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. 2016 Feb 9;6(2):213–227. doi: 10.1016/j.stemcr.2016.01.005

Figure 1.

Figure 1

Re-differentiation of iNKT Cells from iNKT-iPSCs

(A) Surface antigen profiles of re-differentiating T cells from CD4+ iNKT-iPSC (clone 2) on OP9/DL1 feeder cells on day 21 in the presence of the indicated cytokines. The results shown are representative of four independent experiments examining the surface protein expression patterns of the cells gated by CD45, CD3, and 6B11.

(B) Schematic illustration showing the culture protocol for iNKT cell re-differentiation from CD4+ iNKT cell-derived iPSCs. EB differentiation medium: Iscove's modified Dulbecco's medium supplemented with 15% fetal bovine serum and a cocktail of 10 μg/ml human insulin, 5.5 μg/ml human transferrin, 5 ng/ml sodium selenite, 2 mM L-glutamine, 0.45 mM monothioglycerol, and 50 μg/ml ascorbic acid. OP9 medium: α-MEM supplemented with 15% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin.

(C) Fold expansion of parental CD4+ iNKT cells and newly induced re-iNKT cells around 2 weeks after stimulation with α-GalCer-pulsed PBMCs. The result obtained with each indicated cell in three independent experiments is shown as a filled circle. Experiment 1: clone 2, parental clone and subclone of donor A; experiment 2: clone 1, clone 8, and donor A; experiment 3: clone 1, clone 2, clone 8, and parental clones of donor A and B. Horizontal lines indicate medians. NS, not significant.

(D) Co-expression profile of PD-1 and TIGIT on re-iNKT cells and parental iNKT cells after TCR stimulation (gated by CD45, CD3, and 6B11). The results shown are representative of two independent experiments. The numbers in the quadrants indicate percentages of cells.

(E) Telomere length in parental CD4+ iNKT cells and induced re-iNKT cells normalized to the tetraploid leukemic cell line 1301. Datasets from three independent experiments that include data from three different re-iNKT cell clones (1, 2, and 8), and two parental iNKT cell clones (donor A and donor B) are shown as filled diamonds, squares, and triangles, respectively. t Tests for paired data were used to compare results from the same donor; ∗p < 0.05.