Alteration of long and short-term hematopoietic stem cell ratio causes myeloid-biased hematopoiesis

Reviewer #1 (Public Review):

Summary:

In this study, Nishi et al. claim that the ratio of long-term hematopoietic stem cell (LT-HSC) versus short-term HSC (ST-HSC) determines the lineage output of HSCs and reduced ratio of ST-HSC in aged mice causes myeloid-biased hematopoiesis. The authors used Hoxb5 reporter mice to isolate LT-HSC and ST-HSC and performed molecular analyses and transplantation assays to support their arguments. How the hematopoietic system becomes myeloid-biased upon aging is an important question with many implications in the disease context as well. However, their study is descriptive with remaining questions.

Weaknesses:

(1) The authors may need conceptual re-framing of their main argument because whether the ST-HSCs used in this study are functionally indeed short-term "HSCs" is questionable. The data presented in this study and their immunophenotypic definition of ST-HSCs (Lineage negative/Sca-1+/c-Kit+/Flk2-/CD34-/CD150+/Hoxb5-) suggest that authors may find hematopoietic stem cell-like lymphoid progenitors as previously shown for megakaryocyte lineage (Haas et al., Cell stem cell. 2015) or, as the authors briefly mentioned in the discussion, Hoxb5- HSCs could be lymphoid-biased HSCs. The authors disputed the idea that Hoxb5- HSCs as lymphoid-biased HSCs based on their previous 4 weeks post-transplantation data (Chen et al., 2016). However, they overlooked the possibility of myeloid reprogramming of lymphoid-biased population during regenerative conditions (Pietras et al., Cell stem cell., 2015). In other words, early post-transplant ST-HSCs (Hoxb5- HSCs) can be seen as lacking the phenotypic lymphoid-biased HSCs. Thinking of their ST-HSCs as hematopoietic stem cell-like lymphoid progenitors or lymphoid-biased HSCs makes more sense conceptually as well. ST-HSCs come from LT-HSCs and further differentiate into lineage-biased multipotent progenitor (MPP) populations including myeloid-biased MPP2 and MPP3. Based on the authors' claim, LT-HSCs (Hoxb5- HSCs) have no lineage bias even in aged mice. Then these LT-HSCs make ST-HSCs, which produce mostly memory T cells. These memory T cell-producing ST-HSCs then produce MPPs including myeloid-biased MPP2 and MPP3. This differentiation trajectory is hard to accept. If we think Hoxb5- HSCs (ST-HSCs by authors) as a sub-population of immunophenotypic HSCs with lymphoid lineage bias or hematopoietic stem cell-like lymphoid progenitors, the differentiation trajectory has no flaw.

(2) Authors' experimental designs have some caveats to support their claims. Authors claimed that aged LT-HSCs have no myeloid-biased clone expansion using transplantation assays. In these experiments, authors used 10 HSCs and young mice as recipients. Given the huge expansion of old HSC by number and known heterogeneity in immunophenotypically defined HSC populations, it is questionable how 10 out of so many old HSCs can faithfully represent the old HSC population. The Hoxb5+ old HSC primary and secondary recipient mice data (Figure 2C and D) support this concern. In addition, they only used young recipients. Considering the importance of the inflammatory aged niche in the myeloid-biased lineage output, transplanting young vs old LT-HSCs into aged mice will complete the whole picture.

(3) The authors' molecular data analyses need more rigor with unbiased approaches. They claimed that neither aged LT-HSCs nor aged ST-HSCs exhibited myeloid or lymphoid gene set enrichment but aged bulk HSCs, which are just a sum of LT-HSCs and ST-HSCs by their gating scheme (Figure 4A), showed the "tendency" of enrichment of myeloid-related genes based on the selected gene set (Figure 4D). Although the proportion of ST-HSCs is reduced in bulk HSCs upon aging, since ST-HSCs do not exhibit lymphoid gene set enrichment based on their data, it is hard to understand how aged bulk HSCs have more myeloid gene set enrichment compared to young bulk HSCs. This bulk HSC data rather suggests that there could be a trend toward certain lineage bias (although not significant) in aged LT-HSCs or ST-HSCs. The authors need to verify the molecular lineage priming of LT-HSCs and ST-HSCs using another comprehensive dataset.

