Differential Proteomic Analysis Predicts Appropriate Applications for the Secretome of Adipose-Derived Mesenchymal Stem/Stromal Cells and Dermal Fibroblasts

Abstract

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.

Raman spectroscopy as a quick tool to assess purity of extracellular vesicle preparations and predict their functionality

“…As previously suggested, the spectroscopic P/L data can become an additional parameter in the routine quality control of EV preparations and can help exploring the observed differences in EV preparations. Although the investigation of the mechanisms underneath the differences in the functionality of stem‐cell derived secretome and EVs goes beyond the scope of the present work, our findings support the hypothesis that such differences in the biological effect of the secretome of HLSCs and MSCs can be both related to EV and non‐EV components, in accordance with previously reported data demonstrating that not all proteins involved in the regenerative functions of stem cells are released as vesicular cargos [40,41]. As a matter of fact, the 1x UC of HLSC‐derived EVs were demonstrated to have a higher P/L ratio compared to purer 2x UC and SEC samples that might be explained by contaminant protein components that are washed out and removed from the EV preparation when the 2x UC and SEC protocols are applied.…”

“…Taken as a whole, our data showed the ability of RS to perform a bulk biochemical characterisation of EV preparations and suggested the Raman approach as a rapid (≤ 5 min), label‐free method for the investigation of stem cell‐derived secretome. Compared to the single vesicle approach that require even more sophisticated instruments and laborious protocols, the proposed procedure for RS can be considered an economical and fast alternative for other‐omic approaches, such as differential proteomics [40], which already proved to be a successful tool to characterise the secretome of stem cells. It is worth noting that, beside the above‐described advantages, the proposed method has some limitations.…”

Hopes and Limits of Adipose-Derived Stem Cells (ADSCs) and Mesenchymal Stem Cells (MSCs) in Wound Healing

“…Modulation of the inflammatory status of the microenvironment would not be possible without homing and migration of ADSCs and the other immune cells. Finally, controlling the cell enrichment and inflammatory profile of the dermis exhibits a potential advantage in developing strategies in regenerative medicine in both autologous and allogeneic settings [158,159].…”

Adipose-Derived Stem Cells (ADSCs) and Growth Differentiation Factor 11 (GDF11): Regenerative and Antiaging Capacity for the Skin

“…Administrated intradermally to an aged skin, skin texture and wrinkles as well as dermal thickness were found improved 8 weeks after treatment [74]. The extracellular vesicles released in ADSCs' secretome or conditioned media enable the targeted cells to increase the production and deposition of ECM proteins including collagen and elastin [11,16,90,109,110]. Among these autocrine/paracrine factors, TGF-β and GDF11 appeared to be strongly associated (Figure 1).…”