Asymmetric lysosome inheritance predicts activation of haematopoietic stem cells

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Abstract

Haematopoietic stem cells self-renew and differentiate into all blood lineages throughout life, and can repair damaged blood systems upon transplantation. Asymmetric cell division has previously been suspected to be a regulator of haematopoietic-stem-cell fate, but its existence has not directly been shown1. In asymmetric cell division, asymmetric fates of future daughter cells are prospectively determined by a mechanism that is linked to mitosis. This can be mediated by asymmetric inheritance of cell-extrinsic niche signals by, for example, orienting the divisional plane, or by the asymmetric inheritance of cell-intrinsic fate determinants. Observations of asymmetric inheritance or of asymmetric daughter-cell fates alone are not sufficient to demonstrate asymmetric cell division2. In both cases, sister-cell fates could be controlled by mechanisms that are independent of division. Here we demonstrate that the cellular degradative machinery—including lysosomes, autophagosomes, mitophagosomes and the protein NUMB—can be asymmetrically inherited into haematopoietic-stem-cell daughter cells. This asymmetric inheritance predicts the asymmetric future metabolic and translational activation and fates of haematopoietic-stem-cell daughter cells and their offspring. Therefore, our studies provide evidence for the existence of asymmetric cell division in haematopoietic stem cells.

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Fig. 1: Asymmetric mCherry–NUMB inheritance and asymmetric daughter-cell fates in purified HSCs.
Fig. 2: Asymmetric inheritance of mCherry–NUMB predicts asymmetric HSC activation.
Fig. 3: Lysosomes, autophagosomes and mitophagosomes are co-inherited during asymmetric HSC divisions and predict upregulation of CD71.
Fig. 4: Asymmetric lysosome inheritance predicts fate heterogeneity of HSC daughter cells.

Data availability

Source Data for all figures are provided with the paper. The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Code availability

Software used for data acquisition of immunostainings and time-lapse imaging is commercially available (NIS-Elements 4.3.1) or published and open source (YouScope v.2.1, http://langmo.github.io/youscope/). Software for single-cell tracking and fluorescence quantification used in this study is published and open source11. Software used for image segmentation is published and open sourced35. Software used for dimensionality reduction using UMAP is published and open source (https://github.com/lmcinnes/umap.git). Software used for time-series clustering was inspired by (https://github.com/dmattek/shiny-timecourse-inspector). All code is available from the corresponding author on reasonable request.

Change history

  • 13 September 2019

    An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Acknowledgements

We thank S. Ammersdoerfer, G. Camenisch, M. D. Hussherr, V. Jäggin, T. Lopes, H. Oller, C. Raithel, B. Vogel and A. Ziegler for technical support. This work was supported by DFG SFB 684 and the SNF to T.S. T.S. and O.H. acknowledge financial support from SystemsX.ch. We thank J. Arias for providing the cDNA for pHLuorin–DsRed–LC3β.

Author information

D.L. planned and performed experiments and analysed data with A.W., F.S., Y.Z. and N.M.-B. O.H., P.S.H., M.E. and K.D.K. contributed to software development. P.S.H. and M.E. provided support with flow cytometry. T.S. designed and supervised the study, developed and maintained quantitative long-term bioimaging with D.L. and K.D.K. All authors read and commented on the final manuscript.

Correspondence to Timm Schroeder.

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Competing interests

: The authors declare no competing interests.

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Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Peer review information Nature thanks Guy Sauvageau and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Fig. 1 NUMB is asymmetrically inherited during HSC divisions.

a, Freshly isolated HSCs were transduced with fluorescence fusion reporter constructs for 24 h and imaged on OP9 stroma cells. POI, protein of interest. b, Quantification of differences in sister-cell fluorescence intensity during the first division of HSC on OP9 stroma. A sister-cell ratio above 1.5× was considered as asymmetric inheritance. CD63–Venus shows clear asymmetric inheritance, whereas all other candidates are not clearly different from the Venus-only control. The number of independent experiments (n) and total number of analysed HSC divisions (div.) are indicated. Two-sided Fisher’s exact test. c, Representative video frames of dividing HSCs transduced with CD63–Venus. Symmetric and asymmetric inheritance of CD63–Venus can be observed. d, e, Representative quantification of CD63–Venus and mCherry–NUMB fluorescence intensity of HSC daughter cells over time, for symmetric (top) and asymmetric (bottom) segregation during division. Fold differences between sister cells early after division are indicated. Intensity differences between daughter cells can also occur (long) after mitosis, and do not necessarily indicate asymmetric inheritance. The reliable classification of asymmetric inheritance requires continuous observation of single cells. Except for the representative example of tree 1, mother-cell intensities are omitted to improve presentation. n = 3 independent experiments. Source data

