Mesenchymal stem cells (MSCs) have the ability to self-renew and differentiate into multi-lineage cells, thus highlighting the feasibility of using umbilical cord blood-derived MSCs (UCB-MSCs) for cell-therapy and tissueengineering. However, the low numbers of UCB-MSC derived from clinical samples requires that an ex vivo expansion step be implemented. As most stem cells reside in low oxygen tension environments (i.e., hypoxia), we cultured the UCB-MSCs under 3% O 2 or 21% O 2 and the following parameters were examined: proliferation, senescence, differentiation and stem cell specific gene expression. UCB-MSCs cultured under hypoxic conditions expanded to significantly higher levels and showed less senescence compared to UCB-MSCs cultured under normoxic conditions. In regards to differentiation potential, UCB-MSCs cultured under hypoxic and normoxic conditions both underwent similar levels of osteogenesis as determined by ALP and von Kossa assay. Furthermore, UCB-MSCs cultured under hypoxic conditions exhibited higher expression of OCT4, NANOG and SOX2 genes. Moreover, cells expanded under hypoxia maintained a stem cell immnunophenotype as determined by flow cytometry. These results demonstrate that the expansion of human UCB-MSCs under a low oxygen tension microenvironment significantly improved cell proliferation and differentiation. These results demonstrate that hypoxic culture can be rapidly and easily implemented into the clinical-scale expansion process in order to maximize UCB-MSCs yield for application in clinical settings and at the same time reduce culture time while maintaining cell product quality.

Mesenchymal stem cells (MSCs) are one of the most extensively studied stem cell types owing to their capacity for differentiation into multiple lineages as well as their ability to secrete regenerative factors and modulate immune functions. However, issues remain regarding their further application for cell therapy. Here, to demonstrate the superiority of the improvement of MSCs, we divided umbilical cord blood-derived MSCs (UCB-MSCs) from 15 donors into two groups based on efficacy and revealed donor-dependent variations in the anti-inflammatory effect of MSCs on macrophages as well as their immunoregulatory effect on T cells. Through surface marker analyses (242 antibodies), we found that HLA-A2 was positively related to the anti-inflammatory and immunoregulatory function of MSCs. Additionally, HLA-A2 mRNA silencing in MSCs attenuated their therapeutic effects in vitro; namely, the suppression of LPS-stimulated macrophages and phytohemagglutinin-stimulated T cells. Moreover, HLA-A2 silencing in MSCs significantly decreased their therapeutic effects in a rat model of hyperoxic lung damage. The present study provides novel insights into the quality control of donor-derived MSCs for the treatment of inflammatory conditions and diseases.

Hyaluronic acid(HA) is the major glycosaminoglycan component of extracellular matrices. CD44 is a ligand for HA and expresses on a wide variety of cell types. CD44 has been implicated in many cell-cell and cell-matrix interactions. In the present study, we studied the effects of HA on proliferation, differentiation and apoptosis of CD34+ cells during HCB ex vivo expansion using TPO, SCF and FL. CD34+ cells from cord blood MNC were cultured for 4 weeks in the combination of TPO, SCF and FL on HA-coated well and CD34+cells were cultured in identical conditions except HA-coated well. At day 4, 7, 14, 21, 18, we counted the number of expanded cells and analyzed the cell surface markers (CD3, CD14, CD19, CD34, CD44, CD61, CD66b) and apoptosis using 7-AAD staining. To analyze the effects of HA on CD34+ cell differentiation, anti-CD44 antibodies (clone A3D8 and 515) were used. The morphology on the expanded cells was examined using wright’s staining. In results, cell counts were increased in HA-coated well compared with the control group until D7, but proliferation rate was decreased after D7. Apoptosis portion of CD34+ using 7-AAD staining was increased in HA-coated well and % of CD34+ cells was decreased in HA-coated wells compared with that of the control groups. The expanded cells in HA-coated well were negative for CD3 (pan T cell), CD19 (B cell) but CD14 (monocyte), CD61 (megakaryocyte) and CD66b (neutrophil) were highly expressed than those of control groups. Apoptotic portion was also increased in the HA-coated group. CD66b (neutrophil) expression in HA-coated well was significantly higher than those of control groups at D14 and above findings were confirmed by wright’s stain. CD66 expression was reduced by the anti-CD44 antibody clone, A3D8 and however CD66 expression was increased by the clone, 515. Therefore, it might be possible that A3D8 and clone 515 recognize each different epitopes of CD44. In conclusion, CD34+ cell differentiation from HCB into CD66, CD14 and CD61 positive cells was enhanced by the contact of HA during ex vivo expansion using TPO, SCF and FL. This result suggests that the expanded human cord blood graft with HA might be useful as the “post-progenitor” to shorten the severe cytopenia period after myeloablative conditioning for HCB transplantation.

