Dissecting infant leukemia developmental origins with a hemogenic gastruloid model

Reviewer #1 (Public review):

Summary

The authors describe a method for gastruloid formation using mouse embryonic stem cells (mESCs) to study YS and AGM-like hematopoietic differentiation. They characterise the gastruloids during nine days of differentiation using a number of techniques including flow cytometry and single-cell RNA sequencing. They compare their findings to a published data set derived from E10-11.5 mouse AGM. At d9, gastruloids were transplanted under the adrenal gland capsule of immunocompromised mice to look for the development of cells capable of engrafting the mouse bone marrow. The authors then applied the gastruloid protocol to study overexpression of Mnx1 which causes infant AML in humans.

In the introduction, the authors define their interpretation of the different waves of hematopoiesis that occur during development. 'The subsequent wave, known as definitive, produces: first, oligopotent erythro-myeloid progenitors (EMPs) in the YS (E8-E8.5); and later myelo-lymphoid progenitors (MLPs - E9.5-E10), multipotent progenitors (MPPs - E10-E11.5), and hematopoietic stem cells (HSCs - E10.5-E11.5), in the aorta-gonad-mesonephros (AGM) region of the embryo proper.' Herein they designate the yolk sac-derived wave of EMP hematopoiesis as definitive, according to convention, although paradoxically it does not develop from intra-embryonic mesoderm or give rise to HSCs.

General comments

The authors make the following claims in the paper:

(1) The development of a protocol for hemogenic gastruloids (hGx) that recapitulates YS and AGM-like waves of blood from HE.

(2) The protocol recapitulates both YS and EMP-MPP embryonic blood development 'with spatial and temporal accuracy'.

(3) The protocol generates HSC precursors capable of short-term engraftment in an adrenal niche.

(4) Overexpression of MNX1 in hGx transforms YS EMP to 'recapitulate patient transcriptional signatures'.

(5) hGx is a model to study normal and leukaemic embryonic hematopoiesis.

There are major concerns with the manuscript. The statements and claims made by the authors are not supported by the data presented, data is overinterpreted, and the conclusions cannot be justified. Furthermore, the data is presented in a way that makes it difficult for the reader to follow the narrative, causing confusion. The authors have not discussed how their hGx compares to the previously published mouse embryoid body protocols used to model early development and hematopoiesis.

Specific points

(1) It is claimed that HGxs capture cellularity and topography of developmental blood formation. The hGx protocol described in the manuscript is a modification of a previously published gastruloid protocol (Rossi et al 2022). The rationale for the protocol modifications is not fully explained or justified. There is a lack of novelty in the presented protocol as the only modifications appear to be the inclusion of Activin A and an extension of the differentiation period from 7 to 9 days of culture. No direct comparison has been made between the two versions of gastruloid differentiation to justify the changes.

The inclusion of Activin A at high concentration at the beginning of differentiation would be expected to pattern endoderm rather than mesoderm. BMP signaling is required to induce Flk1+ mesoderm, even in the presence of Wnt. FACS analysis of the hGx during differentiation is needed to demonstrate the co-expression of Flk1-GFP and lineage markers such as CD34 to indicate patterning of endothelium from Flk1+ mesoderm. The FACS plots in Figure 1 show c-Kit expression but very little VE-cadherin which suggests that CD34 is not induced. Early endoderm expresses c-Kit, CXCR4, and Epcam but not CD34 which could account for the lack of vascular structures within the hGx as shown in Figure 1E.

(2) The protocol has been incompletely characterised, and the authors have not shown how they can distinguish between either wave of Yolk Sac (YS) hematopoiesis (primitive erythroid/macrophage and erythro-myeloid EMP) or between YS and intraembryonic Aorta-Gonad-Mesonephros (AGM) hematopoiesis. No evidence of germ layer specification has been presented to confirm gastruloid formation, organisation, and functional ability to mimic early development. Furthermore, differentiation of YS primitive and YS EMP stages of development in vitro should result in the efficient generation of CD34+ endothelial and hematopoietic cells. There is no flow cytometry analysis showing the kinetics of CD34 cell generation during differentiation. Benchmarking the hGx against developing mouse YS and embryo data sets would be an important verification.

Single-cell RNA sequencing was used to compare hGx with mouse AGM. The authors incorrectly conclude that ' ..specification of endothelial and HE cells in hGx follows with time-dependent developmental progression into putative AGM-like HE..' And, '...HE-projected hGx cells.......expressed Gata2 but not Runx1, Myb, or Gfi1b..' Hemogenic endothelium is defined by the expression of Runx1 and Gfli1b is downstream of Runx1.

(3) The hGx protocol 'generates hematopoietic SC precursors capable of short-term engraftment' is not supported by the data presented. Short-term engraftment would be confirmed by flow cytometric detection of hematopoietic cells within the recipient bone marrow, spleen, thymus, and peripheral blood that expressed the BFP transgene. This analysis was not provided. PCR detection of transcripts, following an unspecified number of amplification cycles, as shown in Figure 3G (incorrectly referred to as Figure 3F in the legend) is not acceptable evidence for engraftment. Transplanted hGx formed teratoma-like structures, with hematopoietic cells present at the site of transplant only analysed histologically. Indeed, the quality of the images provided does not provide convincing validation that donor-derived hematopoietic cells were present in the grafts.

There is no justification for the authors' conclusion that '... the data suggest that 216h hGx generate AGM-like pre-HSC capable of at least short-term multilineage engraftment upon maturation...'. Indeed, this statement is in conflict with previous studies demonstrating that pre-HSCs in the dorsal aorta of the mouse embryo are immature and actually incapable of engraftment.

The statement '...low-level production of engrafting cells recapitulates their rarity in vivo, in agreement with the embryo-like qualities of the gastruloid system....' is incorrect. Firstly, no evidence has been provided to show the hGx has formed a dorsal aorta facsimile capable of generating cells with engrafting capacity. Secondly, although engrafting cells are rare in the AGM, approximately one per embryo, they are capable of robust and extensive engraftment upon transplantation.

