Immortalized human hair follicle-derived mesenchymal-like stromal cells for the long-term production of scalable Immunomodulatory and regenerative secretome

Abstract Background Mesenchymal stromal cells (MSCs) exert therapeutic effects primarily through their secretome, rich in bioactive factors with immunomodulatory and regenerative properties. However, clinical application of MSC-derived secretome is hindered by donor variability, limited expansion, and replicative senescence. To address these issues, we developed immortalized human hair follicle-derived mesenchymal-like stromal cells (iHF-MSCs) as a consistent and scalable source of therapeutic secretome. Methods HF-MSCs were isolated using a minimally invasive procedure and immortalized with lentiviral SV40 large T antigen. From 576 single-cell clones, C18 and C26 were selected based on proliferation capacity, absence of senescence, and retention of a mesenchymal-like phenotype. Characterization included RT-PCR, flow cytometry for surface markers, trilineage differentiation, and CD56 expression analysis. For immunomodulatory evaluation, cells were licensed with IFN-γ and TNF-α, and the resulting conditioned media (licensed CM) were analyzed by ELISA and cytokine arrays. PBMC proliferation and Treg induction were assessed using licensed CM, while unlicensed CM was tested for its regenerative effects on dermal and epidermal cell functions, including wound repair, tube formation assay as an indicator of angiogenic potential, and oxidative stress response. Results The selected clones (C18 and C26) exhibited long-term stability, rapid expansion, and preserved mesenchymal-like phenotype and multipotency. Secretome profiling revealed an enriched composition of immunoregulatory (e.g., Gal-9, IL-1Ra, TSG-6) and pro-regenerative (e.g., VEGF, PDGF-AA, EGF) factors, with enhanced responsiveness to inflammatory licensing. Notably, iHF-MSCs maintained low immunogenicity and demonstrated superior functional performance: inhibition of PBMC proliferation, Treg induction, and promotion of skin cell proliferation, migration, tube formation, and oxidative stress protection. Conclusion These results position iHF-MSCs as a robust and scalable platform for consistent production of therapeutic secretome, paving the way for innovative, cell-free immunomodulatory and regenerative therapies.