Extended Data Fig. 2 NUMB is asymmetrically inherited during HSPC divisions.

a, Freshly isolated KSL cells were sorted, cultured in 100 ng ml−1 SCF and 100 ng ml−1 TPO, fixed after 44 h and stained for DAPI, α-tubulin and NUMB. α-Tubulin was used to identify mitotic cells. b, Representative examples of fixed mitotic KSL cells, with symmetric (top) and asymmetric (bottom) inheritance of endogenous NUMB. Bar charts indicate quantification of NUMB levels in sister cells, as indicated. Scale bar, 5 μm. c, Examples of different NUMB sister-cell differences in fixed mitotic KSL cells. d, Quantification of level of endogenous NUMB expression, and sister-cell-intensity ratios, in fixed mitotic KSL cells. Differences between sister cells are, in general, below twofold. Low levels of NUMB expression biases towards higher sister-cell ratios. Thus, NUMB staining alone or arbitrary thresholding are not sufficient to discriminate between technical and/or biological noise and functionally relevant asymmetric inheritance. Spearman’s r. n = 16 independent experiments, 974 mitotic cells in total. ei, Correlation of NUMB and DAPI, α-tubulin, PARD3b and AP2A2 sister-cell intensity ratios. Circles in e represent a pair of daughter cells (no. 1 and no. 2 (here denoted #1 and #2, respectively)). PARD3b and AP2A2 are co-inherited into the same daughter cell as is NUMB during asymmetric inheritance. DAPI and α-tubulin sister-cell ratios were used as controls, and are expected to be inherited equally. n = 7, 7, 4 and 3 independent experiments for DAPI, α-tubulin, AP2A2 and PARD3b, respectively. j, Correlation of NUMB sister-cell ratios with sister-cell size ratio. Asymmetric inheritance of NUMB does not rely on differences in cell size upon division. n = 4 independent experiments. k, Frequency of co-inheritance of NUMB and AP2A2 or PARD3b, based on data displayed in fi, Sister-cell ratios of >1.1× for both NUMB and AP2A2, PARD3b, α-tubulin and DAPI were considered as co-inheritance. Co-inheritance of DAPI and α-tubulin was used as control. AP2A2 and PARD3b are co-inherited into the same daughter cell as is NUMB in mitotic KSL cells during asymmetric inheritance of NUMB. Mean ± s.e.m. Two-sided Fisher’s exact test. n = 7, 7, 4 and 3 independent experiments for DAPI, α-tubulin, AP2A2 and PARD3b, respectively. l, Freshly isolated KSL cells were sorted, transduced with mCherry–NUMB or NUMB–Venus and fixed after 44 h of culture. Mitotic cells were identified by α-tubulin staining. m, Representative maximum intensity projections of mitotic KSL cells stained for endogenous NUMB, and transduced with mCherry–NUMB or NUMB–Venus. mCherry–NUMB localizes to endosomes as endogenous NUMB (arrowheads). NUMB–Venus localizes mainly to the cell membrane. Images were acquired using a 100× oil immersion objective (NA = 1.4). Scale bar, 10 μm. n, Representative example of Pearson voxel-intensity correlation of endogenous NUMB and mCherry–NUMB (left) and NUMB–Venus (right), in fixed mitotic KSL cells. Pearson’s r. n = 2 independent experiments. o, Quantification of Pearson voxel-intensity correlation across 70 analysed cells in total. The localization of the N-terminal mCherry–NUMB fusion correlates better with endogenous NUMB than does the C-terminal NUMB–Venus fusion. Randomized voxel intensities were used as control. n = 2 independent experiments with 40 and 30 analysed cells or cell divisions for mCherry–NUMB or NUMB–Venus, respectively. Source data

