Heterotypic interaction promotes asymmetric division of human hematopoietic stem and progenitor cells

  1. Adrian Candelas
  2. Benoit Vianay
  3. Matthieu Gelin
  4. Lionel Faivre
  5. Jerome Larghero
  6. Laurent Blanchoin
  7. Manuel Théry
  8. Stephane Brunet

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Abstract

Hematopoietic Stem and Progenitor Cells (HSPCs) give rise to all cell types of the hematopoietic system through various processes including asymmetric divisions. However, the contribution of stromal cells of the hematopoietic niches in the control of HSPC asymmetric divisions remains unknown. Using polyacrylamide microwells as minimalist niches, we show that heterotypic interaction with osteoblast promotes asymmetric division of human HSPC. Upon interaction, HSPCs polarize in interphase with centrosome, the Golgi apparatus and lysosomes positioned close to the site of contact. Subsequently during mitosis, HSPCs orient their spindle perpendicular to the plane of contact. This division gives rise to siblings with unequal amounts of lysosomes and differentiation markers such as CD34. Such asymmetric inheritance generates heterogeneity in the progeny, which is likely to be a key contributor to the plasticity of the early steps of hematopoiesis.

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    Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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    Reply to the reviewers

    We would like to thank the reviewers for carefully reviewing our manuscript submitted to Review Commons. Their constructive comments have led us to identify key additional experiments to perform (testing endothelial cells, investigating the role of CXCR4/Sdf1 axis in the asymmetric division). They have shed light on specific points of the manuscript that had to be amended (see below).

    First, we are pleased that the reviewers acknowledged the quality of the experiments performed (Reviewer #1: “In general, is a well-done work” ; Reviewer #2: “the evidence is convincing, experiments are rigorously performed with adequate replicates and reproducibility”), as well as interest and large scope our study (Reviewer #1: * “it gives a few more details describing that when HSPCs divide asymmetrically it seems there is an association between centrosome and lysosome distribution”, Reviewer #2: “These results collectively contribute to a deeper understanding of how the hematopoietic niche and interactions with neighboring cells influence HSPC behavior and their commitment to distinct cell fates during division”, “The findings have significance in understanding how the microenvironment influences HSPC behavior. This is broadly significant for stem cell biology”.* Reviewer #3 “The manuscript is likely to have broader interest to not only HSPC researchers, but stem cell biologists and even engineers due to the technologies used”).

    Our detailed answers to the reviewers’ concerns are listed below.

    Reviewer #1 (Evidence, reproducibility and clarity):

    In this manuscript, Candelas and colleagues investigated asymmetric cell division of human hematopoietic stem and progenitor cells (HSPCs) upon heterotypic interactions with osteoblasts. They developed a new in vitro system to test this and found that upon interaction, HSPCs polarized in interphase with centrosome and the Golgi apparatus and lysosomes positioned close to the site of contact. In addition, during mitosis, HSPCs were found to orient their spindle perpendicular to the plane of contact. This division gave rise to siblings with unequal amounts of lysosomes and CD34. In general, is a well-done work and it gives a few more details describing that when HSPCs divide asymmetrically it seems there is an association between centrosome and lysosome distribution.

    Authors: We thank the reviewer for this positive assessment of our work.

    Reviewer #1 (Significance (Required)):

    However, all of these features (described above) have already been independently described even in human HSPCs thus this work does not represent a major advancement in this field (e.g. on the potential molecular mechanism by which heterotypic interactions with osteoblasts promote such behaviours). Overall, this stage is descriptive in nature.

    Authors: We respectfully disagree with the reviewer on this point. The main finding of our work does not simply concern the ability of HSPCs to undergo asymmetric division. This ability has indeed been previously shown in mouse and human models, and we refer to the princeps works in our introduction (page 2 “In vitro HSPCs can undergo asymmetric divisions 11 12 13 14 15 16 17 ”). Our main finding is about the instrumental role of heterotypic interactions with stromal cells of the niches. This had not been shown previously, mostly due to the limitations of classical co-culture systems and of in vivo tracking of HSPCs. We have engineered artificial niches in microwells specifically to overcome these limitations. We are confident that this finding is of interest for of hematopoietic niches biology and more globally for the field of stem cell biology. In that sense, the corresponding biorXiv preprint has just been quoted by the seminal review by Hans Clevers “Hallmarks of stemness in mammalian tissues” (doi: 10.1016/j.stem.2023.12.006).

