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. Author manuscript; available in PMC: 2023 May 1.
Published in final edited form as: Nat Biomed Eng. 2022 Aug 8;6(11):1284–1297. doi: 10.1038/s41551-022-00915-0

Extended Data Fig. 3. Early T lymphoid commitment of WT-TiPS and CAR-TiPS on human Notch ligands.

Extended Data Fig. 3.

a, SFG γRV plasmid design to transduce human Notch ligands (DLL1, DLL4, JAG1 or JAG2) into parental OP9 cells. b, Notch ligand expression on engineered OP9 lines. Filled grey histogram are stained parental OP9 cells, open black histogram are transduced OP9 cells. c, DTX1 induction in WT-TiPS by OP9 expressing indicated Notch ligand. D20 differentiating WT-TiPS cells were co-cultured with indicated OP9. DTX1 induction was measured by ddPCR, relative to endogenous RPL13A. The fold change was calculated relative to 0 h. Data shown is average of n = 2 technical replicates. d, g, Flow cytometric analysis of T lymphoid commitment marker expression (CD7, CD5, TCRαβ and CD56) of WT-TiPS (d) and CAR-TiPS (g) differentiated on OP9 expressing indicated human Notch ligands. Gated on live CD45+ cells. e, Flow cytometric analysis of pluripotency marker expression on CAR-TiPS. Gated on live cells. f, Phosphorylated-ERK1/2 levels in WT-TiPS (blue) and CAR-TiPS (red) on D35 (n = 3 technical replicates). h, Phenotype (left panels) and apoptosis levels (right panels) of WT-TiPS (top) and CAR-TiPS (bottom) from D27 – D35 of differentiation on OP9-DLL4. Percentage of apoptotic cells in each T lineage developmental stage was based on percentage live Annexin-V+ cells. * P<0.05, ** P<0.001, *** P<0.001, Welch’s 2-sample t test, data are means ± s.d (f).