Extended Data Fig. 3 Continuous quantification of mCherry–NUMB, and asymmetric fate marker of dividing HSCs and their daughter cells.

a, Representative examples of trees based on differential CD71 production of sister cells. Differences in CD71 daughter-cell production are frequently associated with asymmetric inheritance of mCherry–NUMB. Comparable CD71 daughter-cell production is associated mostly with symmetric inheritance of mCherry–NUMB. All examples were selected on the basis of differences in CD71. b, Representative examples of continuous simultaneous quantification of mCherry–NUMB and CD71 expression dynamics. Daughter cells that receive less mCherry–NUMB upregulate CD71. c, Quantification of CD41, SCA1, CD48, CD105 and CD71 production in HSC daughter cells. Asymmetric or symmetric inheritance was defined as a >1.8× or <1.2× mCherry–NUMB sister-cell ratio, respectively. Asymmetric daughter cells that receive less mCherry–NUMB (white) produce more CD48, CD105 and CD71 than their sister cells (black). Comparable CD48, CD105 or CD71 production in symmetric mCherry–NUMB daughter cells. Black circles indicate daughter cells that received more mCherry–NUMB. n = 3 independent experiments. Two-sided Mann–Whitney test. In the box plots, centre line is the median; box limits are the upper and lower quartiles; whiskers are Tukey’s 1.5× interquartile range; and points are outliers. d, e, Heat map and quantification of HSC paired daughter-cell expression dynamics of SCA1 (522 cells analysed), CD105 (186 cells analysed), CD48 (186 cells analysed) and CD41 (522 cells analysed) after symmetric and asymmetric inheritance of mCherry–NUMB. Asymmetric and symmetric paired daughter-cell fates can be observed after asymmetric or symmetric mCherry–NUMB inheritance (quantification in e). Source data

Extended Data Fig. 4 Differentiation is accompanied by metabolic activation, upregulation of CD71 and downregulation of stem-cell markers.

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. bd, 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

Extended Data Fig. 5 Lysosomes are asymmetrically inherited during HSC divisions.

a, Freshly isolated HSCs were sorted, transduced with either Venus or LAMP1–Venus and co-cultured on OP9 stroma cells in 100 ng ml−1 SCF and TPO. n = 3 independent experiments. b, Representative video frames of symmetric and asymmetric inheritance of LAMP1–Venus during HSC divisions. c, Quantification of normalized LAMP1–Venus and Venus sister-cell intensity ratio. LAMP1–Venus is asymmetrically inherited during HSC divisions. n = 3 independent experiments with 211 and 92 HSC divisions analysed for Venus and LAMP1–Venus, respectively. Two-sided Mann–Whitney test. In the box plots, centre line is the median; box limits are the upper and lower quartiles; whiskers are Tukey’s 1.5× interquartile range; and points are outliers. d, Mitotic KSL cells fixed after 44 h of in vitro culture, and antibody-stained for the lysosomal marker LAMP2. Endogenous lysosomal LAMP2 is asymmetrically inherited (arrowheads). n = 2 independent experiments with 31 mitotic KSL cells stained in total. e, Quantification of sister-cell ratios of LysoBrite and SCA1 at the first time point after division. LysoBrite sister-cell ratios above 1.5-fold do not correlate with high SCA1 sister-cell ratios. n = 2 independent experiments with 56 analysed HSC divisions in total. f, Quantification of LysoBrite sister-cell ratio and CD71 production ratio of HSC daughter cells. CD71 production was defined as the ratio of CD71 fluorescence intensity at the last time point of a cell cycle, divided by the CD71 fluorescence intensity of the first time point at the beginning of the cell cycle (directly after division). A high LysoBrite sister-cell ratio anti-correlates with the CD71 production ratio of HSC daughter cells; the HSC daughter cell that receives less LysoBrite upregulates CD71, and vice versa. Based on a threshold of 1.5-fold LysoBrite sister-cell ratio, CD71 levels can be predicted with high probability. n = 4 independent experiments with 350 analysed HSC divisions in total. g, Representative examples of continuous simultaneous quantification of LysoBrite and CD71 expression dynamics. Daughter cells that receive less LysoBrite upregulate CD71. n = 6 independent experiments. h, Quantification of CD41, SCA1, CD48, CD105 and CD71 production in HSC daughter cells. Asymmetric or symmetric inheritance was defined as a >1.5× or <1.2× LysoBrite sister-cell ratio, respectively. Asymmetric daughter cells that receive less LysoBrite (white) produce more CD48, CD105 and CD71 than their sister cells (black). Comparable CD48, CD105 or CD71 production in symmetric LysoBrite daughter cells. Black circles indicate daughter cell that received more LysoBrite. n = 3 independent experiments for CD41, SCA1, CD48 and CD105, n = 6 for CD71. Two-sided Mann–Whitney test. In the box plots, centre line is the median; box limits are the upper and lower quartiles; whiskers are Tukey’s 1.5× interquartile range; and points are outliers. i, Paired daughter-cell fate (SCA1 (244 analysed cells), CD105 (258 analysed cells), CD48 (258 analysed cells) or CD41 (244 analysed cells)) cluster (as defined in Extended Data Fig. 4b–e) frequencies after asymmetric or symmetric LysoBrite inheritance. Mean percentage shown. j, Heat map and clustering (top) and cluster frequency (bottom) of paired daughter-cell NOTCH1 dynamics, after symmetric and asymmetric inheritance of LysoBrite. Each row represents one HSC daughter-cell pair (no. 1 and no. 2 (here denoted #1 and #2, respectively)). Daughter cell no. 1 receives more LysoBrite during asymmetric inheritance, which predicts NOTCH1 upregulation. Bottom left, mean fluorescence intensities over time of clusters 1 and 2 with 218 and 209 pooled time series, respectively. Mean ± s.d. n = 3 independent experiments. The numbers of analysed paired daughter cells are indicated. k, Quantification of inheritance of HSC mitotic markers. NOTCH1, CD71 and CD105 are asymmetrically co-inherited with lysosomes, whereas SCA1 and CD41 are not. No correlation between cell size and lysosome inheritance was observed. n = 3 independent experiments. r, Spearman coefficient. Source data

