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

Figure 1 Induced pluripotent stem cells culture and identification. A: Morphological observation and cell cloning of induced pluripotent stem cells (iPSCs) at 2 d, 3 d, 5 d and 6 d and Nanog⁺-iPSCs in culture; B: Quantitative polymerase chain reaction identification; C: Alkaline phosphatase staining; D: Chromosome identification; E: Teratoma formation; red arrow = intestinal epithelium (endoderm), green arrow = muscle tissue (mesoderm), and black arrow = nerve tissue (endoderm). AKP: Alkaline phosphatase.

Figure 2 Morphological changes from induced pluripotent stem cells to induced pluripotent stem cells-dendritic cells. A: The optimal concentration of sinomenine (SN) was 100 μM; B: Morphological changes from induced pluripotent stem cells (iPSCs) to iPSCs-dendritic cells (DCs) at 3 d in the first stage; iPSC-derived hematopoietic cells at 6 d in the second stage; and iPSC-derived imDCs at 13 d in the third stage under the influence of SN. After 48 h of lipopolysaccharide stimulation, the morphology of SN-iPSCs-DCs in phase contrast microscopy, scanning electron microscopy and transmission electron microscopy; C: Expression of the cell surface markers CD45 and CD11b from iPSCs to iPSCs–DCs under the influence of SN on day 6 in the second stage. SN: Sinomenine.

Figure 3 Immature dendritic cells surface phenotypes in differentiation culture. CD80, CD86, MHC-II and CD11c expression from induced pluripotent stem cells (iPSCs) to iPSCs-immature dendritic cells in the presence of sinomenine on days 5, 10 and 13 in stage 3 and after lipopolysaccharide stimulation for 48 h. Compared with iPSCs-dendritic cells group at the same time, ^aP < 0.05. SN: Sinomenine; iPSCs: Induced pluripotent stem cells; imDCs: Immature dendritic cells.

Figure 4 Sinomenine-induced pluripotent stem cells–immature dendritic cells suppress T cell proliferation and enhanced regulatory T cell proliferation. A: Purity of CD3⁺ T cells after sorting; B: Mixed lymphocyte reaction at different proportions of immature dendritic cells: CD3⁺ T cells. Compared with induced pluripotent stem cells-dendritic cells (iPSCs-DCs) group, ^aP < 0.05; C: Purity of CD4⁺ T cells after sorting; D: Mixed culture of iPSCs-DCs or sinomenine (SN)-iPSCs-DCs and CD4⁺ T cells at 1:40. Compared with iPSCs-DCs group, ^aP < 0.05; E: Effects of iPSCs-DCs or SN-iPSCs-DCs on regulatory T (Treg) cell proliferation. Compared with iPSCs-DCs group, ^aP < 0.05; F: Cytokine levels in supernatants from cocultured DCs and Treg cells. Compared with iPSCs-DCs group, ^aP < 0.05. SN: Sinomenine; iPSCs: Induced pluripotent stem cells; DCs: Dendritic cells; IL: Interleukin; IFN: Interferon; TGF-: Transforming growth factor-.

Figure 5 Endocytotic capacity in sinomenine-induced pluripotent stem cells-immature dendritic cells and sinomenine-induced pluripotent stem cells-dendritic cells. Antigen uptake in induced pluripotent stem cells (iPSCs)-immature dendritic cells (imDCs) and sinomenine (SN)-iPSCs-imDCs. Compared with iPSCs-imDCs group, ^aP < 0.05; Antigen uptake in iPSCs-DCs and SN-iPSCs-DCs. Compared with iPSCs-DCs group, ^aP < 0.05. SN: Sinomenine; iPSCs: Induced pluripotent stem cells; DCs: Dendritic cells; imDCs: Immature dendritic cells.

Figure 6 Apoptosis of induced pluripotent stem cells-dendritic cells and sinomenine-induced pluripotent stem cells-dendritic cells stimulated with lipopolysaccharide. A: Apoptosis rate in induced pluripotent stem cells-dendritic cells (iPSCs-DCs) and sinomenine (SN)-iPSCs-DCs stimulated with lipopolysaccharide (LPS). Compared with iPSCs-DCs group, ^aP < 0.05; B: Apoptosis-related protein expression in iPSCs-DCs and SN-iPSCs-DCs stimulated with LPS. Compared with iPSCs-DCs group, ^aP < 0.05. SN: Sinomenine; iPSCs: Induced pluripotent stem cells; DCs: Dendritic cells; PARP: Poly(ADP-ribose) polymerase; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase.

Figure 7 Sinomenine-induced pluripotent stem cells–immature dendritic cells immunization prolonged survival of allogeneic skin grafts. A: Body weight in all groups. Compared with PBS in the same time, ^aP < 0.05. Compared with induced pluripotent stem cells-immature dendritic cells group in the same time, ^bP < 0.05; B: Hematoxylin and eosin staining in all groups; C: Graft scores in all groups; D: Survival curves for all groups. SN: Sinomenine; iPSCs: Induced pluripotent stem cells; DCs: Dendritic cells; imDCs: Immature dendritic cells.

Figure 8 Sinomenine-induced pluripotent stem cells-immature dendritic cells immunization suppressed T cells and increased activated regulatory T cells in allografts. A: Sinomenine (SN)-induced pluripotent stem cells (iPSCs)-immature dendritic cells (imDCs) increased the ratio of CD4⁺CD25⁺ regulatory T (Treg) cells/CD4⁺ T cells in spleen. Compared with PBS group, ^aP < 0.05. Compared with iPSCs-imDCs group, ^bP < 0.05. Compared with SN group, ^cP < 0.05; B: SN-iPSCs-imDCs increased the ratio of CD4⁺CD25⁺FoxP3⁺ Treg cells/CD4⁺CD25⁺ Treg cells in spleen. Compared with iPSCs-imDCs group, ^bP < 0.05. Compared with SN group, ^cP < 0.05; C: Reactivity of recipient lymphocytes to donor or unrelated third-party lymphocytes. Compared to third-party lymphocytes, ^aP < 0.05. Compared with iPSCs-imDCs group, ^bP < 0.05; D: SN-iPSCs-imDCs downregulated proinflammatory cytokines and upregulated anti-inflammatory cytokines. Compared with PBS group, ^aP < 0.05. Compared with iPSCs-imDCs group, ^bP < 0.05. SN: Sinomenine; iPSCs: Induced pluripotent stem cells; DCs: Dendritic cells; imDCs: Immature dendritic cells; IL: Interleukin; IFN: Interferon; TGF-: Transforming growth factor-.