Transcriptional effects of preventing progression past early G₁ during ex vivo culture of LT-HSCs. (A) The cumulative first-division kinetics (excluding dead cells) of UNTR/PD-treated LT-HSCs cultured in a GT_mPB (dark red and light red) or EXPER_CB (dark blue and light blue) system. The curve is a least-squares sigmoidal fit. A representative example is shown (n = 3 UNTR or PD–matched biological replicates: n = 2 UNTR or PD–treated matched biologic replicates). The dashed line indicates the EC₅₀ time to first division. UNTR and PD-treated (200 nM) cells are shown. (B) The divided single cells as a proportion of the total alive cells at 96 hours are shown (EXPER_CB: n = 5 biological replicates, n = 2 UNTR or PD–treated matched biological repeats; GT_mPB: n = 3 matched biological replicates). Paired t tests are shown. (C) Representative example of the flow cytometry plot for pRb (at Ser 807-811) and DAPI staining on 72 hour EXPER_CB cultured UNTR (left) or PD-treated (right) LT-HSCs. (D) Quantification of pRb⁺ (as a percentage of viable cells) in 62 hour GT_mPB cultured UNTR or PD-treated LT-HSCs (n = 3 UNTR or PD-treated matched biological replicates; no lentiviral vector [LV] transduction) or 72 hour EXPER_CB cultured LT-HSCs (n = 3 UNTR or PD-treated matched biological replicates). Paired t tests are shown. (E) UMAP visualization of scRNA-seq of 954 LT-HSCs from the indicated culture conditions (EXPER_CB, 536 single cells; GT_mPB, 418 single cells). Cell cycle regression was applied. (F) The 2-dimensional pseudotime density rank plot of single cells as shown in (E). Cell cycle regression was applied. (G) Pearson correlation coefficient estimates for the comparison of the median expression value of 10903 genes at different time points or conditions for the EXPER_CB data set (union of all differentially expressed genes between any 2 UNTR time points and between PD-treated and UNTR conditions) (available in supplemental Table 5). (H) Pearson correlation coefficient estimates for the comparison of the median expression value of 5469 genes at different time points or conditions for the GT_mPB data set (union of all differentially expressed genes between any 2 UNTR time points and between PD treated and UNTR conditions) (available in supplemental Table 5). (I) Selected Reactome pathways (FDR < 0.05) enriched between PD-treated and UNTR LT-HSCs cultured for matched durations of 24 hours in EXPER_CB (purple), 72 hours in EXPER_CB (pink), and 62 hours in GT_mPB (brown) systems. The full DeSeq2 results and Reactome pathway enrichment are available in supplemental Table 8.