(4) Some data are too weak to fully support their claims. The authors claimed that age-associated extramedullary changes are the main driver of myeloid-biased hematopoiesis based on no major differences in progenitor populations upon transplantation of 10 young HSCs into young or old recipient mice (Figure 7F) and relatively low donor-derived cells in thymus and spleen in aged recipient mice (Figure 7G-J). However, they used selected mice to calculate the progenitor populations in recipient mice (8 out of 17 from young recipients denoted by * and 8 out of 10 from aged recipients denoted by * in Figure 7C). In addition, they calculated the progenitor populations as frequency in c-kit positive cells. Given that they transplanted 10 LT-HSCs into "sub-lethally" irradiated mice and 8.7 Gy irradiation can have different effects on bone marrow clearance in young vs old mice, it is not clear whether this data is reliable enough to support their claims. The same concern applies to the data Figure 7G-J. Authors need to provide alternative data to support their claims.

Reviewer #2 (Public Review):

Summary:

Nishi et al, investigate the well-known and previously described phenomenon of age-associated myeloid-biased hematopoiesis. Using a previously established HoxB5mCherry mouse model, they used HoxB5+ and HoxB5- HSCs to discriminate cells with long-term (LT-HSCs) and short-term (ST-HSCs) reconstitution potential and compared these populations to immunophenotypically defined 'bulk HSCs' that consists of a mixture of LT-HSC and ST-HSCs. They then isolated these HSC populations from young and aged mice to test their function and myeloid bias in non-competitive and competitive transplants into young and aged recipients. Based on quantification of hematopoietic cell frequencies in the bone marrow, peripheral blood, and in some experiments the spleen and thymus, the authors argue against the currently held belief that myeloid-biased HSCs expand with age.

While aspects of their work are fascinating and might have merit, several issues weaken the overall strength of the arguments and interpretation. Multiple experiments were done with a very low number of recipient mice, showed very large standard deviations, and had no statistically detectable difference between experimental groups. While the authors conclude that these experimental groups are not different, the displayed results seem too variable to conclude anything with certainty. The sensitivity of the performed experiments (e.g. Figure 3; Figure 6C, D) is too low to detect even reasonably strong differences between experimental groups and is thus inadequate to support the author's claims. This weakness of the study is not acknowledged in the text and is also not discussed. To support their conclusions the authors need to provide higher n-numbers and provide a detailed power analysis of the transplants in the methods section.

As the authors attempt to challenge the current model of the age-associated expansion of myeloid-biased HSCs (which has been observed and reproduced by many different groups), ideally additional strong evidence in the form of single-cell transplants is provided.

It is also unclear why the authors believe that the observed reduction of ST-HSCs relative to LT-HSCs explains the myeloid-biased phenotype observed in the peripheral blood. This point seems counterintuitive and requires further explanation.

Based on my understanding of the presented data, the authors argue that myeloid-biased HSCs do not exist, as
a) they detect no difference between young/aged HSCs after transplant (mind low n-numbers and large std!); b) myeloid progenitors downstream of HSCs only show minor or no changes in frequency and c) aged LT-HSCs do not outperform young LT-HSC in myeloid output LT-HScs in competitive transplants (mind low n-numbers and large std!).

However, given the low n-numbers and high variance of the results, the argument seems weak and the presented data does not support the claims sufficiently. That the number of downstream progenitors does not change could be explained by other mechanisms, for instance, the frequently reported differentiation short-cuts of HSCs and/or changes in the microenvironment.

Strengths:

The authors present an interesting observation and offer an alternative explanation of the origins of aged-associated myeloid-biased hematopoiesis. Their data regarding the role of the microenvironment in the spleen and thymus appears to be convincing.

Weaknesses:

"Then, we found that the myeloid lineage proportions from young and aged LT-HSCs were nearly comparable during the observation period after transplantation (Figure 3, B and C)."
Given the large standard deviation and low n-numbers, the power of the analysis to detect differences between experimental groups is very low. Experimental groups with too large standard deviations (as displayed here) are difficult to interpret and might be inconclusive. The absence of clearly detectable differences between young and aged transplanted HSCs could thus simply be a false-negative result. The shown experimental results hence do not provide strong evidence for the author's interpretation of the data. The authors should add additional transplants and include a detailed power analysis to be able to detect differences between experimental groups with reasonable sensitivity.

Line 293: "Based on these findings, we concluded that myeloid-biased hematopoiesis observed following transplantation of aged HSCs was caused by a relative decrease in ST-HSC in the bulk-HSC compartment in aged mice rather than the selective expansion of myeloid-biased HSC clones."
Couldn't that also be explained by an increase in myeloid-biased HSCs, as repeatedly reported and seen in the expansion of CD150+ HSCs? It is not intuitively clear why a reduction of ST-HSCs clones would lead to a myeloid bias. The author should try to explain more clearly where they believe the increased number of myeloid cells comes from. What is the source of myeloid cells if the authors believe they are not derived from the expanded population of myeloid-biased HSCs?

