HES4 transcriptional expression confers growth advantage in the transformation of human T-cell progenitors. (A) Flow cytometric analysis of GFP+ and mCherry+ cell abundance in the CB cells doubly transduced, respectively, with NOTCH1-ΔE/GFP or shRNA/mCherry lentiviral constructs. Transduced cells were cultured in vitro on OP9-DL1 feeders and serially passaged every 5 days. GFP+ mCherry+ alive cells were measured at the indicated time points using flow cytometry for DRAQ7 exclusion. The graphs report the result of 2 independent experiments performed in triplicate. ∗∗∗P < .001 (2-way analysis of variance [ANOVA] with Dunnett test, comparing the sh-scramble (shScr) control mean with the other values). (B) Flow cytometric tracking of doubly transduced (GFP+mTagBFP2+) CB cells at each serial passage onto OP9-DL1 feeders every 5 days. The CB cells were transduced with lentiviral particles with NOTCH1-ΔE-R1984A/GFP dimer mutant lentiviruses, in addition to the HES4/mTagBFP2 construct or EV as control on days 0 and 5. The mean values ± range for duplicate experiments are plotted. ∗∗P < .01; ∗∗∗P < .001 (Student t test). (C) Flow cytometric tracking of mTagBFP2+ CB cells, transduced with HES4/mTagBFP2 lentivector or EV as control on days 0 and 5. The transduced cells were serially passaged onto OP9-DL1 feeders every 5 days. The mean values ± range for duplicate experiments are plotted. ∗∗∗P < .001 (Student t test). (D) Limiting dilution growth assays. CB cells were transduced with HES4/mTagBFP2 lentivector or EV as control on day 0, cultured until day 3, and then sorted using FACS into individual wells of a 96-well plate on precoated plastic in T-cell expansion media (StemCell Technologies) and maintained in vitro for 3 weeks. The entire content of each well was assayed by flow cytometry and the frequency of well-initiating cells was calculated using the ELDA software. In the ELDA plot, the cell dose vs the log fraction of negative cultures is represented. Dotted lines represent the confidence interval of 95%. The estimated frequencies are indicated in the box on the right side. P values were calculated using the ELDA tool. (E) t-distributed stochastic neighbour embedding (tSNE) plots based on the flow cytometric tracking of CB cells, singly transduced with NOTCH1ΔE/GFP or HES4/mTagBFP2 lentivectors at day 10 of in vitro coculture on OP9-DL1 feeders. (F) The Kaplan-Meier survival curves of primary NSG mice injected with CB cells transduced with NOTCH1-ΔE, NOTCH1-ΔE-R1984A, or HES4 lentiviruses after 10 days of in vitro growth. The survival curve of secondary recipient mice transplanted with primary HES4 CB leukemia is indicated with a dashed line. The number of recipient animals for each cell type is indicated in parentheses. Data are pooled from 2 separate cohorts of animals.