Clonal analysis of murine HSC self-renewal and differentiation in native hematopoiesis

Reviewer #1 (Public review):

Summary:

You, Zhang et al. comprehensively characterize the long-term fates of mouse HSCs in the unperturbed setting using transposon-based lineage tracing for up to 2 years post-labeling. Their analyses reveal a complex heterogeneity of long-term fates, dominated by two behaviors: i) long-lived differentiation-biased clones, and ii) self-renewal & platelet-biased clones. They further identify two categories of multipotent progenitor clones, with one group showing a markedly reduced differentiation activity.

Strengths:

You et al. present a very comprehensive and high-resolution characterization of mouse hematopoietic clonal dynamics, with robust replicates, and technical prowess. The manuscript is beautifully written, with in-depth and clear explanations of the logic behind experimental design choices, and very well-thought-out interpretations of results.

Some of the results integrate well with past observations in the field, whereas many of them are quite unique and novel.

This will surely be a highly impactful study in the field of hematopoiesis and stem cell biology.

Weaknesses:

The authors trace hematopoiesis in situ, in a fully unbiased way for almost 2-years. They compare this time course with the last few years of Cre-LoxP-based tracing studies and they make an assumption that most hematopoiesis will be derived from some type of HSC at that point in time. They then use this assumption to support that what is being measured in their model are the long-term fates of HSCs (or at least cells that were HSC at the point of labeling). While this is a generally valid assumption, the short-lived nature of certain populations (myeloid cells, megakaryocytes) means that these cells are being produced in the context of a relatively aged environment by the time of sampling, which might change the properties of the system. In other words, the "steady-state" is always changing. It is important to read and interpret this manuscript with this in consideration.

Reviewer #2 (Public review):

Summary:

The work from You et al. elucidates the clonal contribution of ageing stem and progenitor cells to both native and perturbed hematopoiesis. The authors use a previously published in vivo lineage tracing system (Patel et al., 2022) that relies on the random integration of a transposon element in the mouse genome. They barcode all mouse cells and then look at lineage relationships between HSPC and mature populations after ~90 weeks.

Strengths:

This work offers very interesting insights into the clonal behaviour of HSPC in the native and perturbed setting during ageing. Experiments are well-planned and well-executed. Understanding the clonal output of HSPCs in aged mice in a native setting, after 5-FU treatment, and upon transplantation are important findings for the field.

Weaknesses:

We found appraising the graphs, interpreting the findings, and understanding those findings in the main text very difficult to follow. While we have made some suggestions below, we encourage the authors to think carefully about what the core messages are, and how best to visualise those, both in terms of data viz and in a schematic to summarise the key findings, and to use plain language in the text.

Author response:

We genuinely appreciate the reviewers' interest and recognition of our work. The comments and suggestions on the results presentation and interpretation are well taken. We plan to revise the manuscript based on the reviewers' recommendations in the following aspects.

(1) We fully agree with the reviewer that the aged environment indeed would affect the myeloid and megakaryocyte differentiation behaviors of HSC. As a result, the clonal behaviors of HSCs presented in the current manuscript could be different from how HSCs differentiate in young mice. This point will be discussed in the revised manuscript.

(2) We agree with the reviewer that the manuscript was not as easy to follow as many other papers in experimental hematology, primarily because the analyses presented in the current manuscript were not frequently used in previous studies. To address this, we will try to revise the manuscript using plain language to describe the results and conclusions. We will also provide graphical summary schematics where appropriate to present the findings better. We will further discuss our results in the context of previous findings to better illustrate the novelty of the current work.

(3) We will provide more technical details of our analysis in the revised manuscript for readers to better understand how results are obtained and data analyses are performed in the current manuscript.