Sinomenine promotes differentiation of induced pluripotent stem cells into immature dendritic cells with high induction of immune tolerance

BACKGROUND Immature dendritic cells(imDCs)play an important role in the induction of donor-specific transplant immunotolerance.However,these cells have limitations,such as rapid maturation and a short lifespan in vivo.In previous studies,induced pluripotent stem cells(iPSCs)differentiated into imDCs,and sinomenine(SN)was used to inhibit the maturation of imDCs.AIM To study the capacity of SN to maintain iPSC-derived imDCs(SN-iPSCs-imDCs)in an immature state and the mechanism by which SN-iPSCs-imDCs induce immunotolerance.METHODS In this study,mouse iPSCs were induced to differentiate into imDCs in culture medium without or with SN(iPSCs-imDCs and SN-iPSCs-imDCs).The imDCrelated surface markers,endocytotic capacity of fluorescein isothiocyanate Dextran and apoptosis were analyzed by flow cytometry.The effects of iPSCs-imDCs and SNiPSCs-imDCs on T-cell stimulatory function,and regulatory T(Treg)cell proliferative function in vitro were analyzed by mixed lymphocyte reaction.Cytokine expression was detected by ELISA.The apoptosis-related proteins of iPSCs-DCs and SN-iPSCs-DCs were analyzed by western blotting.The induced immunotolerance of SN-iPSCs-DCs was evaluated by treating recipient Balb/c skin graft mice.Statistical evaluation of graft survival was performed using Kaplan–Meier curves.RESULTS Both iPSCs-imDCs and SN-iPSCs-imDCs were successfully obtained,and their biological characteristics and ability to induce immunotolerance were compared.SN-iPSCs-imDCs exhibited higher CD11c levels and lower CD80 and CD86 levels compared with iPSCs-imDCs.Reduced major histocompatibility complex II expression,worse T-cell stimulatory function,higher Treg cell proliferative function and stronger endocytotic capacity were observed with SN-iPSCs-imDCs(P<0.05).The levels of interleukin(IL)-2,IL-12,interferon-γin SN-iPSCs-imDCs were lower than those in iPSCs-imDCs,whereas IL-10 and transforming growth factor-βlevels were higher(P<0.05).The apoptosis rate of these cells was significantly higher(P<0.05),and the expression levels of cleaved caspase3,Bax and cleaved poly(ADP-ribose)polymerase were higher after treatment with lipopolysaccharides,but Bcl-2 was reduced.In Balb/c mice recipients immunized with iPSCsimDCs or SN-iPSCs-imDCs 7 d before skin grafting,the SN-iPSCs-imDCs group showed lower ability to inhibit donor-specific CD4+T-cell proliferation(P<0.05)and a higher capacity to induce CD4+CD25+FoxP3+Treg cell proliferation in the spleen(P<0.05).The survival span of C57bl/6 skin grafts was significantly prolonged in immunized Balb/c recipients with a donor-specific pattern.CONCLUSION This study demonstrated that SN-iPSCs-imDCs have potential applications in vitro and in vivo for induction of immunotolerance following organ transplantation.