The CD4+ T cells were incubated with magnetic beads conjugated with an anti-CD25 monoclonal GSI-IX antibody to separate CD4+ CD25+ and CD4+ CD25− T-cell subpopulations. The purity of the resulting T-cell subpopulations was higher than 95% by flow cytometry. To determine the suppressive capacity of hASC-induced Treg cells, proliferation assays were performed in triplicate by culturing CD4+ CD25− cells (responder, 5 × 104 from splenocytes of EAHL mice), CD4+ CD25+ T cells (suppressor, 5 × 104 from splenocytes of β-tubulin-immunized mice treated with either hASCs or PBS) in 96-well plates with irradiated antigen-presenting cells (5 × 104 from splenocytes
of normal BALB/c mice) for 72 hr at 37° in complete medium. Cultures were stimulated by β-tubulin (10 μg/ml), and some co-cultures were treated with anti-IL-10 antibody (10 μg/ml). After 72 hr, the proliferation of autoreactive T cells was assayed by measuring bromodeoxyuridine-substituted DNA incorporation. Data were analysed using analysis of variance or Student’s t-test to compare differences between the treatment Neratinib manufacturer groups. In the present study, we investigated the potential therapeutic effect of hASCs in an experimental model of murine autoimmune hearing loss. Mice were examined weekly for ABRs for hearing capacity. After three injections (Fig. 1a), the hASC administration
group showed that the ABR threshold to click stimulus and wide range of specific frequencies, in comparison with the PBS control group, significantly decreased. After six injections of hASCs (Fig. 1b), ABR click
and pure tone thresholds of the hASC administration group showed improved hearing level at all frequencies tested from 8 to 32 kHz. The ABRs detected threshold levels similar to those in naive mice that received no treatment (Fig. 1b), and the hASC administration completely restored hearing in deaf mice, whereas the PBS control group developed EAHL. Therefore, electrophysiology tests demonstrated recovery of hearing to click stimulus and a wide range of specific frequencies after six injections of hASCs. We investigated the possible immune-modulating effect of hASCs on T-cell priming and differentiation in vivo by examining the recall old response to β-tubulin in isolated splenocytes from hASC-treated or PBS-treated mice with EAHL in vitro. To determine the ability of hASC treatment to suppress the ongoing inflammatory process, mice with EAHL were treated with PBS or hASCs once a week for 6 consecutive weeks after β-tubulin immunization, and splenocytes that were isolated 10 days after the last treatment with the hASCs were assessed for proliferative responses to β-tubulin. T cells from hASC-treated mice exhibited a significantly decreased stimulation index compared with that in cells from PBS-treated mice (Fig. 2a). Moreover, T cells from hASC-treated non-immunized mice did not develop a xenogenic response to the hASCs in those non-immunized animals (data not shown).