3D Culture and Interferon-γ Priming Modulates Characteristics of Mesenchymal Stromal/Stem Cells by Modifying the Expression of Both Intracellular and Exosomal microRNAs

SIMPLE SUMMARY: With the aim of improving the therapeutic potential of mesenchymal stromal/stem cells (MSCs), we analyzed miRNA expression to investigate the effects of priming on intracellular and exosome (EXO)-derived miRNAs of MSCs. We primed MSCs with 3D culture (3D MSCs) or IFN-γ treatment (γ-MSCs), and EXOs were isolated from the conditioned medium. The miRNA analysis revealed similar expression patterns in intracellular miRNAs among biological replicates, while we observed noticeable variability in EXO miRNAs released even with the same priming condition. Although the MSCs and their EXOs exhibited distinct miRNA profiles following each priming treatment, we found deregulated miRNAs in common between the two sample types. The gene ontology of the deregulated miRNAs obtained after priming showed that MSC and EXO-derived miRNAs were functionally associated with tissue repair/regeneration. Specifically, γ-MSCs, 3D MSC EXOs, and γ-MSC EXOs contained more enriched miRNAs related to immunomodulation compared with 3D MSCs. Moreover, compared with IFN-γ treatment, both cells and EXOs derived from the 3D culture had more enriched miRNAs targeting genes involved in angiogenesis. Our study demonstrates that both 3D culture and IFN-γ treatment are able to modify intracellular and exosomal miRNAs, and our findings might contribute to a better understanding of the molecular mechanisms underlying the miRNA-mediated beneficial effects of MSCs. ABSTRACT: Mesenchymal stromal/stem cells (MSCs) have emerged as a therapeutic tool in regenerative medicine. Recent studies have shown that exosome (EXO)-derived microRNAs (miRNAs) play a crucial role in mediating MSC functions. Additionally, intracellular miRNAs have been found to regulate MSC therapeutic capacities. However, the molecular mechanisms underlying miRNA-mediated MSC effects are not fully understood. We used 3D culture and IFN-γ to prime/enhance the MSC therapeutic effects in terms of functional miRNAs. After priming, our analysis revealed stable variations in intracellular miRNA among the MSC biological replicates. Conversely, a significant variability of miRNA was observed among EXOs released from biological replicates of the priming treatment. For each priming, we observed distinct miRNA expression profiles between the MSCs and their EXOs. Moreover, in both types of priming, gene ontology (GO) analysis of deregulated miRNAs highlighted their involvement in tissue repair/regeneration pathways. In particular, the 3D culture enhanced angiogenic properties in both MSCs and EXOs, while IFN-γ treatment enriched miRNAs associated with immunomodulatory pathways. These findings suggest that 3D culture and IFN-γ treatment are promising strategies for enhancing the therapeutic potential of MSCs by modulating miRNA expression. Additionally, the identified miRNAs may contribute to understanding the molecular mechanisms underlying the miRNA-mediated therapeutic effects of MSCs.