(4) Expression MNX1 transcript and protein in hematopoietic cells in MNX1 rearranged acute myeloid leukaemia (AML) is one cause of AML in infants. In the hGX model of this disease, Mnx1 is overexpressed in the mESCs that are used to form gastruloids. Mnx1 overexpression seems to confer an overall growth advantage on the hGx and increase the serial replating capacity of the small number of hematopoietic cells that are generated. The inefficiency with which the hGx model generates hematopoietic cells makes it difficult to model this disease. The poor quality of the cytospin images prevents accurate identification of cells. The statement that the kit-expressing cells represent leukemic blast cells is not sufficiently validated to support this conclusion. What other stem cell genes are expressed? Surface kit expression also marks mast cells, frequently seen in clonogenic assays of blood cells. Flow cytometric and gene expression analyses using known markers would be required.

(5) In human infant MNX1 AML, the mutation is thought to arise at the fetal liver stage of development. There is no evidence that this developmental stage is mimicked in the hGx model.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors develop an exciting new hemogenic gastruloid (hGX) system, which they claim reproduces the sequential generation of various blood cell types. The key advantage of this cellular system would be its potential to more accurately recapitulate the spatiotemporal emergence of hematopoietic progenitors within their physiological niche compared to other available in vitro systems. The authors present a large set of data and also validate their new system in the context of investigating infant leukemia.

Strengths:

The development of this new in vitro system for generating hematopoietic cells is innovative and addresses a significant drawback of current in vitro models. The authors present a substantial dataset to characterize this system, and they also validate its application in the context of investigating infant leukemia.

Weaknesses:

The thorough characterization and full demonstration that the cells produced truly represent distinct waves of hematopoietic progenitors are incomplete. The data presented to support the generation of late yolk sac (YS) progenitors, such as lymphoid cells, and aortic-gonad-mesonephros (AGM)-like progenitors, including pre-hematopoietic stem cells (pre-HSCs), by this system are not entirely convincing. Given that this is likely the manuscript's most crucial claim, it warrants further scrutiny and direct experimental validation. Ideally, the identity of these progenitors should be further demonstrated by directly assessing their ability to differentiate into lymphoid cells or fully functional HSCs. Instead, the authors primarily rely on scRNA-seq data and a very limited set of markers (e.g., Ikzf1 and Mllt3) to infer the identity and functionality of these cells. Many of these markers are shared among various types of blood progenitors, and only a well-defined combination of markers could offer some assurance of the lymphoid and pre-HSC nature of these cells, although this would still be limited in the absence of functional assays.

The identification of a pre-HSC-like CD45⁺CD41⁻/lo c-Kit⁺VE-Cadherin⁺ cell population is presented as evidence supporting the generation of pre-HSCs by this system, but this claim is questionable. This FACS profile may also be present in progenitors generated in the yolk sac such as early erythro-myeloid progenitors (EMPs). It is only within the AGM context, and in conjunction with further functional assays demonstrating the ability of these cells to differentiate into HSCs and contribute to long-term repopulation, that this profile could be strongly associated with pre-HSCs. In the absence of such data, the cells exhibiting this profile in the current system cannot be conclusively identified as true pre-HSCs.

The engraftment data presented are also not fully convincing, as the observed repopulation is very limited and evaluated only at 4 weeks post-transplantation. The cells detected after 4 weeks could represent the progeny of EMPs that have been shown to provide transient repopulation rather than true HSCs.

Reviewer #3 (Public review):

In this study, the authors employ a mouse ES-derived "hemogenic gastruloid" model which they generated and which they claim to be able to deconvolute YS and AGM stages of blood production in vitro. This work could represent a valuable resource for the field. However, in general, I find the conclusions in this manuscript poorly supported by the data presented. Importantly, it isn't clear what exactly are the "YS" and the "AGM"-like stages identified in the culture and where is the data that backs up this claim. In my opinion, the data in this manuscript lack convincing evidence that can enable us to identify what kind of hematopoietic progenitor cells are generated in this system. Therefore, the statement that "our study has positioned the MNX1-OE target cell within the YS-EMP stage (line 540)" is not supported by the evidence presented in this study. Overall, the system seems to be very preliminary and requires further optimization before those claims can be made.

Specific comments below:

(1) The flow cytometric analysis of gastruloids presented in Figure 1 C-D is puzzling. There is a large % of c-Kit+ cells generated, but few VE-Cad+ Kit+ double positive cells. Similarly, there are many CD41+ cells, but very few CD45+ cells, which one would expect to appear toward the end of the differentiation process if blood cells are actually generated. It would be useful to present this analysis as consecutive gating (i.e. evaluating CD41 and CD45 within VE-Cad+ Kit+ cells, especially if the authors think that the presence of VE-Cad+ Kit+ cells is suggestive of EHT). The quantification presented in D is misleading as the scale of each graph is different.

(2) The imaging presented in Figure 1E is very unconvincing. C-Kit and CD45 signals appear as speckles and not as membrane/cell surfaces as they should. This experiment should be repeated and nuclear stain (i.e. DAPI) should be included.

(3) Overall, I am not convinced that hematopoietic cells are consistently generated in these organoids. The authors should sort hematopoietic cells and perform May-Grunwald Giemsa stainings as they did in Figure 6 to confirm the nature of the blood cells generated.

(4) The scRNAseq in Figure 2 is very difficult to interpret. Specific points related to this:
- Cluster annotation in Figure 2a is missing and should be included.
- Why do the heatmaps show the expression of genes within sorted cells? Couldn't the authors show expression within clusters of hematopoietic cells as identified transcriptionally (which ones are they? See previous point)? Gene names are illegible.
- I see no expression of Hlf or Myb in CD45+ cells (Figure 2G). Hlf is not expressed by any of the populations examined (panels E, F, G). This suggests no MPP or pre-HSC are generated in the culture, contrary to what is stated in lines 242-245. (PMID 31076455 and 34589491).
Later on, it is again stated that "hGx cells... lacked detection of HSC genes like Hlf, Gfi1, or Hoxa9" (lines 281-283). To me, this is proof of the absence of AGM-like hematopoiesis generated in those gastruloids.

