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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.

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.