Inflammatory priming enhances mesenchymal stromal cell secretome potential as a clinical product for regenerative medicine approaches through secreted factors and EV-miRNAs: the example of joint disease
Authors: Ragni Enrico , Perucca Orfei Carlotta , De Luca Paola , Mondadori Carlotta , Viganò Marco , Colombini Alessandra , de Girolamo Laura 
Published: April 2020

Abstract
Background
Mesenchymal stromal cell (MSC)-enriched products showed positive clinical outcomes in regenerative medicine, where tissue restoration and inflammation control are needed. GMP-expanded MSCs displayed an even higher potential due to exclusive secretion of therapeutic factors, both free and conveyed within extracellular vesicles (EVs), collectively termed secretome. Moreover, priming with biochemical cues may influence the portfolio and biological activities of MSC-derived factors. For these reasons, the use of naive or primed secretome gained attention as a cell-free therapeutic option. Albeit, at present, a homogenous and comprehensive secretome fingerprint is still missing. Therefore, the aim of this work was to deeply characterize adipose-derived MSC (ASC)-secreted factors and EV-miRNAs, and their modulation after IFNγ preconditioning. The crucial influence of the target pathology or cell type was also scored in osteoarthritis to evaluate disease-driven potency.

Methods
ASCs were isolated from four donors and cultured with and without IFNγ. Two-hundred secreted factors were assayed by ELISA. ASC-EVs were isolated by ultracentrifugation and validated by flow cytometry, transmission electron microscopy, and nanoparticle tracking analysis. miRNome was deciphered by high-throughput screening. Bioinformatics was used to predict the modulatory effect of secreted molecules on pathologic cartilage and synovial macrophages based on public datasets. Models of inflammation for both macrophages and chondrocytes were used to test by flow cytometry the secretome anti-inflammatory potency.

Results
Data showed that more than 60 cytokines/chemokines could be identified at varying levels of intensity in all samples. The vast majority of factors are involved in extracellular matrix remodeling, and chemotaxis or motility of inflammatory cells. IFNγ is able to further increase the capacity of the secretome to stimulate cell migration signals. Moreover, more than 240 miRNAs were found in ASC-EVs. Sixty miRNAs accounted for > 95% of the genetic message that resulted to be chondro-protective and M2 macrophage polarizing. Inflammation tipped the balance towards a more pronounced tissue regenerative and anti-inflammatory phenotype. In silico data were confirmed on inflamed macrophages and chondrocytes, with secretome being able to increase M2 phenotype marker CD163 and reduce the chondrocyte inflammation marker VCAM1, respectively. IFNγ priming further enhanced secretome anti-inflammatory potency.

Conclusions
Given the portfolio of soluble factors and EV-miRNAs, ASC secretome showed a marked capacity to stimulate cell motility and modulate inflammatory and degenerative processes. Preconditioning is able to increase this ability, suggesting inflammatory priming as an effective strategy to obtain a more potent clinical product which use should always be driven by the molecular mark of the target pathology.