(5) Mapping of scRNA-Seq data onto the dataset by Thambyrajah et al. is not proof of the generation of AGM HE. The dataset they are mapping to only contains AGM cells, therefore cells do not have the option to map onto something that is not AGM. The authors should try mapping to other publicly available datasets also including YS cells.

(6) Conclusions in Figure 3, named "hGx specify cells with preHSC characteristics" are not supported by the data presented here. Again, I am not convinced that hematopoietic cells can be efficiently generated in this system, and certainly not HSCs or pre-HSCs.
- FACS analysis in 3A is again very unconvincing. I do not think the population identified as c-Kit+ CD144+ is real. Also, why not try gating the other way around, as commonly done (e.g. VE-Cad+ Kit+ and then CD41/CD45)?
- The authors must have tried really hard, but the lack of short- or long-engraftment in a number of immunodeficient mouse models (lines 305-313) really suggests that no blood progenitors are generated in their system. I am not familiar with the adrenal gland transplant system, but it seems like a very non-physiological system for trying to assess the maturation of putative pre-HSCs. The data supporting the engraftment of these mice, essentially seen only by PCR and in some cases with a very low threshold for detection, are very weak, and again unconvincing. It is stated that "BFP engraftment of the Spl and BM by flow cytometry was very low level albeit consistently above control (Fig. S4E)" (lines 337-338). I do not think that two dots in a dot plot can be presented as evidence of engraftment.

(7) Given the above, I find that the foundations needed for extracting meaningful data from the system when perturbed are very shaky at best. Nevertheless, the authors proceed to overexpress MNX1 by LV transduction, a system previously shown to transform fetal liver cells, mimicking the effect of the t(7;12) AML-associated translocation. Comments on this section:
- The increase in the size of the organoid when MNX1 is expressed is a very unspecific finding and not necessarily an indication of any hematopoietic effect of MNX1 OE.
- The mild increase of cKit+ cells (Figure 4E) at the 144hr timepoint and the lack of any changes in CD41+ or CD45+ cells suggests that the increase in Kit+ cells % is not due to any hematopoietic effect of MNX1 OE. No hematopoietic GO categories are seen in RNA seq analysis, which supports this interpretation. Could it be that just endothelial cells are being generated?

(8) There seems to be a relatively convincing increase in replating potential upon MNX1-OE, but this experiment has been poorly characterized. What type of colonies are generated? What exactly is the "proportion of colony forming cells" in Figures 5B-D? The colony increase is accompanied by an increase in Kit+ cells; however, the flow cytometry analysis has not been quantified.

(9) Do hGx cells engraft upon MNX1-OE? This experiment, which appears not to have been performed, is essential to conclude that leukemic transformation has occurred.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary

The authors describe a method for gastruloid formation using mouse embryonic stem cells (mESCs) to study YS and AGM-like hematopoietic differentiation. They characterise the gastruloids during nine days of differentiation using a number of techniques including flow cytometry and single-cell RNA sequencing. They compare their findings to a published data set derived from E10-11.5 mouse AGM. At d9, gastruloids were transplanted under the adrenal gland capsule of immunocompromised mice to look for the development of cells capable of engrafting the mouse bone marrow. The authors then applied the gastruloid protocol to study overexpression of Mnx1 which causes infant AML in humans.

In the introduction, the authors define their interpretation of the different waves of hematopoiesis that occur during development. 'The subsequent wave, known as definitive, produces: first, oligopotent erythro-myeloid progenitors (EMPs) in the YS (E8-E8.5); and later myelo-lymphoid progenitors (MLPs - E9.5-E10), multipotent progenitors (MPPs - E10-E11.5), and hematopoietic stem cells (HSCs - E10.5-E11.5), in the aorta-gonadmesonephros (AGM) region of the embryo proper.' Herein they designate the yolk sac-derived wave of EMP hematopoiesis as definitive, according to convention, although paradoxically it does not develop from intraembryonic mesoderm or give rise to HSCs.

The apparent perplexity of the Reviewer with our definition of primitive and definitive waves is somewhat surprising, as it is widely used in the field (e.g. PMID: 18204427; PMID: 28299650; PMID: 33681211). Definitive haematopoiesis, encompassing EMP, MLP, MPP and HSC, highlights their origin from haemogenic hendothelium, generation of mature cells with adult characteristics from progenitors with multilineage potential and direct and indirect developmental contributions to the intra-embryonic and time-restricted generation of HSCs.

General comments

The authors make the following claims in the paper:

(1) The development of a protocol for hemogenic gastruloids (hGx) that recapitulates YS and AGM-like waves of blood from HE.

(2) The protocol recapitulates both YS and EMP-MPP embryonic blood development 'with spatial and temporal accuracy'.

(3) The protocol generates HSC precursors capable of short-term engraftment in an adrenal niche.

(4) Overexpression of MNX1 in hGx transforms YS EMP to 'recapitulate patient transcriptional signatures'.

(5) hGx is a model to study normal and leukaemic embryonic hematopoiesis.

There are major concerns with the manuscript. The statements and claims made by the authors are not supported by the data presented, data is overinterpreted, and the conclusions cannot be justified. Furthermore, the data is presented in a way that makes it difficult for the reader to follow the narrative, causing confusion. The authors have not discussed how their hGx compares to the previously published mouse embryoid body protocols used to model early development and hematopoiesis. the data is presented in a way that makes it difficult for the reader to follow the narrative, causing confusion. The authors have not discussed how their hGx compares to the previously published mouse embryoid body protocols used to model early development and hematopoiesis.

Specific points

(1) It is claimed that HGxs capture cellularity and topography of developmental blood formation. The hGx protocol described in the manuscript is a modification of a previously published gastruloid protocol (Rossi et al 2022). The rationale for the protocol modifications is not fully explained or justified. There is a lack of novelty in the presented protocol as the only modifications appear to be the inclusion of Activin A and an extension of the differentiation period from 7 to 9 days of culture. No direct comparison has been made between the two versions of gastruloid differentiation to justify the changes.

