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The umbilical cord (UC) has become one of the major sources of mesenchymal stem cells (MSCs). The common explant method of isolating UC-derived MSCs (UC-MSCs) involves mincing the UCs into small fragments, which are then attached to a culture dish bottom from which the MSCs migrate. However, the fragments frequently float up from the bottom of the dish, thereby reducing the cell recovery rate. To overcome this problem, we demonstrate an improved explant method for UC-MSC isolation, which involves the use of a stainless steel mesh (Cellamigo(®); Tsubakimoto Chain Co.), to protect the tissue from floating after the minced fragments are aligned at regular intervals in culture dishes. The culture medium was refreshed every 3 days and the adherent cells and tissue fragments were harvested using trypsin. The number of UC-MSCs isolated from 1 g of UC using the explant method with Cellamigo was 2.9 ± 1.4 × 10(6)/g, which was significantly higher than that obtained without Cellamigo (0.66 ± 0.53 × 10(6)/g) (n = 6, p < 0.01) when cells reached 80-90% confluence. In addition, the processing and incubation time required to reach 80-90% confluence was reduced in the improved explant method compared with the conventional method. The UC-MSCs isolated using the improved method were positive for CD105, CD73, CD90, and HLA class I expression and negative for CD45 and HLA class II expression. The isolated UC-MSCs efficiently inhibited the responder T cells induced by allogeneic dendritic cells in a mixed lymphocyte reaction. Conclusively, we demonstrated that the use of Cellamigo improves the explant method for isolating UC-MSCs.

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Recent studies have reported that mesenchymal stromal cells (MSCs) migrate to areas of inflammation and suppress adverse immune reactions. Bone marrow (BM)-derived MSCs have been successfully used in patients with acute graft versus host disease (GVHD), but the harvesting of BM carries certain risks for the donor. To circumvent these, we obtained MSCs from Wharton's jelly (WJ) derived from umbilical cord and investigated their potential for immunosuppression. In a mixed lymphocyte reaction (MLR), responder T cell proliferation triggered by allogeneic dendritic cells was inhibited efficiently by WJ-MSCs derived from the same donor of responder cells or those from a third party donor. These inhibitory effects were reversed in a dose-dependent manner in the presence of 1-methyl-DL-tryptophan, an inhibitor of the soluble factor indoleamine 2, 3-dioxygenase (IDO). Immunosuppression by WJ-MSCs was also attenuated by blocking cell-cell contact between WJ-MSCs and responder T cells using a Transwell chamber. Moreover, IDO gene expression was induced in both WJ- and BM-MSCs by inflammatory cytokine IFN-γ, but HLA-DR was expressed in BM-MSCs and not in WJ-MSCs upon stimulation by a relatively low concentration of IFN-γ. These results indicate that WJ-MSCs exert their immunosuppressive effects by cell-cell contact with activated T cells and in part through IDO, and suggest the need for cells rather than soluble factors secreted from MSCs to achieve immunosuppressive therapy in severe cases of GVHD.

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