RESUMEN
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.
RESUMEN
Mesenchymal stromal/stem cells (MSCs) are believed to function in vivo as a homeostatic tool that shows therapeutic properties for tissue repair/regeneration. Conventionally, these cells are expanded in two-dimensional (2D) cultures, and, in that case, MSCs undergo genotypic/phenotypic changes resulting in a loss of their therapeutic capabilities. Moreover, several clinical trials using MSCs have shown controversial results with moderate/insufficient therapeutic responses. Different priming methods were tested to improve MSC effects, and three-dimensional (3D) culturing techniques were also examined. MSC spheroids display increased therapeutic properties, and, in this context, it is crucial to understand molecular changes underlying spheroid generation. To address these limitations, we performed RNA-seq on human amnion-derived MSCs (hAMSCs) cultured in both 2D and 3D conditions and examined the transcriptome changes associated with hAMSC spheroid formation. We found a large number of 3D culture-sensitive genes and identified selected genes related to 3D hAMSC therapeutic effects. In particular, we observed that these genes can regulate proliferation/differentiation, as well as immunomodulatory and angiogenic processes. We validated RNA-seq results by qRT-PCR and methylome analysis and investigation of secreted factors. Overall, our results showed that hAMSC spheroid culture represents a promising approach to cell-based therapy that could significantly impact hAMSC application in the field of regenerative medicine.