Extended Data Fig. 6 NUMB and lysosomes colocalize partially and are co-inherited.

a, Freshly isolated KSL cells were sorted, cultured in 100 ng ml−1 SCF and TPO, fixed after 44 h and stained for DAPI, α-tubulin, NUMB and LAMP2 as a marker for lysosomes. α-Tubulin was used to identify mitotic cells. b, Frequency of NUMB and LAMP2 co-inheritance into the same daughter cell, based on data shown in ce. Co-inheritance of DAPI and α-tubulin with NUMB were used as control. LAMP2 is co-inherited into the same daughter cell as is NUMB, in fixed mitotic KSL cells during asymmetric inheritance of NUMB. Mean ± s.e.m. n = 3 independent experiments with 172 quantified mitotic KSL cells in total. Two-tailed Fisher’s exact test. c, Representative images of fixed mitotic KSL cells, showing symmetric (top) and asymmetric (bottom) inheritance of NUMB and LAMP2. NUMB and LAMP2 are partially co-localized (arrowheads, quantification in d and e) and are co-inherited asymmetrically into one daughter cell (quantification in b). Images were acquired using a 100× oil immersion objective (NA = 1.4). Bar charts indicate normalized quantification of NUMB and LAMP2 fluorescence signal in daughter cells 1 and 2, respectively. n = 3 independent experiments. Two-sided Mann–Whitney test. In the box plots, centre line is the median; box limits are the upper and lower quartiles; whiskers are Tukey’s 1.5× interquartile range; and points are outliers. f, Representative example of mitotic KSL cells with symmetric (top) and asymmetric (bottom) inheritance of mCherry–NUMB. mCherry–NUMB and LAMP2 co-localize partially, and are asymmetrically co-inherited into the same daughter cell (bottom, arrowheads). Images were acquired using a 100× oil immersion objective (NA = 1.4). Scale bar, 5 μm. n = 2 independent experiments. g, h, Quantification of 3D voxel co-localization of either endogenous NUMB or mCherry–NUMB with LAMP2 in mitotic KSL cells. Frequency of NUMB- or mCherry–NUMB- and LAMP2-positive voxels of all LAMP2-positive voxels is shown, and vice versa. Endogenous NUMB and mCherry–NUMB co-localize partially with LAMP2. Quantification of randomized voxels of NUMB and LAMP2 and mCherry–NUMB and LAMP2 were used as control. n = 2 independent experiments with 30 and 46 mitotic KSL cells in total for NUMB and LAMP2 and mCherry–NUMB and LAMP2 colocalization, respectively. i, Video frames of three representative asymmetric HSC divisions, showing mCherry–NUMB and LysoBrite colocalization (arrows) during mitosis. SCA1–Alexa Fluor 488 staining was used as a more-widely distributed control. Scale bar, 10 μm. j, Pixel colocalization of mCherry–NUMB with LysoBrite and SCA1 in mitotic HSCs. mCherry–NUMB and LysoBrite colocalize strongly. Pearson’s r. n = 3 independent experiments. k, Quantification of mCherry–NUMB and LysoBrite, and mCherry–NUMB and SCA1, fluorescence-intensity correlation and colocalization in mitotic HSCs. Cellular localization of mCherry–NUMB and LysoBrite correlate strongly. n = 3 independent experiments, 89 HSC divisions were analysed. Two-sided Mann–Whitney test. In the box plots, centre line is the median; box limits are the upper and lower quartiles; whiskers are Tukey’s 1.5× interquartile range; and points are outliers. ns = not significant. Source data