    One major caveat of this work is that CD34+ HSPCs represent a very heterogeneous population. Although the underlying features described in this work could be similar between different cell populations, the frequency of these events (e.g. % magnupodium; polarization index, % of asymmetric inheritance, etc) is likely to be different between stem and progenitor cells. This may also explain the wide spread of their data points. Experiments should also be conducted with different cell populations, in particular with HSCs (either with the CD34+CD38-CD45RA-CD90+ or CD34+CD38-CD45RA-CD90+CD49f+ HSC enriched fraction or the highly CD34+CD38-CD45RA-EPCR+ HSCs).

    Authors: We agree that we are dealing with an heterogenous population of stem and progenitor cells; we cannot exclude that more pronounced effects could be obtained by analyzing separately stem cells, lymphoid and myeloid progenitors. The use of CD34+ heterogenous population has been a strategical choice. We are working with human primary cells, harvested from cord blood, a rare and precious material, who’s access has been in addition reduced during the COVID period and since. Working with the global CD34+ population was giving us the ability to work with a higher number of cells and avoid material limitations to perform experiments. Importantly, in our initial publication (Bessey et al doi:10.1083/jcb.202005085), we both used CD34+, or CD34+/CD38- versus CD38+/CD34+ subpopulations (see figure 4): the effects we described using global CD34+ population were significant, validating a posteriori this choice. Similarly, the effects observed in the present work with CD34+ population have always been validated by appropriate statistic tests. So, we are confident that the use of the CD34+ population, despite its heterogeneity, did not alleviate the validity and pertinence of our analyzes and conclusions.

    Results shown in Figure 2 were obtained with a very limited number of cells; also data obtained for Figure 4; this should be substantiated;

    Authors: We acknowledge that the analyses performed on fixed mitotic cells are based on relatively small samples. We have decided to fix and analyze the cells at 40h of culture which corresponds to the peak of mitosis, instead of synchronizing HSPCs in mitosis with available drugs, in order to avoid potential perturbations of these drugs. The consequence was the scarcity of the mitotic HSPCs in each experiment. This limitation has also been encountered by other laboratories working on HSPCs : our samples sizes are in fact within the range of the samples analyzed in other works (see Hinge et al., 2020, doi:10.1016/j.stem.2020.01.016: around 20 cells; Florian, et al., 2018: doi: 10.1371/journal.pbio.2003389. from 8 to 40 cells). However, in order to improve this limitation, few additional experiments and analyses have been performed to increase the number of mitotic cells (Figure 3: from 27 to 38 cells).

    In addition, data from Figure 2 adds little and should be combined with Figures 3 and 4 to make a single stronger message.

    __Authors __: We agree on this comment: Figures 2 and 3 have now been merged

    It is/was unclear why the authors investigated the distribution of CD34 and CD33 and the major and important question remained to be answered: whether the symmetric cell divisions were/are differentiating or self-renewal in nature hence, the asymmetric cell divisions promoted by the osteoblasts may represent asymmetric self-renewal ones; this needs to be investigated further and potential molecular mechanisms for such promotion should be highlighted.

    Authors: CD34 and CD33 were chosen as our “gold standards” according to the princeps work of Loeffler et al (Loeffler et al., 2019, and 2022). Our selection is based on the need for markers that encompass this diverse population. We focused on CD34 and CD33 as they are among the most prevalent CD markers on HSPCs, particularly when working with the CD34+ population, which is broad and necessitates widely expressed CD markers. Importantly, previous works have reported that CD34 anti-correlates with lysosome asymmetric inheritance after the first division, whereas CD33 does not (see Fig3E, Loeffler et al., 2022). So using these markers we can compare two markers: one that follows lysosome inheritance after the first division, and one which doesn’t. We found that osteoblasts inducing asymmetric lysosome inheritance in HSPC do increase asymmetric production of CD34 but not CD33, in concordance with the literature. This has been now explained in more detail in the text.