The Reviewer paradoxically claims that the protocol is not novel and that it differs from a previous publication in at least 2 ways – the patterning pulse and the length of the protocol. Of these, the patterning pulse is key. As documented in Fig. S1, we cannot obtain Flk1-GFP expression in the absence of Activin A. Expression of Flk1 is a fundamental step in haemato-endothelial specification and, accordingly, we do not see CD41 or CD45+ cells in the absence of Activin A. Also, in our hands, there is a clear time-dependent progression of marker expression, with sequential acquisition of CD41 and CD45, with the latter not detectable until 192h (Fig. 1C-D), another key difference relative to the Rossi et al (2022) protocol. The 192h-timepoint, we argue in the manuscript, and present further evidence for in this rebuttal, corresponds to the onset of AGM-like haematopoiesis. We have empirically extended the protocol to maximise the CD45+ cell output (Fig. S1B-D).

The inclusion of Activin A at high concentration at the beginning of differentiation would be expected to pattern endoderm rather than mesoderm. BMP signaling is required to induce Flk1+ mesoderm, even in the presence of Wnt.

Again, we call the Reviewer’s attention to Fig. S1 which clearly shows that Activin A (with no BMP added) is required for induction of Flk1 expression, in the presence of Wnt. Activin A in combination with Wnt, is used in other protocols of haemato-endothelial differentiation from pluripotent cells, with no BMP added in the same step of patterning and differentiation (PMID: 39227582; PMID: 39223325). In the latter protocol, we also call the Reviewer’s attention to the fact that a higher concentration of Activin A precludes the need for BMP4 addition. Finally, one of us has recently reported that Activin A, on its own, will induce FLK1, as well as other anterior mesodermal progenitors (https://www.biorxiv.org/content/10.1101/2025.01.11.632562v1). In addressing the Reviewer’s concerns with the dose of Activin A used, we titrated its concentration against activation of Flk1, confirming optimal Flk1-GFP expression at the 100ng/ml dose used in the manuscript.

Author response image 1.

Dose-dependent requirement of Activin A for induction of Flk1 expression in haemogenic gastruloids. Composite GFP and brightfield live imaging of Flk1-GFP haemogenic gastruloids at 96h. Images were acquired using a Cytation5 instrument (Thermo). Images are representative of 12 gastruloids per condition.

FACS analysis of the hGx during differentiation is needed to demonstrate the co-expression of Flk1-GFP and lineage markers such as CD34 to indicate patterning of endothelium from Flk1+ mesoderm. The FACS plots in

Fig. 1 show c-Kit expression but very little VE-cadherin which suggests that CD34 is not induced. Early endoderm expresses c-Kit, CXCR4, and Epcam, but not CD34 which could account for the lack of vascular structures within the hGx as shown in Fig. 1E.

We were surprised by the Reviewer’s comment that there are no endothelial structures in our gastruloids. The presence of a Flk1-GFP+ network is visible in the GFP images in Fig.1B, from 144h onwards, also shown in Author response image 2A. In addition, our single-cell RNA-seq data, included in the manuscript, confirms the presence of endothelial cells with a developing endothelial, including arterial, programme. This can be seen in Fig. 2B, F of the manuscript and is represented in Author response image 2B. In contrast with the Reviewer’s claims that no endothelial cells are formed, the data show that Kdr (Flk1)+ cells co-express Cdh5/VE-Cadherin and indeed Cd34, attesting to the presence of an endothelial programme. Arterial markers Efnb2, Flt1, and Dll4 are present. A full-blown programme, which also includes haemogenic markers including Sox17, Esam, Cd44 and Mecom is clear at early (144h) and, particularly at late (192h) timepoints in cells sorted on detection of surface c-Kit (Author response image 2B). Further to the data shown in B, already present in the manuscript, we also document co-expression of Flk1-GFP and CD34 by flow cytometry (Author response image 2C).

Author response image 2.

Haemogenic gastruloids have a branched vascular network. A. Whole-mount confocal imaging of 144h-haemogenic gastruloids. B. Differentiation of an arterial endothelial programme in haemogenic gastruloids; singlecell RNA-seq data of differentiating haemogenic gastruloids, sorted on cell surface expression of c-Kit at 144 and 192h; gene expression colour scale from yellow (low) to orange (high); grey = no detectable expression. C. Flow cytometry plots of 216h-haemogenic gastruloids showing detection of haemato-endothelial marker CD34.

(2) The protocol has been incompletely characterised, and the authors have not shown how they can distinguish between either wave of Yolk Sac (YS) hematopoiesis (primitive erythroid/macrophage and erythro-myeloid EMP) or between YS and intraembryonic Aorta-Gonad-Mesonephros (AGM) hematopoiesis. No evidence of germ layer specification has been presented to confirm gastruloid formation, organisation, and functional ability to mimic early development. Furthermore, differentiation of YS primitive and YS EMP stages of development in vitro should result in the efficient generation of CD34+ endothelial and hematopoietic cells. There is no flow cytometry analysis showing the kinetics of CD34 cell generation during differentiation. Benchmarking the hGx against developing mouse YS and embryo data sets would be an important verification.

The Reviewer is correct that we have not provided detailed characterisation of the different germ layers, as this was not the focus of the study. In that context, we were surprised by the earlier comment assuming co-expression of c-kit, Cxcr4 and Epcam, which we did not show, while overlooking the endothelial programme reiterated above, which we have presented.

Given our focus on haemato-endothelial specification, we have started the single-cell RNA-seq characterisation of the haemogenic gastruloid at 120h and have not looked specifically at earlier timepoints of embryo patterning.

This said, we show the presence of neuroectodermal cells in cluster 9; on the other hand, cluster 7 includes hepatoblast-like cells, denoting endodermal specification. We are happy to include this characterisation, to the extent that it is present, in a revised version of the manuscript. However, in the absence of earlier timepoints and given the bias towards mesodermal specification, we expect that specification of ectodermal and endodermal programmes may be incomplete.

In respect of the contention regarding the capture of YS-like and AGM-like haematopoiesis, we have presented evidence in the manuscript that haemogenic cells generated during gastruloid differentiation, particularly at late 192h and 216h timepoints project onto highly purified c-Kit+ CD31+ Gfi1-expressing cells from mouse AGM (PMID: 38383534), providing support for the recapitulation of the corresponding developmental stage. In distinguishing between YS-like and AGM-like haematopoiesis, we call the Reviewer’s attention to the replotting of the single-cell RNA-seq data already in the manuscript, which we provided in response to point 1 (Author response image 2B), which highlights an increase in Sox17, but not Sox18, expression in the 192h haemogenic endothelium, which suggests an association with AGM haematopoiesis (PMID: 20228271). A significant association of Cd44 and Procr expression with the same time-point (Fig. 2F in the manuscript), further supports an AGM-like endothelial-to-haematopoietic transition at the 192h timepoint.