Extended Data Fig. 7 Autophagosomes and mitophagosomes are asymmetrically inherited during HSC divisions.

a, To test for intra- versus extra-lysosomal localization of NUMB and mitochondria, HSCs were transduced with GFP–mCherry double-fluorescence fusion reporters for (1) lysosomal outside surface (LAMP1; negative control), (2) autophagosomes (LC3β; positive control), (3) NUMB and (4) mitochondria (COX8a). The differences in maturation time and pH stability between GFP (which matures faster and is pH-unstable) and mCherry (which matures slower and is pH-stable) enable the identification of the reporter in nascent (green), mature (yellow) or lysosomal (red) form, and the inheritance of these forms upon mitosis. b, Representative images of GFP–mCherry–NUMB expressing cells during mitosis. Asymmetric inheritance can be observed in the mCherry and GFP channels. n = 5 independent experiments. Scale bar, 5 μm c, Quantification mCherry and GFP inheritance. LAMP1–GFP–mCherry is localized outside the lysosomal lumen and serves as a control (diagonal yellow line, indicative of equal amount of mCherry and GFP signal that is asymmetrically inherited). Nascent and mature asymmetrically inherited GFP–mCherry–NUMB is mostly found outside the lysosome. Autophagosomes and mitochondria are asymmetrically inherited mostly in lysosomes. n = 4, 3, 3 and 3 independent experiments with 236, 132, 183 and 185 analysed mitotic cells for lysosomes, NUMB, autophagosomes and mitochondria, respectively. Source data

Extended Data Fig. 8 Translational activity precedes upregulation of CD71, and is predicted by asymmetric inheritance of lysosomes.

a, HSCs were imaged in 100 ng ml−1 SCF and TPO, supplemented with LysoBrite and fluorescent anti-CD71. After 44 h, cells were incubated for 1 h with puromycin. After fixation, puromycin incorporated into nascent proteins was then stained to quantify translational activity. b, Representative video frames of HSC daughter cells stained with puromycin and CD71. Asymmetric translational activity correlates with asymmetric upregulation of CD71. Scale bar, 10 μm. n = 6 independent experiments. c, Quantification of asymmetric translational activity and asymmetric upregulation of CD71 in daughter cells fixed at different times after division. Translational (first) and CD71 (later) daughter-cell differences increase over time. n = 6 independent experiments. One-way analysis of variance. In the box plots, centre line is the median; box limits are the upper and lower quartiles; whiskers are Tukey’s 1.5× interquartile range; and points are outliers. d, Increased translation precedes upregulation of CD71. e, Quantification of CD71, translational activity (puromycin) and LysoBrite inheritance differences between HSC daughter cells. CD71 and translational activity are upregulated in the daughter cell (daughter no. 2) that receives less LysoBrite during mitosis. Daughter cells with symmetric upregulation of CD71 upregulate puromycin symmetrically. n = 6 independent experiments. Two-sided Mann–Whitney test. In the box plots, centre line is the median; box limits are the upper and lower quartiles; whiskers are Tukey’s 1.5× interquartile range; and points are outliers. f, Quantification of upregulation of CD71 and translational activity after symmetric and asymmetric inheritance of LysoBrite and translational activity. CD71 and translation are upregulated in the daughter cell that asymmetrically inherits less LysoBrite, and are upregulated in both daughter cells after symmetric inheritance of lysosomes. Two-sided χ2 test against the hypothesis of random distribution of CD71 upregulation and translational activity. n = 6 independent experiments, with 155 and 109 analysed HSC daughter-cell pairs for symmetric and asymmetric LysoBrite and translational activity. Source data