    In addition, higher lysosome inheritance has been previously demonstrated to be associated with the most stem cell (Loeffler et al., 2019), implying a more stem-like profile for the proximal cell during division in our work. However, recent studies using single-cell sequencing approaches challenge the notion of discrete differentiation within this stem cell model (reviewed by Zhang et al., 2018; Laurenti and Gottgens, 2018; Cheng et al., 2020; Liggett and Sankaran, 2020). Our study contributes to this discussion by providing insights into the relationship between CD markers, lysosome inheritance, and the stem cell profile during asymmetric division, and presenting this heterogeneity observed in single-cell sequencing works.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    The paper titled "Heterotypic Interaction Promotes Asymmetric Division of Human Hematopoietic Stem and Progenitor Cells" presents research on how interactions between Hematopoietic Stem and Progenitor Cells (HSPCs) and osteoblasts impact cell division symmetry. The use of polyacrylamide microwells to simulate the hematopoietic niche and study osteoblast interactions is a novel and valuable approach. The paper effectively explains the experimental design, making it easy for others to replicate and verify the results. The paper provides detailed insights into how interactions with osteoblasts influence HSPCs, particularly in terms of cell polarization, spindle orientation, and organelle distribution during cell division. The evidence is convincing, experiments are rigorously performed with adequate replicates and reproducibility.

    Authors: We thank the reviewer for this positive evaluation of our work.

    • The study acknowledges that not all HSPCs respond to heterotypic interaction, suggesting that individual variability in HSPC behavior plays a role. Future research could explore the factors that determine which HSPCs undergo asymmetric divisions. This raises questions about the heterogeneity in HSPC responses. The researcher starts culturing CD34+ which is a heterogeneous group with osteoblast. This heterogeneity by default will give different cells progeny which mimics the in vivo BM status. But, to minimize the invitro experiment variables examining a more pure HSC population such as isolated CD34+CD38-CD90+ cells would strengthen the findings?

    Authors: We agree that we are dealing with an heterogenous population of stem and progenitor cells; we cannot exclude that more pronounced effects could be obtained by analyzing separately stem cells, lymphoid and myeloid progenitors. The use of CD34+ heterogenous population has been a strategical choice. We are working with human primary cells, harvested from cord blood, a rare and precious material, who’s access has been in addition reduced during the COVID period and since. Working with the global CD34+ population gave us the ability to work with a higher number of cells and avoid material limitations to perform experiments. Importantly, in our initial publication by Bessy et al (doi:10.1083/jcb.202005085), we used both CD34+, or in some cases CD34+/CD38- versus CD38+/CD34+ subpopulations (see figure 4), to investigate HSPC mode of polarization. The effects observed in the case of total CD34+ were significant, validating a posteriori this choice. Similarly, the effects observed in the present work with CD34+ population have always been validated by appropriate statistic tests. Our study indicates that the mechanism we described is conserved throughout the entire HSPC population.

    In conclusion, we are confident that the use of the CD34+ population, despite its heterogeneity, did not alleviate the validity and pertinence of our analyzes and conclusions.

    What was the Osteoblast/HSCs ratio used in the experimental setup? Does this ratio affect the results?

    Authors: The loading of HSPC has been optimized to get one HPSC per microwell, as indicated in the result section (page 3 line 3). This has now been clarified in the material and methods section. However, we observe some wells with more than one HSPC on the stromal cell, but we did not notice any clear impact on cell division asymmetry and did not investigate further this parameter.

    • The paper primarily focuses on lysosome inheritance and CD34 expression, leaving room to explore other lineage-specific markers and their correlation with asymmetric division. Do osteoblast/ HSCPs interactions have also an effect on mitochondrial inheritance?

    Authors: We fully agree that other markers could have been analyzed to comfort our observations.

    A far as mitochondria are concerned, recent publications suggest that lysosomes can be considered as a “inversed” readout of mitochondria inheritance: (1) mitophagy has been shown to take place in quiescent hematopoietic stem cells (Ito et al., 2016), suggesting that lysosomes do participate in the regulation mitochondria levels in HSPCs. (2) Loeffler et al. (2019) has shown an opposite pattern between mitochondria and lysosomes, lysosomes co-inheriting with mitophagosomes. Considering that this mutual or opposite inheritances were already described, we did not investigate them further. However we agree that it could be the focus for future works.

    • While the study hints at the clinical implications of its findings, it does not show the practical applications in detail. How the knowledge gained from these interactions could be translated into clinical practices or therapies is not explicitly discussed. Bridging this gap is necessary for understanding the practical utility of these results.

