Intravenous injection of umbilical cord-derived mesenchymal stromal cells attenuates reactive gliosis and hypomyelination in a neonatal intraventricular hemorrhage model.

Intraventricular hemorrhage (IVH) is a frequent complication of preterm newborns, resulting in cerebral palsy and cognitive handicap as well as hypoxic ischemic encephalopathy and periventricular leukomalacia. In this study, we investigated the restorative effect on neonatal IVH by umbilical cord-derived mesenchymal stromal cells (UC-MSCs) cultured in serum-free medium (RM medium) for clinical application. UC-MSCs were cultured with αMEM medium supplemented with FBS or RM. A neonatal IVH mouse model at postnatal day 5 was generated by intraventricular injection of autologous blood, and mice were intravenously administered 1×105 UC-MSCs two days after IVH. Brain magnetic resonance imaging was performed at postnatal day 15, 22 and neurological behavioral measurements were performed at postnatal day 23, accompanied by histopathological analysis and cytokine bead assays in serum after IVH with or without UC-MSCs. Both UC-MSCs cultured with αMEM and RM met the criteria of MSCs and improved behavioral outcome of IVH mice. Moreover the RM group exhibited significant behavioral improvement compared to the control group. Histopathological analysis revealed UC-MSCs cultured with RM significantly attenuated periventricular reactive gliosis, hypomyelination, and periventricular cell death observed after IVH. Furthermore, human brain-derived neurotrophic factor and hepatocyte growth factor were elevated in the serum, cerebrospinal fluid and brain tissue of neonatal IVH model mice 24h after UC-MSCs administration. These results suggest UC-MSCs attenuate neonatal IVH by protecting gliosis and apoptosis of the injured brain, and intravenous injection of UC-MSCs cultured in RM may be feasible for neonatal IVH in clinic.

Neurosphere formation enhances the neurogenic differentiation potential and migratory ability of umbilical cord-mesenchymal stromal cells.

BACKGROUND AIMS: The human umbilical cord (UC) is a rich source of mesenchymal stromal cells (MSCs), which have been reported to have multi-lineage potential. The objectives of this study were to investigate the characteristics and capacity of UC-MSC neurosphere formation and whether this event enhances the propensity of UC-MSCs to undergo neural differentiation. METHODS: UC-MSCs were collected by the improved explant method. UC-MSCs and neurosphere-forming UC-MSCs (UC-MSC-neurospheres) were induced to undergo neurogenic differentiation, the latter of which were induced by suspension culturing in the presence of epidermal growth factor and basic fibroblast growth factor. The differentiation and migratory capacities of the individual cultures were then compared on the basis of the expression of neural markers, as measured by immunocytochemistry, immunoblotting and quantitative real-time polymerase chain reaction and transwell assays, respectively. RESULTS: Both UC-MSCs and UC-MSC-neurospheres were capable of differentiating into neurogenic cells when cultured in neurogenic differentiation medium. However, pre-conditioned UC-MSC-neurospheres exhibited significantly higher expression of neural markers--including microtubule-associated protein (MAP2), MUSASHI1, glial fibrillary acidic protein (GFAP), and NESTIN--compared with those derived from UC-MSCs directly. Moreover, UC-MSC-neurospheres expressed significantly higher levels of the stemness markers NANOG, KLF4 and OCT4 than did UC-MSCs. Migration assays also revealed that both UC-MSCs and UC-MSC-neurospheres actively migrate toward glucose-depleted cells. CONCLUSIONS: Neurogenic differentiation potential probably is greater in UC-MSC-neurospheres than in UC-MSCs. Thus, UC-MSC-neurospheres may serve as a better source of cells for neurogenic regenerative medicine.

Immunosuppressive properties of Wharton's jelly-derived mesenchymal stromal cells in vitro.

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.

Serum- and xeno-free cryopreservation of human umbilical cord tissue as mesenchymal stromal cell source.

BACKGROUND AIMS: Human umbilical cord (UC) has become a notable source for mesenchymal stromal cells (MSCs) that can migrate to areas of inflammation and damaged tissue and can suppress excess immune reactions and to repair, respectively. Although UC is a solid tissue, there are several advantages, including repeatable uses from the same donor sample when needed and the possibility of future explorations for cells with unknown potential, if we could cryopreserve the UC as a living tissue material. However, because the cryoprotectants in the previous reports included animal- or allogeneic human-derived serum or no serum, the frozen-thawed UC-MSCs were inferior to fresh UC-MSCs in cell proliferation. The objective of this study was to find a suitable cryopreservation method of UC for clinical use. METHODS: The UC was cut in cross-section and incised longitudinally, immersed in the cryoprotectant and frozen slowly. Later, it was thawed and minced rapidly, and the fragments of UC were cultured by improved explant method. RESULTS: The highest yield of cells was obtained from frozen-thawed UC with serum- and xeno-free cryoprotectant, STEM-CELLBANKER, when compared with others. The cells derived from frozen-thawed UC stored in STEM-CELLBANKER expressed the phenotypes of MSCs, retained the immunosuppressive properties in allogeneic mixed lymphocyte reactions and the differentiation potentials (into adipocyte and chondrocytes) comparable to those derived from fresh UC. CONCLUSIONS: UC can be cryopreserved in serum- and xeno-free cryoprotectant as a living tissue while keeping its growth and functions equivalent to fresh UC. Our method is simple and feasible for clinical use.

Improved explant method to isolate umbilical cord-derived mesenchymal stem cells and their immunosuppressive properties.

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.