Complement-Driven MicroRNA Modulation in Mesenchymal Stem Cells Insights into Immune Crosstalk and Niche Localization.

Abstract

Multipotent stem cells (MSCs) serve as key modulators within the bone marrow microenvironment, orchestrating hematopoietic support and immune modulation through direct cellular interactions and secretion of bioactive factors. This investigation specifically evaluated how complement system components C3a and C5a influence the expression profile of immunoregulatory microRNAs (miRNAs) in Mel-1-derived MSCs, a well-characterized stem cell model. Quantitative real-time PCR analysis revealed distinct modulation patterns across the three targeted miRNAs (miR-628-3p, miR-20a-5p, miR-21-5p) under complement stimulation. C3a consistently upregulated all three miRNAs, while C5a exposure suppressed miR-628-3p and miR-21-5p but induced no change in miR-20a-5p, with miR-628-3p and miR-21-5p demonstrating particularly robust responses. These coordinated expression changes suggest complement fragments act as previously unrecognized modulators of MSC immunomodulatory capacity through post transcriptional control mechanisms. Complementary immunofluorescence studies of unstimulated murine bone marrow, via characterization of endothelial markers expression patterns to provide anatomical context for MSC niche localization. CD31 exhibited extensive vascular network staining reflecting its role in maintaining vascular integrity, while E-selectin showed restricted distribution domains consistent with its compartmentalized expression within the marrow vasculature. The distinct spatial distribution of these markers suggests compartmentalized regulation relevant to leukocyte trafficking within the marrow microenvironment. Our integrated findings demonstrate complement-dependent MSC plasticity, with C3a promoting tissue repair functions (including enhanced angiogenic potential), while C5a preferentially induced immunomodulatory responses. Furthermore, these differential effects highlight how complement activation products can selectively program MSC functional states. The identified miRNA signatures (miR-628-3p, miR-20a-5p, and miR-21-5p) provide mechanistic insights into MSC. immunoregulation and represent potential biomarkers for monitoring therapeutic potential. These findings would advance regenerative medicine by elucidating how complement signaling influences MSC behavior. The demonstration of complement-mediated miRNA regulation offers new opportunities for developing targeted preconditioning strategies to enhance MSC therapeutic efficacy. Additionally, our endothelial marker analysis provides anatomical insight into MSC localization within the bone marrow niche, laying the groundwork for future investigations into MSC-microenvironment interactions.