A1. Conclusions These biological and molecular evidences of ASC-CM beneficial action on CH with an induced OA phenotype may lay the basis for its future clinical translation as a cell-free therapeutic in the management of OA.

The procedure to obtain MSCs from interdental papilla is less invasive than that used for both bone marrow and adipose tissue, GinPa-MSCs are easy to expand and can be efficiently loaded with PTX. Taken together these qualities suggest that GinPa-MSCs may prove to be a good tool for cell-mediated drug delivery in cancer, particularly if related to stomatognathic system.

The adult stem cell secretome is currently under investigation as an alternative to cell-based therapy in regenerative medicine, thanks to the remarkable translational opportunity and the advantages in terms of handling and safety. In this perspective, we recently demonstrated the efficient performance of the adipose-derived mesenchymal stem/stromal cell (ASC) secretome in contrasting neuroinflammation in a murine model of diabetic neuropathy, where the administration of factors released by dermal fibroblasts (DFs) did not exert any effect. Up to now, the complex mixture of the constituents of the conditioned medium from ASCs has not been fully deepened, although its appropriate characterization is required in the perspective of a clinical use. Herein, we propose the differential proteomic approach for the identification of the players accounting for the functional effects of the cell secretome with the aim to unravel its appropriate applications. Out of 967 quantified proteins, 34 and 62 factors were found preponderantly or exclusively secreted by ASCs and DFs, respectively. This approach led to the recognition of distinct functions related to the conditioned medium of ASCs and DFs, with the former being involved in the regulation of neuronal death and apoptosis and the latter in bone metabolism and ossification. The proosteogenic effect of DF secretome was validated in vitro on human primary osteoblasts, providing a proof of concept of its osteoinductive potential. Besides discovering new applications of the cell type-specific secretome, the proposed strategy could allow the recognition of the cocktail of bioactive factors which might be responsible for the effects of conditioned media, thus providing a solid rationale to the implementation of a cell-free approach in several clinical scenarios involving tissue regeneration.

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