P. Robb, K. | bioRxiv Science

bioRxiv2026

Combined computational and experimental analysis confirm donor-dependent optimization of critical processing parameters for improving mesenchymal stromal cell potency and expansion attributes

Mesenchymal Stromal Cells (MSC) face several heterogeneity challenges hindering clinical and commercial success. Employing a multiple response model, interplay between donor heterogeneity, and critical processing parameters (CPPs), effects on MSC potency and cell expansion attributes were investigated through computed composite attribute scores. Twelve unique CPP combinations were tested in thirteen marrow-derived MSC(M) and five adipose-tissue MSC(AT) training and test datasets, respectively. Donor heterogeneity and select CPP conditions affected a curated gene panel (surrogate for MSC potency); while MSC expansion was primarily influenced by CPPs. Model performances were evaluated against clinical effectiveness data from a previously deployed clinical trial; top-performing model predicted donor rankings coincided with clinical effectiveness data, validating the modeling approach used. Our model predicted that only 8% of tested donors were agnostic to CPPs; a majority (62%) of donors showed CPP-dependent optimal composite quality attributes, with MSC seeding density as a key driver; medium supplementation and oxygen preferences were highly donor dependent. Approximately 30% of donors performed poorly at all conditions tested and may be prospectively identified using a subset of genes (TGFB, VEGF, PDCD1LG1, PDCD1LG2, IDO). Model predicted optimal parameters worked for 69% of tested donors, while sub-optimal parameters worked for only 23% of donors and were confirmed in an independent CD14+ macrophage assay. Our integrated computational and experimental framework predictably identified interactive effects of donor heterogeneity and CPP conditions to optimize MSC potency attributes.

bioengineering↗

bioRxiv2025

Mesenchymal Stromal Cells Immunosuppress Osteoarthritis Synovial Fluid Tolerized Monocytes via IL-6

BackgroundMesenchymal stromal cell (MSC) interactions with monocytes/macrophages are central to their therapeutic effects in knee osteoarthritis (KOA); however, mechanisms of these interactions are not fully understood. HypothesisWe hypothesize that MSC soluble factors, particularly interleukin-6 (IL-6) and C-C motif chemokine ligand (CCL2) modulate monocytes in KOA environment. MethodsUsing healthy donor CD14+ monocytes exposed to KOA synovial fluid (SF) in the presence or absence of marrow-derived MSC(M) directly or conditioned medium (CM), we evaluated cell surface markers and signaling via signal transducer and activator of transcription (STAT3), nuclear factor kappa-light-chain-enhancer of activated B (NF-{kappa}B) and c-Jun Terminal Kinase (JNK); functional responses were measured by secretion of tumor necrosis factor (TNF) and IL-1, and by phagocytosis of pHrodo Red E-coli. ResultsCD14+ monocytes demonstrated a mixed phenotype in KOA SF with increased CD163, CD206 and unchanged HLA-DR, CD86 marker expression. This was accompanied by activated STAT3, JNK and NF-{kappa}B signaling. TNF and IL-1 secreted levels were unchanged, but phagocytosis was impaired, indicative of a net dysfunctional repair phenotype and functionality. CD14+ monocytes in KOA SF were hyporesponsive to additional lipopolysaccharide re-challenge, based on TNF and IL-1 secretion. Addition of MSC(M) to KOA SF programmed CD14+ monocytes resolved the dysfunctional phenotype and functionality, with significant increases in CD163, CD206; significant reductions in HLA-DR and CD86 expression; this was accompanied by significantly increased activated STAT3, and decreased activated JNK and NF-{kappa}B. TNF and IL-1 secretion were also significantly reduced, and phagocytic capacity restored. Blocking IL-6, or to a lesser extent, CCL2, partially abrogated MSC(M) soluble factor effects. MSC(M) experienced apoptosis in KOA SF; however, apoptotic bodies did not fully recapitulate MSC(M) soluble factor effects. ConclusionIL-6, CCL2, other soluble factors and apoptotic bodies from MSC(M) secretome mitigate the dysfunctional effects of KOA SF on CD14+ monocytes resulting in immunosuppressed phenotype and functionality.

immunology↗