Reviewer #3 (Public Review):

In this manuscript, Nishi et al. propose a new model to explain the previously reported myeloid-biased hematopoiesis associated with aging. Traditionally, this phenotype has been explained by the expansion of myeloid-biased hematopoietic stem cell (HSC) clones during aging. Here, the authors question this idea and show how their Hoxb5 reporter model can discriminate long-term (LT) and short-term (ST) HSC and characterized their lineage output after transplant. From these analyses, the authors conclude that changes during aging in the LT/ST HSC proportion explain the myeloid bias observed.

Although the topic is appropriate and the new model provides a new way to think about lineage-biased output observed in multiple hematopoietic contexts, some of the experimental design choices, as well as some of the conclusions drawn from the results could be substantially improved. Also, they do not propose any potential mechanism to explain this process, which reduces the potential impact and novelty of the study. Specific concerns are outlined below.

Major

(1) As a general comment, there are experimental details that are either missing or not clear. The main one is related to transplantation assays. What is the irradiation dose? The Methods sections indicates "recipient mice were lethally irradiated with single doses of 8.7 or 9.1 Gy". The only experimental schematic indicating the irradiation dose is Figure 7A, which uses 8.7 Gy. Also, although there is not a "standard", 11 Gy split in two doses is typically considered lethal irradiation, while 9.5 Gy is considered sublethal. Is there any reason for these lower doses? Same question for giving a single dose and for performing irradiation a day before transplant.

(2) The manuscript would benefit from the inclusion of references to recent studies discussing hematopoietic biases and differentiation dynamics at a single-cell level (e.g., Yamamoto et. al 2018; Rodriguez-Fraticelli et al., 2020). Also, when discussing the discrepancy between studies claiming different biases within the HSC pool, the authors mentioned that Montecino-Rodriguez et al. 2019 showed preserved lymphoid potential with age. It would be good to acknowledge that this study used busulfan as the conditioning method instead of irradiation.

(3) When representing the contribution to PB from transplanted cells, the authors show the % of each lineage within the donor-derived cells (Figures 3B-C, 5B, 6B-D, 7C-E, and S3 B-C). To have a better picture of total donor contribution, total PB and BM chimerism should be included for each transplantation assay. Also, for Figures 2C-D and Figures S2A-B, do the graphs represent 100% of the PB cells? Are there any radioresistant cells?

(4) For BM progenitor frequencies, the authors present the data as the frequency of cKit+ cells. This normalization might be misleading as changes in the proportion of cKit+ between the different experimental conditions could mask differences in these BM subpopulations. Representing this data as the frequency of BM single cells or as absolute numbers (e.g., per femur) would be valuable.

(5) Regarding Figure 1B, the authors argue that if myeloid-biased HSC clones increase with age, they should see increased frequency of all components of the myeloid differentiation pathway (CMP, GMP, MEP). This would imply that their results (no changes or reduction in these myeloid subpopulations) suggest the absence of myeloid-biased HSC clones expansion with age. This reviewer believes that differentiation dynamics within the hematopoietic hierarchy can be more complex than a cascade of sequential and compartmentalized events (e.g., accelerated differentiation at the CMP level could cause exhaustion of this compartment and explain its reduction with age and why GMP and MEP are unchanged) and these conclusions should be considered more carefully.

(6) Within the few recipients showing good donor engraftment in Figure 2C, there is a big proportion of T cells that are "amplified" upon secondary transplantation (Figure 2D). Is this expected?

(7) Do the authors have any explanation for the high level of variability within the recipients of Hoxb5+ cells in Figure 2C?

(8) Can the results from Figure 2E be interpreted as Hoxb5+ cells having a myeloid bias? (differences are more obvious/significant in neutrophils and monocytes).

(9) Is Figure 2G considering all primary recipients or only the ones that were used for secondary transplants? The second option would be a fairer comparison.

(10) When discussing the transcriptional profile of young and aged HSCs, the authors claim that genes linked to myeloid differentiation remain unchanged in the LT-HSC fraction while there are significant changes in the ST-HSCs. However, 2 out of the 4 genes shown in Figure S4B show ratios higher than 1 in LT-HSCs.

(11) When determining the lymphoid bias in ST-HSCs, the authors focus on the T-cell subtype, not considering any other any other lymphoid population. Could the authors explain this?

(12) Based on the reduced frequency of donor cells in the spleen and thymus, the authors conclude "the process of lymphoid lineage differentiation was impaired in the spleens and thymi of aged mice compared to young mice". An alternative explanation could be that differentiated cells do not successfully migrate from the bone marrow to these secondary lymphoid organs. Please consider this possibility when discussing the data.