Recently, we have reported on two different types of EPCs from human umbilical cord blood(ASH 2005, Abstract #1706). These EPCs had different biologic properties in angiogenic capabilities during the HCB ex vivo expansion. In this present study, the aim is to examine the synergism by mixing the conditioned medium from the early EPCs into the late EPCs on neovascularization. The mononuclear cells from the HCB were cultures using an EGM-2 medium with VEGF, IGF-1 and FGF for 21 days. We found early spindle-shaped cells(early EPCs), which were grown in the first week of the culture and late cobblestone shaped cells(late EPCs) which peaked in their growth in the third week of the culture. First, we compared the two types of cells in terms of phenotypic expressions and migration ability. Next, we examined their proliferation capacity and tube formations in the Matrigel plate under the conditions that the early outgrowing cells-contained medium was added to the cobblestone shaped cells. The late-appearing cobblestone shaped cells were positive for VEGFR2, VE-cadherin, CD31, CD34 and CXCR-4 but not for CD14 and CD54. These late outgrowing cells expressed high levels of mRNA on the endothelial marker genes and effectively formed capillary tubes in the Matrigel plates. The early spindle cells excreted more angiogenic cytokines and had more migratory ability. When the early spindle-shaped cell-conditioned medium was added to the late cobblestone shaped outgrowing cells, significantly higher proliferation and tube formation measured by the area and length of tubes were found. These results suggested that the two types of cells raised with different biologic properties during the ex vivo HCB expansion and the angiogenic capacity of late EPCs were augmented by a mutual interaction via the excreted cytokines from early EPCs. These finding may have potential applications for a “cell therapy” in situations such as vascular injuries (ie, hindlimb ischemia/myocardial infarction). Murine models are being tested to see whether the injections of two different EPCs will result in synergic noevascularization in our Lab.

In respect to B lymphocyte-mediated immunity, characteristics of human cord blood are low counts of mature B lymphocytes, deficient expression of CD40L and cytokine production in CD4+ T lymphocytes, defect in the isotype switch of immunoglobulin and the activation of B lymphocytes, and low IgG production of B lymphocytes. These characteristics of the B lymphocyte from human cord blood lead to a delayed B lymphocyte-mediated immune reconstitution and an increased susceptibility to infections after a cord blood transplantation. The mechanism of immunological recostitution after cord blood transplantation has been examined from a variety of viewpoints in experimental models as well as clinical studies. However, problems of sustained immunodeficiency after cord blood transplantation remain to be resolved. The aim of the present study is to establish culture conditions that support the effective B lymphocyte expansion of human cord blood using IL-4, IL-10, and CD40L, to which cytokines are defected in B lymphocyte of human cord blood, and established conditions are compared to previously established cytokine combinations, TPO+SCF+FL in our Lab (Br J Haematol 107:176–185, 1999 & Stem Cells 21:228–235, 2003). To elucidate the effective B lymphocyte-mediated immune reconstitution of cord blood after ex vivo expansion, mononuclear cells, separated from density gradient of Ficoll system, and CD34+ purified cells, isolated from immunomicrobead(MiniMACS) system, were cultured with various combinations of cytokines (TPO+FL+SCF and/or IL-4, IL-10 and CD40L) for 2 weeks or 4 weeks. This then allowed for cytometric analysis after immunofluorescence stain with CD34, CD38 (for HSC analysis) and CD19, IgG and IgM (for B lymphocyte-mediated immune reconstitution) and CD4 (for T helper cell) and CD25 (for lymphocyte activation assay) to be performed. In the B lymphocyte expansion aspect, the immunoglobulin expression, and functional activity, expansion with the TPO+FL+SCF+IL-4+IL-10 combination showed best results in the expression of CD19, CD25, IgG, and IgM. However, the addition of CD40L to those culture condition did not increase expression of CD19, CD25, IgG, and IgM after the expansion of human cord blood. Expansion of CD34+ purified cells was superior to MNCs in the expression of CD19, CD25, IgG, and IgM. In consideration for the duration of cultures, the 2 week culture was superior to the 4 week culture with respect to graft stemness (CD34+CD38- fraction). Our data suggests most superior results were observed from the ex vivo expansion of CD34+ purified cells cultured for 2 weeks with TPO+FL+SCF+IL-4+IL-10, in the B lymphocyte-mediated immune reconstitution and graft stemness aspect. The results of this study warrant further investigation on effective B lymphocyte-mediated immune reconstitution after cord blood transplantation in vivo using ex vivo expanded cord blood.