a, Representative images of HSC-derived colonies (after 2.5 days) stained with fluorescent CD71 antibody, and TMRM or CellRox DeepRed (for ROS). CD71low (arrowheads) of CD71high cells express low or high levels of GFP–MYC, TMRM and ROS, respectively. n = 3 independent experiments. b–d, Correlation of GFP–MYC, TMRM and ROS with CD71 production in HSCs and daughter cells. Fold changes of >2 were considered as activation. Metabolically inactive, freshly isolated HSCs are low for CD71, GFP–MYC, TMRM and ROS. HSC daughter cells show correlated upregulation of GFP–MYC, TMRM, ROS and CD71. Mean ± s.e.m. n = 6, 3 and 3 independent experiments with 141, 162 and 179 HSCs and 282, 632 and 356 daughter cells for GFP–MYC, TMRM and ROS, respectively. Mean ± s.d. Two-sided Fisher’s exact test. Spearman’s r. e, GFP–MYC expression in freshly isolated HSCs and multipotent progenitors (MPP1–MPP5) analysed by flow cytometry. GFP–MYC expression is low in HSCs. MPP1–MPP5 have increased levels of GFP–MYC expression. n = 3 independent experiments. f, g, Representative examples of the quantification of the fluorescence dynamics of HSC daughter cells. HSC daughter cells that upregulate CD71 also upregulate GFP–MYC, and TMRM or ROS. In the case of the asymmetric onset of CD71, CD71low daughter cells remain low for GFP–MYC, TMRM and ROS. n = 3 independent experiments. h, Representative images of HSC-derived colony after four days. Fixation and immunostaining for MYC and CD71. Cells with low levels of CD71 expression express low levels of GFP–MYC (arrowheads). n = 3 independent experiments. i, Image cytometric quantification of GFP–MYC mean fluorescence intensity over time. MPP1–MPP5 upregulate GFP–MYC faster than do HSCs. n = 3 independent experiments. Mean ± s.e.m. Error bars and individual data points not displayed, for ease of reading. Data are from all cells (without known cell identity) in culture at specific time points. j, Image cytometric quantification of ROS in HSCs and MPP1–MPP5 over time. ROS production increased in differentiated cells. n = 3 independent experiments. Mean ± s.e.m. Error bars and individual data points are not displayed, for ease of reading. k, Image cytometric quantification of mitochondrial activity with TMRM 8 h after the start of the video. n = 4 independent experiments. l, Image cytometric quantification of CD71 mean fluorescence intensity of cells derived from TMRMhigh and TMRMlow HSCs over time. The progeny of HSCs with active mitochondria upregulates CD71 earlier than does the progeny of HSCs with inactive mitochondria. n = 4 independent experiments, 2,060 quantified data points (cells) across 5 measured time points total with 1,131 TMRMhigh and 929 TMRMlow HSCs analysed. P = 5.4 × 10−3, 2.7 × 10−3 and 4.9 × 10−3 for time points 0, 12 and 24 h, respectively. Mean ± s.e.m. Two-sided multiple t-tests, false-discovery-rate-corrected q = 0.01 (Benjamini–Yekuteli). m, Representative images of HSC-derived colonies after three days. Cells that express high levels of CD71 (arrowheads) have downregulated SCA1, and partially downregulated CD105. There is no clear correlation between levels of CD41 and CD71 expression. n = 3 independent experiments. Scale bar, 20 μm. n, Representative image cytometric quantification of all segmented cells in culture over time for CD71 versus CD41 expression, and SCA1 and CD105 expression. SCA1 and CD105 are downregulated during CD71 upregulation. n = 3 independent experiments. o, Image cytometric quantification of mean fluorescence intensity of CD71, SCA1, CD105 and CD41 expression over time in HSCs. At the population average, SCA1 and CD105 are downregulated during CD71 upregulation. Mean ± s.e.m. Error bars and individual data points not shown, for ease of reading. n = 3 independent experiments with 723, 450, 372 and 401 HSCs analysed for CD71, SCA1, CD105 and CD41, respectively and ≥9.3 × 105 quantified data points (cells) across 96 time points in total. p, Image cytometric quantification of CD71 mean fluorescence intensity over time in HSCs, MPP1, MPP2 and MPP3. MPP1–MPP3 upregulate CD71 earlier and stronger than do HSCs, which indicates differentiation. Mean ± s.e.m. Error bars and individual data points are not shown, for ease of reading. n = 3 independent experiments with 528, 519, 543 and 557 analysed HSCs, MPP1, MPP2 and MPP3, respectively, with ≥4.5 × 106 quantified data points (cells) across 96 time points in total. Source data