Immunomodulation By Therapeutic Mesenchymal Stromal Cells (MSC) Is Triggered Through Phagocytosis of MSC By Monocytic Cells.

Mesenchymal stem or stromal cells (MSC) are under investigation as a potential immunotherapy. MSC are usually administered via intravenous infusion, after which they are trapped in the lungs and die and disappear within a day. The fate of MSC after their disappearance from the lungs is unknown and it is unclear how MSC realize their immunomodulatory effects in their short lifespan. We examined immunological mechanisms determining the fate of infused MSC and the immunomodulatory response associated with it. Tracking viable and dead human umbilical cord MSC (ucMSC) in mice using Qtracker beads (contained in viable cells) and Hoechst33342 (staining all cells) revealed that viable ucMSC were present in the lungs immediately after infusion. Twenty-four hours later, the majority of ucMSC were dead and found in the lungs and liver where they were contained in monocytic cells of predominantly non-classical Ly6Clow phenotype. Monocytes containing ucMSC were also detected systemically. In vitro experiments confirmed that human CD14++ /CD16- classical monocytes polarized toward a non-classical CD14++ CD16+ CD206+ phenotype after phagocytosis of ucMSC and expressed programmed death ligand-1 and IL-10, while TNF-α was reduced. ucMSC-primed monocytes induced Foxp3+ regulatory T cell formation in mixed lymphocyte reactions. These results demonstrate that infused MSC are rapidly phagocytosed by monocytes, which subsequently migrate from the lungs to other body sites. Phagocytosis of ucMSC induces phenotypical and functional changes in monocytes, which subsequently modulate cells of the adaptive immune system. It can be concluded that monocytes play a crucial role in mediating, distributing, and transferring the immunomodulatory effect of MSC. Stem Cells 2018;36:602-615.

Epigenetic changes in umbilical cord mesenchymal stromal cells upon stimulation and culture expansion.

BACKGROUND: Mesenchymal stromal cells (MSCs) are studied for their immunotherapeutic potential. Prior to therapeutic use, MSCs are culture expanded to obtain the required cell numbers and, to improve their efficacy, MSCs may be primed in vitro. Culture expansion and priming induce phenotypical and functional changes in MSCs and thus standardisation and quality control measurements come in need. We investigated the impact of priming and culturing on MSC DNA methylation and examined the use of epigenetic profiling as a quality control tool. METHODS: Human umbilical cord-derived MSCs (ucMSCs) were cultured for 3 days with interferon (IFN)γ, transforming growth factor (TGF)ß or a multi-factor combination (MC; IFNγ, TGFß and retinoic acid). In addition, ucMSCs were culture expanded for 14 days. Phenotypical changes and T-cell proliferation inhibition capacity were examined. Genome-wide DNA methylation was measured with Infinium MethylationEPIC Beadchip. RESULTS: Upon priming, ucMSCs exhibited a different immunophenotype and ucMSC(IFNγ) and ucMSC(MC) had an increased capacity to inhibit T-cell proliferation. DNA methylation patterns were minimally affected by priming, with only one significantly differentially methylated site (DMS) in IFNγ- and MC-primed ucMSCs associated with autophagy activity. In contrast, 14 days after culture expansion, ucMSCs displayed minor phenotypical and functional changes but showed >4000 significantly DMSs, mostly concerning genes involved in membrane composition, cell adhesion and transmembrane signalling. DISCUSSION: These data show that DNA methylation of MSCs is only marginally affected by priming, whereas culture expansion and subsequent increased cellular interactions have a large impact on methylation. On account of this study, we suggest that DNA methylation analysis is a useful quality control tool for culture expanded therapeutic MSCs.

Aging of bone marrow- and umbilical cord-derived mesenchymal stromal cells during expansion.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) are used as experimental immunotherapy. Extensive culture expansion is necessary to obtain clinically relevant cell numbers, although the impact on MSCs stability and function is unclear. This study investigated the effects of long-term in vitro expansion on the stability and function of MSCs. METHODS: Human bone marrow-derived (bmMSCs) and umbilical cord-derived (ucMSCs) MSCs were in vitro expanded. During expansion, their proliferative capacity was examined. At passages 4, 8 and 12, analyses were performed to investigate the ploidy, metabolic stability, telomere length and immunophenotype. In addition, their potential to suppress lymphocyte proliferation and susceptibility to natural killer cell lysis was examined. RESULTS: BmMSCs and ucMSCs showed decreasing proliferative capacity over time, while their telomere lengths and mitochondrial activity remained stable. Percentage of aneuploidy in cultures was unchanged after expansion. Furthermore, expression of MSC markers and markers associated with stress or aging remained unchanged. Reduced capacity to suppress CD4 and CD8 T-cell proliferation was observed for passage 8 and 12 bmMSCs and ucMSCs. Finally, susceptibility of bmMSCs and ucMSCs to NK-cell lysis remained stable. CONCLUSIONS: We showed that after long-term expansion, phenotype of bmMSCs and ucMSCs remains stable and cells exhibit similar immunogenic properties compared with lower passage cells. However, immunosuppressive properties of MSCs are reduced. These findings reveal the consequences of application of higher passage MSCs in the clinic, which will help increase the yield of therapeutic MSCs but may interfere with their efficacy.

Cytokine treatment optimises the immunotherapeutic effects of umbilical cord-derived MSC for treatment of inflammatory liver disease.

BACKGROUND: Mesenchymal stromal cells (MSC) possess immunomodulatory properties and low immunogenicity, both crucial properties for their development into an effective cellular immunotherapy. They have shown benefit in clinical trials targeting liver diseases; however the efficacy of MSC therapy will benefit from improvement of the immunomodulatory and immunogenic properties of MSC. METHODS: MSC derived from human umbilical cords (ucMSC) were treated for 3 days in vitro with various inflammatory factors, interleukins, vitamins and serum deprivation. Their immunogenicity and immunomodulatory capacity were examined by gene-expression analysis, surface-marker expressions, IDO activity, PGE2 secretion and inhibition of T cell proliferation and IFNγ production. Furthermore, their activation of NK cell cytotoxicity was investigated via CD107a expression on NK cells. The immunomodulatory capacity, biodistribution and survival of pre-treated ucMSC were investigated in a CCl4-induced liver disease mouse model. In addition, capacity of pre-treated MSC to ameliorate liver inflammation was examined in an ex vivo liver inflammation co-culture model. RESULTS: IFN-γ and a multiple cytokine cocktail (MC) consisting of IFN-γ, TGFß and retinoic acid upregulated the expression of immunomodulatory factor PD-L1 and IDO activity. Subsequently, both treatments enhanced the capacity of ucMSC to inhibit CD4 and CD8 T cell proliferation and IFN-γ production. The susceptibility of ucMSC for NK cell lysis was decreased by IFN-ß, TGFß and MC treatment. In vivo, no immunomodulation was observed by the ucMSC. Four hours after intravenous infusion in mice with CCl4-induced inflammatory liver injury, the majority of ucMSC were trapped in the lungs. Rapid clearance of ucMSC(VitB6), ucMSC(Starv + VitB6) and ucMSC(MC) and altered bio-distribution of ucMSC(TGFß) compared to untreated ucMSC was observed. In the ex vivo co-culture system with inflammatory liver slices ucMSC(MC) showed significantly enhanced modulatory capacity compared to untreated ucMSC. CONCLUSIONS: The present study demonstrates the responsiveness of ucMSC to in vitro optimisation treatment. The observed improvements in immunomodulatory capacity as well as immunogenicity after MC treatment may improve the efficacy of ucMSC as immunotherapy targeted towards liver inflammation.