Anti-miR-195-engineered mesenchymal stem cells promote migration, vascularization, and ECM protein expression in vivo

Advanced therapy medicinal products (ATMPs) involving mesenchymal stromal/stem cells (MSCs) have emerged as a promising therapeutic approach for a wide spectrum of diseases, including several musculoskeletal disorders and bone fractures. Despite their potential, clinical translation has often led to inconsistent outcomes and the therapeutic efficacy of unmodified MSCs remains suboptimal. To overcome these challenges, strategies to enhance the functional properties of MSCs are needed. MicroRNAs (miRNAs) are master regulators of gene expression and have emerged as powerful tools for engineering MSCs to enhance their pro-regenerative capacity and potentially improve clinical outcomes. High-throughput RNA sequencing was performed to characterize transcriptional changes induced by anti-miR-195 modulation. Anti-miR-195-modulated MSCs were seeded onto collagen/hydroxyapatite scaffolds and their potential was evaluated in vivo using a heterotopic bone formation mouse model. Histological and molecular analyses were performed at 14 and 28 days post-implantation to assess cell migration, osteogenic marker expression, collagen deposition and vascularization. Anti-miR-195-modulated MSCs displayed transcriptional profiles associated with enhanced migration, invasion, and cell cycle progression, together with reduced senescence, cell death and apoptosis. Expression of the bone mineral density-associated transcript IBSP was significantly increased. In vivo, scaffolds seeded with anti-miR-195-treated MSCs showed increased cell migration at day 14 post-implantation. At day 28 post-implantation, enhanced collagen deposition and increased expression of osteogenic markers, including RUNX2 and SPP1, were observed. In addition, the scaffold area covered by blood vessels and the number of larger vessels were significantly higher in anti-miR-195-MSC scaffolds compared with controls. These findings demonstrate that anti-miR-195 promotes cell migration and vascularization in vivo, supporting miRNA-based engineering as a promising strategy to improve the efficacy of MSC-based ATMPs for musculoskeletal diseases and bone defects.