Although the central nervous system is relatively immunotole

Although the central nervous system is relatively immunotolerant (Barker and Billingham, 1977) and human fetus-derived NS/PCs and hESC-derived NS/PCs (hESC-NS/PCs) showed weak immunogenicity (Odeberg et al., 2005; Åkesson et al., 2009; Liu et al., 2013a), no studies to date have investigated the extent to which HLA-matching plays a role in hiPSC-NS/PC transplantation. To enable safer cell transplantation, it is imperative to assess the immunogenicity of hiPSC-NS/PCs and establish a system to predict immune rejection prior to transplantation. Here, we examined the clinical significance of HLA matching in hiPSC-NS/PC transplantation using a modified mixed lymphocyte reaction (MLR) assay, which is extensively employed for assessing immune rejection in vitro (Mittal et al., 2009; Levitsky et al., 2011; Karlsson et al., 2012; Hu et al., 2013; Sakai et al., 2014).
Materials and methods
Results
Discussion These phenomena are explained by the weak leukotriene receptor agonist of surface markers involved in the inflammatory response on hiPSC-NS/PCs. The current findings are consistent with a previous report in which the expression of HLA class II and co-stimulatory molecules on both human fetal and hESC-NS/PCs was low (Liu et al., 2013a). Low immunogenicity and the low antigen-presenting function of hiPSC-NS/PCs may underlie the lack of any significant differences between the autologous and allogeneic conditions or the allogeneic HLA-matched and -mismatched conditions in the NS/PC-PBMC MLR assay. Although a stimulation index >2 indicates a significant positive response (Richman et al., 1976; Ringdén et al., 1976), none of the MLR indexes obtained in the NS/PC-PBMC MLR in either HLA-matched or -mismatched conditions was >2. As hiPSC-NS/PCs suppressed the activation of PBMCs in a dose-dependent manner, hiPSC-NS/PCs clearly exerted an immunomodulatory effect and depressed the immune response, even in allogeneic HLA-mismatched conditions. In previous studies, human fetal NS/PCs and hESC-NS/PCs were also observed to exert immunomodulatory effects (Einstein et al., 2003; Odeberg et al., 2005; Ben-Hur, 2008; Wang et al., 2009; Bonnamain et al., 2012; Liu et al., 2013a). Transforming growth factor-β, nitric oxide, prostaglandin E2 and heme oxygenase-1 secreted or expressed by fetal NS/PCs or ESC-NS/PCs suppress the activation of T cells (Odeberg et al., 2005; Ben-Hur, 2008; Wang et al., 2009; Bonnamain et al., 2012; Liu et al., 2013a). Since conditioned medium derived from hiPSC-NS/PCs did not display an inhibitory effect on the proliferation of mixed PBMCs, hiPSC-NS/PCs likely secrete or express immunomodulatory factors involved in cell-cell interactions with PBMCs. Thus, hiPSC-NS/PCs may possess immunomodulatory characteristics similar to those of fetal NS/PCs and ESC-NS/PCs. While the expression of HLA molecules on hiPSC-NS/PCs has been studied previously (Liu et al., 2013b), the correlation between the expression of immune-related surface antigens on hiPSC-NS/PCs and the immune response remains largely unclear. To address whether the expression of immune-related surface antigens affects the immune response in NS/PC-PBMC MLR, we compared the proliferative response of PBMCs towards allogeneic HLA-mismatched hiPSC-NS/PCs and human fetal NS/PCs. However, the two stimulation indexes were similar and were <2 (Fig. S5). These data suggested that the differences in surface antigen expression observed via flow cytometry were not sufficient to explain the differences of the immune reactions in NS/PC-PBMC MLR. The immunomodulatory effect of human fetal NS/PCs (Åkesson et al., 2009; Liu et al., 2013a) may overcome the potential immune reaction mediated by the differential expression of surface antigens between hiPSC-NS/PCs and human fetal NS/PCs. In addition, the low expression of co-stimulatory molecules on human fetal NS/PCs may further explain the weak immune reaction of PBMCs co-cultured with human fetal NS/PCs. Although the expression of co-stimulatory molecules on human fetal NS/PCs was higher than that on hiPSC-NS/PCs, the average percentage of co-stimulatory molecule-positive cells was <15% even in human fetal NS/PCs treated with TNFα and IFNγ. These data indicated that human fetal NS/PCs also exhibit low immune antigen-presenting functions.