Following on the Reviewer’s comments about CD34, we also inspected co-expression of CD34 with CD41 and CD45, the latter co-expression present in, although not necessarily exclusive to, AGM haematopoiesis.

Reassuringly, we observed clear co-expression with both markers (Author response image 3), in addition to a CD41+CD34-population, which likely reflects YS EMP-independent erythropoiesis. Interestingly, marker expression is responsive to the levels of Activin A used in the patterning pulse, with the 100ng/ml Activin A used in our protocol superior to 75ng/ml.

Author response image 3.

Association of CD34 with CD41 and CD45 expression is Activin A-responsive and supports the presence of definitive haematopoiesis. A. Flow cytometry analysis of CD34 and CD41 expression in 216h-haemogenic gastruloids; two doses of Activin A were used in the patterning pulse with CHI99021 between 48-72h. FMO controls shown. B. Flow cytometry analysis of CD34 and CD45 at 216h in the same experimental conditions.

We agree that it remains challenging to identify markers exclusive to AGM haematopoiesis, which is operationally equated with generation of transplantable haematopoietic stem cells. While HSC generation is a key event characteristic of the AGM, not all AGM haematopoiesis corresponds to HSCs, an important point in evaluating the data presented in the manuscript, and indeed acknowledged by us.

Author response image 4.

Clustering of haemogenic gastruloid cells sorted on the basis of haemato-endothelial surface markers CD41, C-Kit and CD45. A. Leiden clustering to single-cell RNA-seq data. B. Time stamps of sorted haemogenic gastruloid cells in A. C. Surface marker stamps of cells in A.

Given the centrality of this point in comments by all the Reviewers, we have conducted projections of our single-cell RNA-seq data against two studies which (1) capture arterial and haemogenic specification in the para-splanchnopleura (pSP) and AGM region between E8.0 and E11 (Hou et al, PMID: 32203131), and (2) uniquely capture YS, AGM and FL progenitors and the AGM endothelial-to-haematopoietic transition (EHT) in the same scRNA-seq dataset (Zhu et al, PMID: 32392346).

Focusing the analysis on the subsets of haemogenic gastruloid cells sorted as CD41+ (144h) CKit+ (144h and 192h) and CD45+ (192h and 216h) (Author response image 4AC), we show:

(1) That a subset of haemato-endothelial cells from haemogenic gastruloids at 144h to 216h project onto intra-embryonic cells spanning E8.25 to E10 (Author response image 5A-B). This is in agreement with our interpretation that 216h are no later than the MPP/pre-HSC state of embryonic development, requiring further maturation to generate long-term engrafting HSC.

(2) That haemogenic gastruloids contain YS-like (including EMP-like) and AGM-like haematopoietic cells (Author response image 6A-B). Significantly, some of the cells, particularly c-Kit-sorted cells with a candidate endothelial and HE-like signature project onto AGM pre-HE and HE, as well as IAHC, and later, predominantly 216h cells, have characteristics of MPP/LMPP-like cells from the FL.

Altogether, the data support the notion that haemogenic gastruloids capture YS and AGM haematopoiesis until E10, as suggested by us in the manuscript. We thought it was important to share this preliminary data with the Editors at an early stage, and we will incorporate a deeper analysis in a revised version of the manuscript.

Single-cell RNA sequencing was used to compare hGx with mouse AGM. The authors incorrectly conclude that ' ..specification of endothelial and HE cells in hGx follows with time-dependent developmental progression into putative AGM-like HE..' And, '...HE-projected hGx cells.......expressed Gata2 but not Runx1, Myb, or Gfi1b..' Hemogenic endothelium is defined by the expression of Runx1 and Gfli1b is downstream of Runx1.

As a hierarchy of regulation, Gata2 precedes and drives Runx1 expression at the specification of HE (PMID: 17823307; PMID: 24297996), while Runx1 drives the EHT, upstream of Gfi1b in haematopoietic clusters (PMID: 34517413).

Author response image 5.

Projection of sorted haemogenic gastruloid cells onto Hou et al dataset (PMID: 32203131) analysing development of mouse intra-embryonic haematopoiesis. A. Time signatures of Hou et al data. B. Projection of Leiden clusters in Author response image 4A. Methodology as described in our manuscript; 68% gastruloid cells projected.

Author response image 6.

Projection of sorted haemogenic gastruloid cells onto Zhu et al dataset (PMID: 32392346), capturing arterial endothelial and haemogenic endothelial development, in reference to YS, AGM and FL haematopoietic progenitors. A. Functional cluster classification as per Zhu et al. B. Projection of Leiden clusters in Author response image 4A. Methodology as detailed in our manuscript; 58% gastruloid cells projected. Haematopoietic clusters annotated as in A.

(3) The hGx protocol 'generates hematopoietic SC precursors capable of short-term engraftment' is not supported by the data presented. Short-term engraftment would be confirmed by flow cytometric detection of hematopoietic cells within the recipient bone marrow, spleen, thymus, and peripheral blood that expressed the BFP transgene. This analysis was not provided. PCR detection of transcripts, following an unspecified number of amplification cycles, as shown in Figure 3G (incorrectly referred to as Figure 3F in the legend) is not acceptable evidence for engraftment.

We provide the full flow cytometry analysis of spleen engraftment in the 5 mice which received implantation of 216h-haemogenic gastruloids in the adrenal gland; an additional (control) animal received adrenal injection of PBS (Author response image 7). The animals were analysed at 4 weeks. In this experiment, the bone marrow collection was limiting, and material was prioritised for PCR.

