iPSCs were previously reported to induce
iPSCs were previously reported to induce T cell-dependent immune response by direct transplantation of undifferentiated purchase Madecassoside into syngeneic mice. However, a more detailed investigation proved that autologous transplantation of terminally differentiated cells derived from iPSCs or embryonic stem cells elicits only negligible immunogenicity (Araki et al., 2013; Okano et al., 2013). In our study, we did not observe excess inflammatory responses around transplanted cells under treatment of low-dose immunosuppression, suggesting that even if transplanted cells were not autologous, we could control the immune responses of the recipients by immunosuppressant treatment. The increase in the levels of neurotrophic factors had been commonly observed in transplantation therapy of ALS models (Nizzardo et al., 2014; Teng et al., 2012). We observed that the transplanted hiPSC-GRNPs produced VEGF, and expressions of endogenous VEGF and other neurotrophic factors in the host mice were upregulated. A previous study showed that VEGF retrograde delivery with lentiviral vector could prolong the survival of ALS model mice by 30% (Azzouz et al., 2004) and that activated AKT signaling, which is downstream of VEGF, is important for cell survival in ALS (Lunn et al., 2009). Similarly in this study, transplanted hiPSC-GRNPs increased the VEGF level and prolonged the survival of mSOD1 mice. We could observe positive immunostaining for VEGF and phosphorylated AKT in both remaining motor neurons and astrocytes. However, we could not observe any morphological difference in motor neurons between control and transplanted groups at end-stage. We also speculated that, as shown in previous studies (Howland et al., 2002), transplanted hiPSC-GRNPs differentiated into astrocytes expressing glutamate transporter 1 (GLT1) might restore glutamate homeostasis in our study. In our study, the males in both the control and hiPSC-GRNPs transplantation groups died long before the females, and this result is consistent with the previous reports of ALS model mice (Cervetto et al., 2013; Choi et al., 2008). However, improvement in male mice was greater than in females (Figure S1) Interestingly, a similar gender-dependent difference of therapeutic efficacy was reported in ALS model mice (Cervetto et al., 2013; Li et al., 2012). The epidemiological studies of sporadic ALS have shown that both incidence and prevalence of ALS are greater in men than in women and onset of the disease is also earlier for men than it is for women (McCombe and Henderson, 2010). Sex steroids are suggested to be involved in the gender difference in ALS, but the direct importance of estrogen is still controversial (Choi et al., 2008; Li et al., 2012). Moreover, male neural cells are reported to be more vulnerable to oxidative stress, induced by mutant SOD1 overexpression, than female neural cells (Li et al., 2012). In our study, transplanted cells were mainly differentiated into GFAP-positive astrocytes and upregulated VEGF. Furthermore, astrocytes can play neuroprotective roles from oxidative stress via VEGF (Chu et al., 2010). These findings suggest that hiPSC-GRNPs transplantation may ameliorate male-specific vulnerability to oxidative stress and improve the survival lifespan of male mice. Further analysis would be necessary to elucidate VEGF-associated mechanisms in transplantation therapy. In regard to safety, the potential tumorigenicity of grafts is a predominant concern. We used the human iPSC line “201B7,” which was previously reported to be safe from the viewpoint of tumorigenesis (Kobayashi et al., 2012). Furthermore, we found no signs of tumor formation or Ki67-positive grafts (Figure S2). However, a very small proportion of grafts remained positive for the neural progenitor marker NESTIN at 3 months posttransplantation. We cannot exclude the risk of tumor formation from the remaining NESTIN-positive NPCs. It is important to evaluate tumorigenicity by longer-term observations for future clinical trials.