    Authors: we are confident that microwell technology could find, in the long term, more medical applications. However, we did not wish to speculate on this point in the present manuscript. The words “clinical” and “therapy” show no occurrence in our text. The potential implications of our findings in the field of leukemia are now mentioned in the discussion section with the corresponding references (page 6 line 10) “Such spatial control of the division is a conserved feature of asymmetric divisions in other stem cell niches 6. It may account for in vivo observations of organization into clusters of blood cell differentiation 44, and may play an important role in competition mechanisms at play within the hematopoietic niches in physiological 45 and pathological 46 contexts.”

    __Reviewer #2 (Significance (Required)): __

    • Investigating the process of HSPC asymmetric division is highly important to understand HSPC fate decisions and functions. The findings have significance in understanding how the microenvironment influences HSPC behavior. This is broadly significant for stem cell biology.
    • The paper's observation of uneven lysosome distribution and its potential impact on differentiation markers, including CD34 and CD33, contribute to our understanding of HSPC division and lineage determination.
    • Upon interaction with osteoblasts, HSPCs polarize during interphase, with centrosomes, the Golgi apparatus, and lysosomes positioned close to the site of contact. This reorganization of intracellular architecture plays a significant role in asymmetric division.
    • These results collectively contribute to a deeper understanding of how the hematopoietic niche and interactions with neighboring cells influence HSPC behavior and their commitment to distinct cell fates during division.

    Authors: We thank the reviewer for this quite positive evaluation of our work.

    Reviewer #3 (Evidence, reproducibility and clarity):

    Summary: In this study, the authors systematically work to identify the contribution of hematopoietic niche stromal cells in the control of HSPC asymmetric divisions. Using live cell imaging techniques, the authors show that heterotypic interaction with osteoblasts promotes asymmetric division of human HSPCs. The authors find that HSPCs polarize in interphase with centrosome, the Golgi apparatus and lysosomes positioned close to the site of contact. Moreover, during mitosis, HSPCs orient their spindle perpendicular to the plane of osteoblast contact and the subsequent division gives rise to siblings with unequal amounts of lysosomes and differentiation markers such as CD34. Taken together, the authors suggest that this asymmetric inheritance generates heterogeneity in progeny, which is likely to contribute to plasticity during the early steps of hematopoiesis.

    Major Comments:

    1. The study that uses polyacrylamide microwells as minimalist niches to evaluate the role of heterotypic interactions in the asymmetric division of HSPCs. Using this model system, the data clearly indicate that osteoblasts promote HSPC polarization when compared to fibronectin or skin fibroblasts. However, osteoblasts weren't compared to other HSPC stromal niche components. For example, is the same polarization observed with endothelial cells? This would help to determine whether this observation is specific to only osteoblasts in the niche or if there's a broader role for heterotypic interactions, which is the claim made in the discussion.

    Authors: We have now performed additional experiments using endothelial cells (see Figure 1 and 2, Page 3 and 4) and found that endothelial cells do also promote asymmetric division.

    This is primarily a descriptive study that doesn't attempt to address or even postulate the type of receptors potentially responsible for the polarization-driving heterotypic interactions. Perhaps inhibitors to candidate receptors could be incorporated or at least candidate receptors could be mentioned and described in the discussion as future directions.

    Authors: We have now performed additional experiments to address this point: We have analyzed the effect of the CXCR4 antagonist AMD3100 on HSPC division upon interaction with osteoblast and found that blocking these receptors reduce the asymmetry of daughter cells to the level of isolated HSPC (see Figure 3 E -G; Figure S3 H-J; page 4).

    __Minor Comments: __

    There is no reference to Figures 2C and D in the text. Authors : This oversight has been corrected.

    Page 3: “HSPC cultured on fibronectin exhibited spindle oriented parallel to the well bottom, (Figure 2A-C and S3C and D)”.

    Page 4: In contrast, both equal and unequal LysoBriteTM segregations could be observed (Figure 2D and S3E).

    The different circle colors in Figure 4C and D are a bit difficult to distinguish.

    Authors: The figure has been modified accordingly

    It's difficult to identify where the sites of cell contact are in Figure 2. Perhaps contact sites could be indicated in the images.

    Authors: The sites of contact have now been systematically indicated using dashed lines, in Figure 2 and 3.

    It's not clear how spindle orientation is being identified in Figure 3.

    Authors: We are aware of the fact that spindle orientation is not easy to visualize in the still images presented in Figure 3A and B. These images are extracted from the movies that were used to determine the cell division plane visually. This plane, following the long-axis rule, is perpendicular to the spindle orientation, which could therefore be determined. Movies 6 and 7 are shown to support these observations and illustrate how amenable these movies are to support spindle orientation analysis.