We had previously provided only representative plots of flow cytometry analysis of bone marrow and spleen in Fig. S4E, which we described as low-level engraftment. The analysis was complemented with genomic DNA PCR, where detection was present in only some of the replicates tested per animal. We confirm that PCR analysis used conventional 40 cycles; the sensitivity was shown in Fig. S4F. As shown in Fig. 3 A-C, no more than 7 CD45+CD144+ multipotent cells are present per haemogenic gastruloid, with 3 haemogenic gastruloids implanted in the adrenal gland of each transplanted animal. We argue that the low level of cytometric and molecular engraftment at 4 weeks, from haemogenic gastruloid-derived progenitors that have not progressed beyond a stage equivalent to E10 Author response image 5A-B) and that we have described as requiring additional maturation in vivo, are not surprising.

Author response image 7.

BFP engraftment of Nude recipient mice 4 weeks after unilateral adrenal implantation of 216h-haemogenic gastruloids. Flow cytometry analysis of spleen engraftment. Genomic PCR analysis is shown in Fig. 3G of the manuscript.

Transplanted hGx formed teratoma-like structures, with hematopoietic cells present at the site of transplant only analysed histologically. Indeed, the quality of the images provided does not provide convincing validation that donor-derived hematopoietic cells were present in the grafts.

As stated in the text, the images mean to illustrate that the haemogenic gastruloids developed in situ. The observation of donor-derived blood cells in the implanted haemogenic gastruloids would not correspond to engraftment, as we have amply demonstrated that they have generated blood cells in vitro. There is no evidence that there are remaining pluripotent cells in the haemogenic gastruloid after 9 days of differentiation, and it is therefore not clear that these are teratomas

There is no justification for the authors' conclusion that '... the data suggest that 216h hGx generate AGM-like pre-HSC capable of at least short-term multilineage engraftment upon maturation...'. Indeed, this statement is in conflict with previous studies demonstrating that pre-HSCs in the dorsal aorta of the mouse embryo are immature and actually incapable of engraftment.

We have clearly stated that we do not see haematopoietic engraftment through transplantation of dissociated haemogenic gastruloids, which reach the E10 state containing pre-HSC (Author response image 5). Instead, we observed rare myelo-erythroid (in the manuscript) and myelo-lymphoid (Author response image 9 below, in response to Reviewer 2) engraftment upon in vivo maturation of haemogenic gastruloids with preserved 3D organisation. These statements are not contradictory.

The statement '...low-level production of engrafting cells recapitulates their rarity in vivo, in agreement with the embryo-like qualities of the gastruloid system....' is incorrect. Firstly, no evidence has been provided to show the hGx has formed a dorsal aorta facsimile capable of generating cells with engrafting capacity. Secondly, although engrafting cells are rare in the AGM, approximately one per embryo, they are capable of robust and extensive engraftment upon transplantation.

We are happy to rephrase the statement to simply say that “…the data suggest that 216h haemogenic gastruloids contain candidate AGM-like progenitors with some short-term engraftment potential but incomplete functional maturation.” To be clear, with our existing statement we meant to highlight that the production of definitive AGM-like haematopoietic progenitors (not all of which are engrafting) in haemogenic gastruloids does not correspond to non-physiological single-lineage programming. We did not claim that we achieved production of HSC, which would be long-term engrafting.

(4) Expression MNX1 transcript and protein in hematopoietic cells in MNX1 rearranged acute myeloid leukaemia (AML) is one cause of AML in infants. In the hGX model of this disease, Mnx1 is overexpressed in the mESCs that are used to form gastruloids. Mnx1 overexpression seems to confer an overall growth advantage on the hGx and increase the serial replating capacity of the small number of hematopoietic cells that are generated. The inefficiency with which the hGx model generates hematopoietic cells makes it difficult to model this disease. The poor quality of the cytospin images prevents accurate identification of cells. The statement that the kit-expressing cells represent leukemic blast cells is not sufficiently validated to support this conclusion. What other stem cell genes are expressed? Surface kit expression also marks mast cells, frequently seen in clonogenic assays of blood cells. Flow cytometric and gene expression analyses using known markers would be required.

The haemogenic gastruloid model generates haematopoietic and haemato-endothelial cells. MNX1 expands Kit+ cells at 144h, which we show to have a haemato-endothelial signature (manuscript Fig. 2B, which we replotted in Author response image 2B).

Serial replating of CFC assays is a conventional in vitro assay of leukaemia transformation. Critically, colony replating is not maintained in EV control cells, attesting to the transformation potential of MNX1.

Although we have not fully-traced the cellular hierarchy of MNX1-driven transformation in the haemogenic gastruloid system, the in vitro replating expands a Kit+ cell (Fig. 5E), which reflects the surface phenotype of the leukaemia, also recapitulated in the mouse model initiated by MNX1-overexpressing FL cells. Importantly, it recapitulates the transcriptional profile of MNX1-leukaemia patients (Fig. 6C), which is uniquely expressed by MNX1144h and replated colony cells, but not to MNX1 216h gastruloid cells, arguing against a generic signature of MNX1 overexpression (Fig. 6B). Importantly, the MNX1-transformation of haemogenic gastruloid cells is superior to the FL leukaemia model at capturing the unique transcriptional features of MNX1-driven leukaemia, distinct from other forms of AML in the same age group (Fig S7). It is possible that this corresponds to a preleukaemia event, and we will explore this in future studies, which are beyond the proof-of-principle nature of this paper.

(5) In human infant MNX1 AML, the mutation is thought to arise at the fetal liver stage of development. There is no evidence that this developmental stage is mimicked in the hGx model.

We never claim that the haemogenic gastruloid model mimics the foetal liver. We propose that susceptibility to MNX1 is at the HE-to-EMP transition. Moreover, and importantly, contrary to the Reviewer’s statement, there is no evidence in the literature that the mutation arises in the foetal liver stage, just that the mutation arises before birth (PMID: 38806630), which is different. In a mouse model of MNX1 overexpression, the authors achieve leukaemia engraftment upon MNX1 overexpression in foetal liver, but not in bone marrow cells (PMID: 37317878). This is in agreement with a vulnerability of embryonic / foetal, but not adult cells to the MNX1 expression caused by the translocation. However, haematopoietic cells in the foetal liver originate from YS and AGM precursors, so the origin of the MNX1-susceptible cells can be in those locations, rather than the foetal liver itself.

