CP DCL cells protect hESC-derived teratomas from allogeneic immune rejection. (a) Teratomas weight and representative image of teratomas formed by HUES3 hESCs in NSG mice, in Hu-mice injected with DCL cells differentiated from CP HUES3 ESCs (HUES3 CP DCL cells), and in Hu-mice that didn`t receive any DCL cells. The DCL cells were delivered via the tail vein 7 days prior to the subcutaneous transplantation of hESCs. Teratomas were collected 6 to 8 weeks after transplantation. No DCL group n = 6, CP DCL group n = 8, NSG group n = 6. Data are presented as mean values ± SEM. Data from different groups were compared using Kruskal-Wallis test with Dunn`s multiple comparison test, **p<0.01. (b) Representative images of CD4+T cell infiltration into the HUES3 teratomas formed in Hu-mice with or without prior HUES3 CP DCL cell transplantation. The presence of CD4+T cells was revealed by immunofluorescent staining. Scale bar 200 μm. (c) CD3+ T cell infiltration into the HUES3 teratomas formed in Hu-mice with or without prior HUES3 CP DCL cell transplantation was evaluated by flow cytometry. The plots show representative data obtained from 5 different batches of Hu-mice (5 different donors of human fetal liver and thymus). No DCL group n = 6, CP DCL group n = 8. Data are presented as mean values ± SEM. The two groups were compared using non-parametric Mann-Whitney test. *** p<0.001. (d) The infiltration of human monocyte and B cells into the HUES3 teratomas formed in Hu-mice with or without prior HUES3 CP DCL cell transplantation. No DCL group n = 6, CP DCL group n = 8. Data are presented as mean values ± SEM. The two groups were compared using non-parametric Mann-Whitney test. ** P<0.01. (e) Relative expression of CD3, FOXP3, TGFB and IL-10 genes in HUES3 teratomas formed in Hu-mice with or without prior HUES3 CP DCL cell transplantation. The levels of CD3 mRNA were normalized with those of GAPDH mRNA, FOXP3, TGFB and IL-10 mRNA levels were normalized with those of CD3 mRNA. The fold change difference in gene expression was tested using Wilcoxon signed rank test, n = 7 for each group, * P<0.05.