Characterization of the secretome from Spheroids of Adipose derived Stem Cells (SASCs) and its potential for tissue regeneration

Introduction: Adipose-derived Stem Cells (ADSCs), a population of Mesenchymal Stem Cells (MSCs), showed very important features in the field of regenerative medicine such as high multilineage differentiation potential and immunomodulatory activity. In the last years, MSC properties have been attributed to their secreted factors such as soluble proteins, cytokines and growth factors. Moreover, a key role is played by the extracellular vesicles (EVs) which lead a heterogeneous cargo of proteins, lipids, mRNAs and small and long non-coding RNAs that interfere with the pathways of the recipient cells.Purpose: The aim of this work was to characterize the composition of the secretome from3D Spheroids of Adipose-derived Stem Cells (SASCs) by screening it for a pool of mRNAs and miRNAs. In addition, the effect of the total secretome and the isolated exosomes on endothelial cells, fibroblasts and osteoblasts was tested in in vitro wound healing assays.Material and Methods: Adipose tissue and/or lipoaspirate samples were collected from healthy individuals after signing informed consent. Cells were extracted and cultured forone week. Then, their secretomes were isolated every 72 hours. The exosomes were extracted and characterized by DLS, SEM and Western Blotting analysis and the expression of a panel of mRNAs and miRNAs as internal cargo were evaluated through Real-timePCR. Lastly, the wound healing assay was performed to investigate the regenerative potential of the total secretome and/or isolated exosomes on endothelial cells, fibroblastsand osteoblasts.Results: The exosomes secreted from SASCs showed an up-regulation of NANOG and SOX2 mRNAs, typical of stemness maintenance, as well as miR-126 and miR-146a, related to angiogenic and osteogenic processes. Moreover, the isolated exosomes showed an enhanced pro-regenerative effect on endothelial cells, fibroblasts and osteoblasts if compared to the total secretome in in vitro wound healing assay. Conclusions: The secretome from SASCs carried paracrine signals involved in stemnessmaintenance, pro-angiogenic and pro-osteogenic differentiation, immune system regulation and regeneration.