Reviewer #2 (Public review):
Summary:
In this manuscript, the authors develop an exciting new hemogenic gastruloid (hGX) system, which they claim reproduces the sequential generation of various blood cell types. The key advantage of this cellular system would be its potential to more accurately recapitulate the spatiotemporal emergence of hematopoietic progenitors within their physiological niche compared to other available in vitro systems. The authors present a large set of data and also validate their new system in the context of investigating infant leukemia.
Strengths:
The development of this new in vitro system for generating hematopoietic cells is innovative and addresses a significant drawback of current in vitro models. The authors present a substantial dataset to characterize this system, and they also validate its application in the context of investigating infant leukemia.
Weaknesses:
The thorough characterization and full demonstration that the cells produced truly represent distinct waves of hematopoietic progenitors are incomplete. The data presented to support the generation of late yolk sac (YS) progenitors, such as lymphoid cells, and aortic-gonad-mesonephros (AGM)-like progenitors, including pre-hematopoietic stem cells (pre-HSCs), by this system are not entirely convincing. Given that this is likely the manuscript's most crucial claim, it warrants further scrutiny and direct experimental validation. Ideally, the identity of these progenitors should be further demonstrated by directly assessing their ability to differentiate into lymphoid cells or fully functional HSCs. Instead, the authors primarily rely on scRNA-seq data and a very limited set of markers (e.g., Ikzf1 and Mllt3) to infer the identity and functionality of these cells. Many of these markers are shared among various types of blood progenitors, and only a well-defined combination of markers could offer some assurance of the lymphoid and pre-HSC nature of these cells, although this would still be limited in the absence of functional assays.
The identification of a pre-HSC-like CD45⁺CD41⁻/lo c-Kit⁺VE-Cadherin⁺ cell population is presented as evidence supporting the generation of pre-HSCs by this system, but this claim is questionable. This FACS profile may also be present in progenitors generated in the yolk sac such as early erythro-myeloid progenitors (EMPs). It is only within the AGM context, and in conjunction with further functional assays demonstrating the ability of these cells to differentiate into HSCs and contribute to long-term repopulation, that this profile could be strongly associated with pre-HSCs. In the absence of such data, the cells exhibiting this profile in the current system cannot be conclusively identified as true pre-HSCs.

At this preliminary response stage, we present 2 additional pieces of evidence to support our claims that we capture YS and AGM stages of haematopoietic development. In future experiments, we can complement these with functional assays, including co-culture with OP9 and OP9-DL stroma.

Author response image 8.

EZH2 inhibition affects CD41+ cellular output in haemogenic gastruloids at 144, but not 216h. A. Flow cytometry analysis of CD41 expression in 144h-haemogenic gastruloid treated with 0.5μM EZH2 inhibitor GSK126 from 120h. DMSO (0.05%), vehicle. 1 of 2 independent experiments (average CD41+: DMSO, 21.20%; GSK126, 12.10%; CD45 not detected). B. Flow cytometry analysis of CD41 and CD45 expression in 216h gastruloids, treated with DMSO or GSK216. (DMSO: average CD41+, 15.28%; average CD45+ 0.46%. GSK126: average CD41+, 23.78%; average CD45+, 2.08%).

In Author response images 5 and 6, we project our single-cell RNA-seq data onto (1) developing intra-embryonic pSP and AGM between E8 and E11 (Author response image 5) and (2) a single-cell RNA-seq study of HE development which combines haemogenic and haematopoietic cells from the YS, the developing HE and IAHC in the AGM, and FL (Author response image 6). Our data maps E8.25-E10 (Author response image 5) and captures YS EMP and erythroid and myeloid progenitors, as well as AGM pre-HE, HE and IAHC, with some cells matching HSPC and LMPP (Author response image 6), as suggested by the projection onto the Thambyrajah et al data set (Fig. S3 in the manuscript).

Given the difficulty in finding markers that specifically associate with AGM haematopoiesis, we inspected the possibility of capturing different regulatory requirements at different stages of gastruloid development mirroring differential effects in the embryo. Polycomb EZH2 is specifically required for EMP differentiation in the YS, but does not affect AGM-derived haematopoiesis; it is also not required for primitive erythroid cells (PMID: 29555646; PMID: 34857757). We treated haemogenic gastruloids from 120h onwards with either DMSO (0.05%) or GSK126 (0.5μM), and inspected the cellularity of gastruloids at 144h, which we equate with YS-EMP, and 216h – putatively AGM haematopoiesis (Author response image 8). We show that EZH2 inhibition / GSK126 treatment specifically reduces %CD41+ cells at 144h (Author response image 8A), but does not reduce %CD41+ or %CD45+ cells at 216h (Author response image 8B).

Although preliminary, these data, together with the scRNA-seq projections described, provide evidence to our claim that 144h haemogenic gastruloids capture YS EMPs, while CD41+ and CD45+ cells isolated at 216h reflect AGM progenitors. We cannot conclude as to the functional nature of the AGM cells from this experiment.

The engraftment data presented are also not fully convincing, as the observed repopulation is very limited and evaluated only at 4 weeks post-transplantation. The cells detected after 4 weeks could represent the progeny of EMPs that have been shown to provide transient repopulation rather than true HSCs.

We clearly state that there is low level engraftment and do not claim to have generated HSC. We describe cells with short-term engraftment potential. Although the cells we show in the manuscript at 4 weeks could be EMPs (Author response image 7 and Fig. 3 and S3), we now have 8-week analysis of implant recipients, in which we observed, again low-level, engraftment of the recipient bone marrow in 1:3 animals (Author response image 9). This engraftment is myeloid-lymphoid and therefore likely to have originated in a later progenitor. To be clear, we do not claim that this corresponds to the presence of HSC. It nevertheless supports the maturation of progenitors with engraftment potential.

Author response image 9.

Flow cytometry BFP engraftment of recipient bone marrow 8-weeks post implantation of 216hhaemogenic gastruloids in the adrenal gland of Nude mice. 1:3 animals show BFP CD45+ engraftment in the myeloid (Mac1+) and B-lymphoid (B220+) lineages. 3 haemogenic gastruloids were implanted unilaterally in the adrenal gland of each animal. A. Engrafted animal, showing CD45+ BFP cells of myeloid (CD11b) and B-lymphoid affiliation (B220). B. Non-engrafted mouse recipient of haemogenic gastruloid implants.