Extended Data Fig. 9 Cell features used for cell-state clustering and dynamics quantification.

a, Clustering of single-cell dynamics b, Heat-map overlay of quantification of single-cell dynamics used for clustering and cell-fate assignment to cell-lineage tree projected onto UMAP. n = 4 independent experiments. c, Quantification of cell features per cluster. n = 4 independent experiments. In the box plots, centre line is the median; box limits are the upper and lower quartiles; whiskers are Tukey’s 1.5× interquartile range; and points are outliers. d, Quantification of cluster frequencies per generation in cell-lineage trees after symmetric and asymmetric inheritance of lysosomes. Later differentiation is heterogeneous. Mean ± s.e.m. n = 4 independent experiments. e, Quantification of subcolonies derived from HSC daughter cells. Asymmetric inheritance of lysosomes correlates with increased overall heterogeneity in generation 1, but not in later generations. Black circles indicate daughter cell that received more LysoBrite. n = 4 independent experiments. In the box plots, centre line is the median; box limits are the upper and lower quartiles; whiskers are Tukey’s 1.5× interquartile range; and points are outliers. f, g, Lineage contribution and colony size of paired HSC daughter-cell colony assay after 12 days of in vitro culture. Black circles indicate daughter cell that received more LysoBrite. n = 5 independent experiments. Two-sided Wilcoxon matched-pairs signed-rank test. In the box plots, centre line is the median; box limits are the upper and lower quartiles; whiskers are Tukey’s 1.5× interquartile range; and points are outliers. Source data

Extended Data Figure 10 Graphical abstract.

a, Unequivocal classification into symmetric and asymmetric daughter cells fates requires continuous quantitative single cell observation. b, Asymmetric inheritance of the autophagosomal and lysosomal degradative machinery predicts which HSC daughter cell becomes metabolic active.

Supplementary information

Supplementary Figures Supplementary Data Fig. 1 | Flow cytometric gating strategy for isolation of HSC and MPP1-5s. Flow cytometric sorting scheme used to isolate HSC and MPP1-5. Supplementary Data Fig. 2 | Flow cytometric gating strategy used in paired daughter cell colony assay. Prog. - Progenitor; GMP - granulocyte-monocyte progenitor; Gm - granulocyte-monocyte (myeloid) lineage; MegE - megakaryocyte-erythrocyte lineage; Macro.- macrophage; Granu. - granulocyte; Megak. - megakaryocyte; Ery. - erythocyte

Reporting Summary

Supplementary Table Supplementary Table S1 | Antibodies used in this study. List of antibodies used in this study

Video 1

Symmetric inheritance of mCherryNUMB. Symmetric inheritance of mCherryNUMB during HSC division. Compare Fig. 3a and b (left). n = 3 independent experiments

Video 2

Asymmetric inheritance of mCherryNUMB. Asymmetric inheritance of mCherryNUMB during HSC division. Compare Fig. 3a and b (right). n = 3 independent experiments

Video 3

Symmetric inheritance of mCherryNUMB followed by symmetric CD71 upregulation. CD71 is upregulated in both HSC daughter cells after symmetric inheritance of mCherryNUMB. n = 3 independent experiments

Video 4 Asymmetric inheritance of mCherryNUMB predicts CD71 upregulation. CD71 is upregulated in HSC daughter cells receiving less mCherryNUMB after its asymmetric inheritance. n = 3 independent experiments.

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