Comparison of in-vitro immunomodulatory capacity between large and small apoptotic bodies from human bone marrow mesenchymal stromal cells

Abstract

Background: Mesenchymal stromal cell (MSC) apoptosis is essential for their therapeutic effects, including immunomodulation. Previous studies have shown that MSC-derived apoptotic bodies (ApoBDs) also possess immunomodulatory properties. However, compared to small extracellular vesicles, the preparation, characterization, and biological properties of ApoBDs remain underexplored.

Results: ApoBDs were isolated from the conditioned medium of staurosporine-induced apoptotic human MSCs and categorized into large (∼700 nm) and small (∼500 nm) groups. Both types expressed CD90, CD44, and CD73, with low levels of PD-L1, CD11b, and HLA-DR, mirroring their parental MSCs. Functional assays revealed that both ApoBDs inhibited allogeneic T-cell proliferation, with large ApoBDs demonstrating superior efficacy. In macrophage co-culture experiments, both ApoBDs polarized M1 macrophages toward an M2-like phenotype, with large ApoBDs more effectively upregulating CD163 expression. Additionally, both ApoBDs suppressed the proliferation of murine primary T cells. Furthermore, large ApoBDs exhibited enhanced macrophage uptake, as confirmed by flow cytometry and immunocytochemistry. Importantly, no cytotoxicity was observed for either ApoBD type following staurosporine treatment.

Conclusions: Staurosporine-induced ApoBDs are non-cytotoxic and exhibit significant immunomodulatory potential in vitro. Large ApoBDs are more effective than small ApoBDs in T-cell suppression and M2 macrophage polarization, suggesting their potential as an alternative to MSC-based therapies in future studies.

Keywords: Apoptotic bodies (ApoBDs); Immunomodulation; Large extracellular vesicles; Macrophage polarization; Mesenchymal stromal cells (MSCs); Staurosporine; T-cell proliferation.