    Some grammatical errors need to be addressed throughout the manuscript.

    Authors: We thank the reviewer for drawing our attention on this point: the modified text has been carefully examined, and hopefully improved.

    Reviewer #3 (Significance):

    Significance: Overall, this is straightforward study that presents data illustrating the polarization driving capacity of osteoblast interactions with HPSC. The findings are important to the field, as little is known about asymmetric vs symmetric division in HSPCs. However, in its current form, the manuscript lacks clarity with respect to whether specifically osteoblasts promote HSPC polarization, or any stromal niche player has this capacity. Moreover, there is a lack of mechanistic data and/or discussion about how osteoblasts may be driving the observed polarization.

    Authors: We have now added data showing the similar role of endothelial cells and the implication of CXCR4 receptors.

    The manuscript is likely to have broader interest to not only HSPC researchers, but stem cell biologists and even engineers due to the technologies used.

    Authors: We thank the reviewer for his/her enthusiasm for our work

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #3

    Evidence, reproducibility and clarity

    Title: Heterotypic interaction promotes asymmetric division of human hematopoietic stem and progenitor cells

    Authors: Adrian Candelas, Benoit Vianay, Matthieu Gelin, Lionel Faivre, Jerome Larghero, Laurent Blanchoin, Manuel Théry, Stephane Brunet

    Summary: In this study, the authors systematically work to identify the contribution of hematopoietic niche stromal cells in the control of HSPC asymmetric divisions. Using live cell imaging techniques, the authors show that heterotypic interaction with osteoblasts promotes asymmetric division of human HSPCs. The authors find that HSPCs polarize in interphase with centrosome, the Golgi apparatus and lysosomes positioned close to the site of contact. Moreover, during mitosis, HSPCs orient their spindle perpendicular to the plane of osteoblast contact and the subsequent division gives rise to siblings with unequal amounts of lysosomes and differentiation markers such as CD34. Taken together, the authors suggest that this asymmetric inheritance generates heterogeneity in progeny, which is likely to contribute to plasticity during the early steps of hematopoiesis.

    Major Comments:

    1. The study that uses polyacrylamide microwells as minimalist niches to evaluate the role of heterotypic interactions in the asymmetric division of HSPCs. Using this model system, the data clearly indicate that osteoblasts promote HSPC polarization when compared to fibronectin or skin fibroblasts. However, osteoblasts weren't compared to other HSPC stromal niche components. For example, is the same polarization observed with endothelial cells? This would help to determine whether this observation is specific to only osteoblasts in the niche or if there's a broader role for heterotypic interactions, which is the claim made in the discussion.
    2. This is primarily a descriptive study that doesn't attempt to address or even postulate the type of receptors potentially responsible for the polarization-driving heterotypic interactions. Perhaps inhibitors to candidate receptors could be incorporated or at least candidate receptors could be mentioned and described in the discussion as future directions.

    Minor Comments:

    1. There is no reference to Figures 2C and D in the text.
    2. The different circle colors in Figure 4C and D are a bit difficult to distinguish.
    3. It's difficult to identify where the sites of cell contact are in Figure 2. Perhaps contact sites could be indicated in the images.
    4. It's not clear how spindle orientation is being identified in Figure 3.
    5. Some grammatical errors need to be addressed throughout the manuscript.

    Significance

    Overall, this is straightforward study that presents data illustrating the polarization driving capacity of osteoblast interactions with HPSC. The findings are important to the field, as little is known about asymmetric vs symmetric division in HSPCs. However, in its current form, the manuscript lacks clarity with respect to whether specifically osteoblasts promote HSPC polarization, or any stromal niche player has this capacity. Moreover, there is a lack of mechanistic data and/or discussion about how osteoblasts may be driving the observed polarization.

    The manuscript is likely to have broader interest to not only HSPC researchers, but stem cell biologists and even engineers due to the technologies used.

    I have experience with questions of HSPC regulation.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    The paper titled "Heterotypic Interaction Promotes Asymmetric Division of Human Hematopoietic Stem and Progenitor Cells" presents research on how interactions between Hematopoietic Stem and Progenitor Cells (HSPCs) and osteoblasts impact cell division symmetry. The use of polyacrylamide microwells to simulate the hematopoietic niche and study osteoblast interactions is a novel and valuable approach. The paper effectively explains the experimental design, making it easy for others to replicate and verify the results. The paper provides detailed insights into how interactions with osteoblasts influence HSPCs, particularly in terms of cell polarization, spindle orientation, and organelle distribution during cell division. The evidence is convincing, experiments are rigorously performed with adequate replicates and reproducibility.

