Immunosuppressive Diversity of Umbilical Cord-Derived Mesenchymal Stromal Cells
Authors: Nagamura-Inoue Tokiko , Takahashi Atsuko , Yamamoto Yuki , Hori Akiko , Miharu Yuta , Ogami Kazuo , Narita Miwako , Nagamura Fumitaka , Tojo Arinobu 
Published: November 2019

Recently, umbilical cord (UC) has become attracted source of mesenchymal stromal cells (MSCs) for immunotherapy such as treatment of severe acute graft versus host disease, because of abundant sources and ease of collection without invasive process. In previous reports on bone marrow-derived MSCs, there is diversity of immunosuppressive mechanisms of MSCs, which have not yet fully clarified in UC-MSCs. Here we studied the immunosuppressive properties of UC-MSCs on the activated immune system in vitro.
Methods; UC was collected after obtaining informed consent from the mother. UC-MSCs were obtained from frozen-thawed UC tissue with explant method and expanded in culture medium. Mixed lymphocyte reaction (MLR) consisted with mononuclear cells (MNCs) stained with CFSE and allogeneic dendritic cell (DC) line (PMDC05) co-cultured with or without UC-MSCs was carried out, and the proliferation of CD4 and CD8-positive lymphocytes were analyzed by flowcytometry followed by FlowJo software. Migratory activities of UC-MSCs toward activated lymphocytes in MLR were evaluated by counting the migrating cells stained with DAPI through the insert filter under the microscope. The percentage of CD4+CD25+FOXP3+ regulatory T cells (Treg) before and after co-culture with UC-MSCs was analyzed by flowcytometry. Monocyte phenotypes co-cultured with UC-MSCs directly or separately by insert filter were analyzed with anti-CD14, CD206, and CD168 antibodies.
Results: The UC-MSCs were positive for CD105, CD73, CD90, and negative for CD45 and HLA-DR. HLA-DR, CD80, CD86, and CD40 were negative even in the high concentration of IFN-γ, while BM-MSCs became positive for HLA-DR. PD-L2 was constitutively expressed in UC-MSC, while PD-L1 was induced upon the addition of IFN-γ. In MLR, responder T cell proliferation triggered by allogeneic DC was inhibited efficiently by 3rd party derived UC-MSCs. Mean+/- SD distribution index of proliferation in CD4- and CD8- positive cells co-cultured with UC-MSCs showed 7.6+/-4.4% and 8.8+/-3.9% compared to those without UC-MSCs (n=4, as different donor derived MSCs). This inhibition was attenuated by the addition of 1-Methyl-dl-tryptophan, Indoleamine 2, 3-dioxygenase (IDO-1) inhibitor, in a dose-dependent manner. The supernatant of MLR showed the increase of IFN-γ and TNF-α, which were also suppressed in MLR co-cultured with UC-MSCs. UC-MSCs migrated toward activated lymphocytes in MLR compared with those of non-activated lymphocytes significantly, which was inhibited by the addition of AG1296, known as platelet-derived growth factor inhibitor; and PPP as insulin-like growth factor (P&lt;0.05). Cytokine beads array indicated the increase of RANTES, IL-8, and MIP-1. Regulatory T cells in CD4-positive T cells were significantly increased in the MNCs co-cultured with UC-MSCs. Interestingly, CD206-positive M2 monocytes were significantly increased in the MNCs cu-cultured with UC-MSC. Mean +/- SD percentage of CD206+CD80- M2 cells indicated 28.9+/-15.3% in MNCs and 83.3+/-11.2% in MNCs cu-cultured with UC-MSC (n=4, P&lt;0.05). On the other hand, CD206-CD80+ M1 monocytes significantly increased in addition of LPS, which were inhibited by the co-culture with UC-MSCs (P&lt;0.05). Furthermore IFN-γ and TNF-α secreted in the MNCs with LPS were suppressed by the co-culture of UC-MSCs.
Discussions and Conclusion: These results demonstrated that UC-MSCs have the diverse immunosuppressive potency through migration toward the activated lymphocytes, suppressing activated T cells proliferation, regulatory T cells induction, and transition of monocyte phenotype. And UC-MSCs are the feasible and abundant source for immunotherapy.

Disclosures
Nagamura-Inoue: AMED: Research Funding. Nagamura:AMED: Research Funding. Tojo:AMED: Research Funding; Torii Pharmaceutical: Research Funding.