Preventing progression past early G₁ during ex vivo culture of LT-HSCs dampens the establishment of differentiation programs but does not affect the loss of long-term repopulation capacity. (A) GSVA scores for the indicated lineage gene expression signatures from Laurenti et al⁴⁰ at the indicated time points for the LT-HSC culture. The GSVA score is generated per cell, and the line indicates the median. EXPER_CB, n = 536 single cells; GT_mPB, n = 418 single cells. (B) The cell diameters of single EXPER_CB cultured LT-HSCs (n = 2 biological replicates representing n = 469 total single cells). Unpaired t tests are shown. ∗P < .001. (C) Tetramethylrhodamine (TMRM) staining of bulk EXPER_CB cultured LT-HSCs (n = 5 biological replicates for 0 hours; n = 4 matched biological replicates for UNTR or PD-treated cells for 24 hours; n = 3 matched biological replicates for UNTR or PD-treated cells for 48 hours). Unpaired t tests are shown. (D) Workflow of in vivo transplantation of EXPER_CB cultured LT-HSCs (24 hours and 72 hours) and CD34⁺/CD38⁻ cells cultured in the GT_mPB system (62 hours). UNTR or PD-treated cells were transplanted in matched cell dose experiments. (E) Graft size (percentage of human CD45⁺⁺ and GlyA⁺) at 18 weeks after transplantation of UNTR or PD-treated EXPER_CB LT-HSCs cultured for 72 hours (n = 5 biological experiments; the graph is representative of engrafted mice only; n = 42 PD mice; n = 38 UNTR mice). Two-way analysis of variance (ANOVA) with Sidak multiple comparisons performed (50 cells UNTR vs 50 cells PD; P = .9552; 300 cells UNTR vs 300 cells PD; P = .4084; 700 cells UNTR vs 700 cells PD; P = .971). (F) Graft size (percentage of human CD45⁺⁺ and GlyA⁺) at 18 weeks post transplantation of mPB CD34⁺CD38⁻ cells after GT protocol culture for 62 hours including LV transduction (n = 3 biological replicates; the graph is representative of engrafted mice only; n = 25 mice UNTR; n = 26 mice PD-treated). Two-way ANOVA with Sidak multiple comparisons performed (all cell doses UNTR vs PD; P > .9). (G) The percentage of LTRC in EXPER_CB cultured LT-HSCs UNTR or PD-treated, determined at 24 hours (n = 31 mice UNTR; n = 30 mice PD) and 72 hours (n = 42 mice PD; n = 40 mice UNTR). Numerical estimates for LTRC frequency available in supplemental Table 2. ELDA statistics (24 hours UNTR vs 24 hours PD; P = .405; 72 hours UNTR vs PD; P = .426). (H) LDA of secondary transplantation experiment from EXPER_CB cultured LT-HSCs UNTR or PD-treated from the 72 hour primary mice cohort. Secondary animals were transplanted with sorted CB CD45⁺⁺ from primary recipients (n = 20 mice total; 10 UNTR, 10 PD; n = 1 experiment) (supplemental Table 10). An ELDA statistical test was performed (P = .190). (I) LDA of secondary transplantation experiment from GT_mPB UNTR or PD-treated from the 62 hour primary mice cohort. Secondary animals were transplanted with whole mouse bone marrow (BM) isolated from primary recipients (n = 21 mice total; UNTR = 11 mice; PD = 10 mice; n = 1 experiment) (supplemental Table 10). An ELDA statistical test was performed (P = .860). (D) was created with BioRender.com (license agreement, KE26QKHW50).