    • The study acknowledges that not all HSPCs respond to heterotypic interaction, suggesting that individual variability in HSPC behavior plays a role. Future research could explore the factors that determine which HSPCs undergo asymmetric divisions. This raises questions about the heterogeneity in HSPC responses. The researcher starts culturing CD34+ which is a heterogeneous group with osteoblast. This heterogeneity by default will give different cells progeny which mimics the in vivo BM status. But, to minimize the invitro experiment variables examining a more pure HSC population such as isolated CD34+CD38-CD90+ cells would strengthen the findings? What was the Osteoblast/HSCs ratio used in the experimental setup ? Does this ratio affect the results?
    • The paper primarily focuses on lysosome inheritance and CD34 expression, leaving room to explore other lineage-specific markers and their correlation with asymmetric division. Do osteoblast/ HSCPs interactions have also an effect on mitochondrial inheritance ?
    • While the study hints at the clinical implications of its findings, it does not show the practical applications in detail. How the knowledge gained from these interactions could be translated into clinical practices or therapies is not explicitly discusssed. Bridging this gap is necessary for understanding the practical utility of these results.

    Significance

    • Investigating the process of HSPC asymmetric division is highly important to understand HSPC fate decisions and functions. The findings have significance in understanding how the microenvironment influences HSPC behavior. This is broadly significant for stem cell biology.
    • The paper's observation of uneven lysosome distribution and its potential impact on differentiation markers, including CD34 and CD33, contribute to our understanding of HSPC division and lineage determination.
    • Upon interaction with osteoblasts, HSPCs polarize during interphase, with centrosomes, the Golgi apparatus, and lysosomes positioned close to the site of contact. This reorganization of intracellular architecture plays a significant role in asymmetric division.
    • These results collectively contribute to a deeper understanding of how the hematopoietic niche and interactions with neighboring cells influence HSPC behavior and their commitment to distinct cell fates during division.
  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    In this manuscript, Candeleas and colleagues investigated asymmetric cell division of human hematopoietic stem and progenitor cells (HSPCs) upon heterotypic interactions with osteoblasts. They developed a new in vitro system to test this and found that upon interaction, HSPCs polarized in interphase with centrosome and the Golgi apparatus and lysosomes positioned close to the site of contact. In addition during mitosis, HSPCs were found to orient their spindle perpendicular to the plane of contact. This division gave rise to siblings with unequal amounts of lysosomes and CD34. In general, is a well-done work and it gives a few more details describing that when HSPCs divide asymmetrically it seems there is an association between centrosome and lysosome distribution.

    Significance

    However, all of these features (described above) have already been independently described even in human HSPCs thus this work does not represent a major advancement in this field (e.g. on the potential molecular mechanism by which heterotypic interactions with osteoblasts promote such behaviours). Overall, this stage is descriptive in nature.

    One major caveat of this work is that CD34+ HSPCs represent a very heterogeneous population. Although the underlying features described in this work could be similar between different cell populations, the frequency of these events (e.g. % magnupodium; polarization index, % of asymmetric inheritance, etc) is likely to be different between stem and progenitor cells. This may also explain the wide spread of their data points. Experiments should also be conducted with different cell populations, in particular with HSCs (either with the CD34+CD38-CD45RA-CD90+ or CD34+CD38-CD45RA-CD90+CD49f+ HSC enriched fraction or the highly CD34+CD38-CD45RA-EPCR+ HSCs).

    Results shown in Figure 2 were obtained with a very limited number of cells; also data obtained for Figure 4; this should be substantiated; in addition, data from Figure 2 adds little and should be combined with Figures 3 and 4 to make a single stronger message.

    It is/was unclear why the authors investigated the distribution of CD34 and CD33 and the major and important question remained to be answered: whether the symmetric cell divisions were/are differentiating or self-renewal in nature hence, the asymmetric cell divisions promoted by the osteoblasts may represent asymmetric self-renewal ones; this needs to be investigated further and potential molecular mechanisms for such promotion should be highlighted.

  5. Version published to 10.1101/2023.08.31.555706v2 on bioRxiv
  6. Version published to 10.1101/2023.08.31.555706v1 on bioRxiv