Allogeneic mesenchymal stromal cells (MSCs) have been used clinically for decades, without cross‐matching, on the assumption that they are immune‐privileged. In the equine model, we demonstrate innate and adaptive immune responses after repeated intra‐articular injection with major histocompatibility complex (MHC) mismatched allogeneic MSCs, but not MHC matched allogeneic or autologous MSCs. We document increased peri‐articular edema and synovial effusion, increased synovial cytokine and chemokine concentrations, and development of donor‐specific antibodies in mismatched recipients compared with recipients receiving matched allogeneic or autologous MSCs. Importantly, in matched allogeneic and autologous recipients, but not mismatched allogeneic recipients, there was increased stromal derived factor‐1 along with increased MSC concentrations in synovial fluid. Until immune recognition of MSCs can be avoided, repeated clinical use of MSCs should be limited to autologous or cross‐matched allogeneic MSCs. When non–cross‐matched allogeneic MSCs are used in single MSC dose applications, presensitization against donor MHC should be assessed.

BackgroundWe recently demonstrated that intracellular xenogen-contaminated autologous MSCs (FBS) and non-xenogen-contaminated allogeneic (ALLO) MSCs caused an adverse clinical response after repeated intra-articular injection in horses, whereas autologous (AUTO) MSCs did not. Our current objective was to use clinical data from the previous study to compare MSC stemness against adverse response indicated by synovial total nucleated cell count (TNCC) following intra-articular MSC injection.MethodsStemness, quantified by a trilineage differentiation (TLD) score; immunomodulation, quantified by mixed lymphocyte reactions (MLRs); and degree of MHCI expression, quantified by mean fluorescent intensity (MFI); were correlated to the synovial TNCC 24 h after naïve and primed injection.ResultsThere was a trend of a negative correlation (p = 0.21, r = − 0.44) between TLD score and TNCC after primed injection in the ALLO group. Within the ALLO group only, there was a significant positive correlation (p = 0.05, r = 0.77) between MHCI MFI and TNCC after naïve injection and a trend (p = 0.16, r = 0.49) of a positive association of MHCI MFI to TNCC after primed injection. Within the FBS group only, there was a positive correlation (p = 0.04, r = 1) between TNCC and lymphocyte proliferation after both injections.ConclusionsThe trend of a negative correlation of TLD score and TNCC in the ALLO, but not the FBS group, together with the association of MHCI expression and TNCC in the ALLO group, indicates that improved stemness is associated with reduced MSC immunogenicity. When inflammation was incited by xenogen, there was a strong correlation of lymphocyte activation in vitro to adverse response in vivo, confirming that MLRs in vitro reflect MSC immunomodulatory activity in vivo. The relationship of stemness in vitro, suppression of lymphocyte activation in vitro, MHCI expression in vitro, and clinical response in vivo should be further investigated.

Fetal bovine serum (FBS) is used for MSC preparation in pre-clinical animal models and veterinary applications, recently in US clinical trials, and for MSC products with current foreign market authorizations. The effect of anti-bovine titers, which are common in animals and humans, has not been investigated. In the equine model, where anti-bovine titers are universally high due to routine vaccination, we evaluated the recipient immune response to autologous MSCs prepared with and without FBS. Preparation of MSCs with FBS resulted in post injection inflammation and antibody mediated cytotoxicity of MSCs when compared to MSCs prepared without FBS. Importantly, synovial MSC concentrations were reduced and LPS induced pain was higher, when FBS was used to prepare MSCs, demonstrating reduced efficacy of FBS prepared MSCs. Fetal bovine serum should no longer be utilized for MSC preparation in pre-clinical study, clinical study, or veterinary applications. The use of FBS in previously reported studies, and in MSC therapeutics with current foreign market authorization, should be considered when interpreting results.

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