Fig. 2

Tissue distributions, transcriptome characterization, and TCRα/β diversities of ESC-derived T Cells. a Flow cytometry analysis of mature iT cells in spleen (SP), lymph node (LN), and PB of B-NDG mice transplanted with ESC-derived hematopoietic cells. Each B-NDG mouse was transplanted with one million iHPCs collected at day 21. Representative mouse was sacrificed and analyzed at 5 and 6 weeks after transplantation. Data from two representative mice are shown. b Flow cytometry analysis of iDN cells in the thymus of B-NDG mice transplanted with ESC-derived hematopoietic cells. Each B-NDG mouse was transplanted with one million iHPCs at day 21. Representative mouse was sacrificed and analyzed at 4 and 5 weeks after transplantation. Data from four representative mice of two independent experiments are shown. Lin was defined as Ter119⁻CD11b⁻Gr1⁻CD19⁻B220⁻NK1.1⁻TCRγδ⁻. c Flow cytometry analysis of iHPC derivatives in bone marrow (BM). Each B-NDG mouse was transplanted with one million iHPCs collected at day 10 in the presence of OP9-DL1 feeder cells. Representative mouse was sacrificed and analyzed 5 weeks and 6 weeks after transplantation. The BM-derived iHPCs (CD45.2⁺Lin⁻c-kit^midSca1⁺) were sorted for the 2nd transplantation. Data from two mice are shown. d Flow cytometry analysis of iT and iNK cells in PB, spleen (SP) and bone marrow (BM) 6 weeks after the 2nd transplantation. Five hundred LSK cells from primary iT mice were used as input for secondary transplantation. The secondary recipients were sacrificed and analyzed 6 weeks after transplantation. Data from one mouse are shown. e Characterization of surface markers on CD4SP and CD8SP iT cells. CD4SP and CD8SP iT cells were sorted from the spleens of B-NDG mice transplanted with ESC-derived hematopoietic cells at week 5. One biological replicate per column. Myeloid cells (n = 2 sample repeats): Ter119^–CD3^–CD19^–CD11b⁺; B cells (n = 4 sample repeats): Ter119^–CD11b^–CD3^–CD19⁺; CD4⁺ cells (n = 3 sample repeats): Ter119^–CD19^–CD11b^–CD4⁺; CD8⁺ cells (n = 3 sample repeats): Ter119^–CD19^–CD11b^–CD8⁺; iCD4⁺ cells (n = 3 sample repeats): CD45.2⁺Ter119^–CD19^–CD11b^–CD4⁺; iCD8⁺ cells (n = 3 sample repeats): CD45.2⁺Ter119^–CD19^–CD11b^–CD8⁺. f Characterization of transcription factors in CD4SP and CD8SP iT cells. g Chord diagram of TCRα diversity in thymus iT cells. h Chord diagram of TCRβ diversity in thymus iT cells. i Chord diagram of TCRα diversity in spleen iT cells. j Chord diagram of TCRβ diversity in spleen iT cells. Aliquots of sorted 15,000 naïve CD4SP and CD8SP iT cells from either thymus or spleen of iT-B-NDG mice were used as cell inputs for TCRαβ sequencing