Kinetics of cell cycle progression, survival, and loss of long-term repopulation capacity of LT-HSCs during ex vivo culture. (A) The cumulative quiescence exit kinetics of GT_mPB LT-HSCs are shown, determined by phosphorylation of Rb (pRb; at Ser 807-811) using flow cytometry analysis. The curve is a least-squares sigmoidal fit with half maximal effective concentration (EC₅₀) = 24.7 hours; there are n = 3 biological replicates for 0 hours, 24 hours, 62 hours, and 72 hours and n = 4 biological replicates for 6 hours. Standard error ± 5.186; R² = 0.9797. (B) The cumulative quiescence exit kinetics of EXPER_CB LT-HSCs are shown, determined by pRb (at Ser 807-811) using flow cytometry analysis. The curve is a least-squares sigmoidal fit with an EC₅₀ = 24.72 hours; there are n = 3 biological replicates for 0 hours, 6 hours, 24 hours, and 48 hours and n = 4 biological replicates for 72 hours. Standard error ± 3.944; R² = 0.9844. (A-B) The dashed line indicates the EC₅₀ at the time of quiescence exit. (C) Cell cycle phase assignment of GT_mPB LT-HSCs determined by pRb/DAPI flow cytometry analysis. Equivalent repeats as in panel A. (D) The cell cycle phase assignment of EXPER_CB LT-HSCs determined by pRb/DAPI flow cytometry analysis. Equivalent repeats as in panel B. (E) The cumulative first-division kinetics (excluding dead cells) of GT_mPB LT-HSCs are shown. The curve is a least-squares sigmoidal fit. Representative examples are shown (n = 3 biological replicates). The dashed line indicates the EC₅₀. The EC₅₀ = 64.83 hours; 95% confidence interval (CI) = 62.83-66.87; R² = 0.9976. (F) The cumulative first-division kinetics (excluding dead cells) of EXPER_CB LT-HSCs are shown. The curve is a least-squares sigmoidal fit. Representative examples shown (n = 4 biological replicates). EC₅₀ = 55.44 hours; 95% CI = 54.28-56.70; R² = 0.9995. (G) The time to first-division kinetics summary of LT-HSCs cultured in (E) GT_mPB (F) and EXPER_CB systems (n = 3 biological replicates for GT_mPB; n = 4 biological replicates for EXPER_CB). Unpaired t tests are shown. (H) The percentage of LTRC in GT_mPB CD34⁺CD38⁻ cells at each time point, as determined by LDA analysis in the transplanted population ± 95% CIs is shown. The LTRC frequency estimates for GT_mPB are as follows: 0 hours, 1 in 939 (29 mice); 6 hours, 1 in 1211 (21 mice); 24 hours, 1 in 3371 (23 mice); 62 hours, 1 in 2510 (23 mice). Extreme Limiting Dilution Analysis (ELDA) statistical tests are shown. The data are shown in supplemental Table 1. (I) The percentage of LTRC in LT-HSCs cultured in EXPER_CB systems at each time point, based on LDA analysis in the transplanted population ± 95% CIs is shown. The LTRC frequency estimates for EXPER_CB are as follows: 0 hours, 1 in 14.8 (31 mice); 6 hours, 1 in 15.2 (19 mice); 24 hours, 1 in 80.6 (31 mice); and 72 hours, 1 in 293.7 (40 mice). ELDA statistical tests are shown. The data are shown in supplemental Table 1. (J) The survival of LT-HSCs cultured in GT_mPB systems, as determined by Annexin-V/7-AAD flow cytometry, is shown for n = 3 biological replicates at each time point. The mean ± SD is shown. (K) Survival of LT-HSCs cultured in EXPER_CB systems, as determined by Annexin-V/7-AAD flow cytometry, is shown for n = 3 biological replicates at each time point. Paired t tests are shown for the 6 hours and 24 hours comparison. The mean ± SD is shown. SD, standard deviation.