Isolation and Characterization of Marrow-Isolated Adult Multilineage Inducible (MIAMI) Cell-Derived Extracellular Vesicles Demonstrate Multifunctional Therapeutic Potential in Tissue Regeneration and Anti-Inflammatory Immunomodulation

Marrow-isolated adult multilineage inducible (MIAMI) cells are a subpopulation of mesenchymal stem/stromal cells (MSC) with enhanced self-renewal, multilineage plasticity, and anti-inflammatory properties, suggesting that their extracellular vesicles (MIA-EVs) may confer advantages over conventional MSC-EVs. MIAMI cells were transcriptionally profiled and expressed regenerative markers, including PDGFRB, CDX2, and TERT. We report the first successful isolation and characterization of MIA-EVs. EVs were isolated by ultracentrifugation and characterized by nanoparticle tracking analysis, transmission electron microscopy, flow cytometry, and surface markers. Cargo analysis identified growth factors (IGFBP-1, HGF, VEGF-D) and 19 highly expressed miRNA targeting survival, regenerative, and immune regulatory pathways. MIA-EVs were efficiently internalized, enhanced keratinocyte wound closure and suppressed osteosarcoma proliferation in vitro. Conditioned MIA-EVs reshaped pathway weighting without altering core regulatory identity, as a conserved 15-miRNA backbone persisted across naïve, irradiated, and cytokine-primed states. In contrast, a 9-miRNA core shared with MSC-EVs defined a basal mesenchymal framework, while MIA-EVs expanded regenerative, survival, and immune network connectivity. Similar to embryonic stem cell (ESC)-EVs, both MIA- and cytokine-primed EVs promoted M2 macrophage polarization, selectively upregulating IL1R2 and PPARG/STAT1, respectively. Meanwhile, MSC-EVs induced heterogeneous responses. These findings establish MIA-EVs as a conditioning-resistant, systems-regulated, cell-free platform with regenerative, immunomodulatory, and cytoprotective potential under hostile microenvironments.