Reviewer #3 (Public review):
In this study, the authors employ a mouse ES-derived "hemogenic gastruloid" model which they generated and which they claim to be able to deconvolute YS and AGM stages of blood production in vitro. This work could represent a valuable resource for the field. However, in general, I find the conclusions in this manuscript poorly supported by the data presented. Importantly, it isn't clear what exactly are the "YS" and the "AGM"-like stages identified in the culture and where is the data that backs up this claim. In my opinion, the data in this manuscript lack convincing evidence that can enable us to identify what kind of hematopoietic progenitor cells are generated in this system. Therefore, the statement that "our study has positioned the MNX1-OE target cell within the YS-EMP stage (line 540)" is not supported by the evidence presented in this study. Overall, the system seems to be very preliminary and requires further optimization before those claims can be made.
Specific comments below:
(1) The flow cytometric analysis of gastruloids presented in Figure 1 C-D is puzzling. There is a large % of c-Kit+ cells generated, but few VE-Cad+ Kit+ double positive cells. Similarly, there are many CD41+ cells, but very few CD45+ cells, which one would expect to appear toward the end of the differentiation process if blood cells are actually generated. It would be useful to present this analysis as consecutive gating (i.e. evaluating CD41 and CD45 within VE-Cad+ Kit+ cells, especially if the authors think that the presence of VE-Cad+ Kit+ cells is suggestive of EHT). The quantification presented in D is misleading as the scale of each graph is different.

Fig. 1C-D provide an overview of haemogenic markers during the timecourse of haemogenic gastruloid differentiation, and does indeed show a late up-regulation of CD45, as the Reviewer points out would be expected. The %CD45+ cells is indeed low. However, we should point out that the haemogenic gastruloid protocol, although biased towards mesodermal outputs, does not aim to achieve pure haematopoietic specification, but rather place it in its embryo-like context. Consecutive gating at the 216h-timepoint is shown and quantified in Fig. 3A-B. We refute that the scale is misleading. It is a necessity to represent the data in a way that is interpretable by the reader: the gates (in C) are truly representative and annotated, as are the plot axes (in D).

(2) The imaging presented in Figure 1E is very unconvincing. C-Kit and CD45 signals appear as speckles and not as membrane/cell surfaces as they should. This experiment should be repeated and nuclear stain (i.e. DAPI) should be included.

We include the requested images below (Author response image 10).

Author response image 10.

Confocal images of haematopoietic production in haemogenic gastruloids. Wholemount, cleared haemogenic gastruloids were stained for CD45 (pseudo-coloured red) and c-Kit antigens (pseudo-coloured yellow) with indirect staining, as described in the manuscript. Flk1-GFP signal is shown in green. Nuclei are contrasted with DAPI. (A) 192h. (B) 216h.

(3) Overall, I am not convinced that hematopoietic cells are consistently generated in these organoids. The authors should sort hematopoietic cells and perform May-Grunwald Giemsa stainings as they did in Figure 6 to confirm the nature of the blood cells generated.

It is factual that the data are reproducible and complemented by functional assays shown in Fig. 3, which clearly demonstrate haematopoietic output. The single-cell RNA-seq data also show expression of a haematopoietic programme. Nevertheless, we include Giemsa-Wright’s stained cytospins obtained at 216h to illustrate haematopoietic output (Reviewer Fig. 11). Inevitably, the cytospins will be inconclusive as to the presence of endothelial-to-haematopoietic transition or the generation of haematopoietic stem/progenitor cells, as these cells do not have a distinctive morphology.

Author response image 11.

Cytospin of dissociated haemogenic gastruloids at 216h. Cytospins were stained with Giemsa-Wright’s stain and are visualised with a 40x objective. Annotated are cells in the monocytic (dashed open arrow), granulocytic (solid open arrow), megakaryocytic (solid arrow) and erythroid (asterisk) lineages; arrowheads indicate cells with a non-specific blast-like morphology. Representative image.

(4) The scRNAseq in Figure 2 is very difficult to interpret. Specific points related to this:
- Cluster annotation in Figure 2a is missing and should be included.
- Why do the heatmaps show the expression of genes within sorted cells? Couldn't the authors show expression within clusters of hematopoietic cells as identified transcriptionally (which ones are they? See previous point)? Gene names are illegible.
- I see no expression of Hlf or Myb in CD45+ cells (Figure 2G). Hlf is not expressed by any of the populations examined (panels E, F, G). This suggests no MPP or pre-HSC are generated in the culture, contrary to what is stated in lines 242-245. (PMID 31076455 and 34589491).
Later on, it is again stated that "hGx cells... lacked detection of HSC genes like Hlf, Gfi1, or Hoxa9" (lines 281-283). To me, this is proof of the absence of AGM-like hematopoiesis generated in those gastruloids.

Author response image 12.

Expression of endothelial, haemogenic and haematopoietic genes in haemogenic gastruloid cells sorted at 144h, 192h and 216h. UMAP as in Author response image 4. Pecam (CD31) and CD34 represent endothelial genes also detected in haemogenic endothelium. CD44 is specifically enriched at the endothelial-to-haemogenic transition. Mecom is detected in haemogenic endothelium and haematopoietic progenitors. Mllt3 and Runx1 are haematopoietic markers. Hoxa9 and Hlf are associated with haematopoietic stem and progenitor cells and their detection is rare in haemogenic gastruloids at 216h.

For a combination of logistic and technical reasons, we performed single-cell RNA-seq using the Smart-Seq2 platform, which is inherently low throughput. We overcame the issue of cell coverage by complementing whole-gastruloid transcriptional profiling at successive time-points with sorting of subpopulations of cells based on individual markers documented in Fig. 1. We clearly stated which platform was used as well as the number and type of cells profiled (Fig. S2A and lines 172-179 of the manuscript), and our approach is standard. We will review our representation of the data in a revised manuscript. Nevertheless, at this stage, we provide plots of the expression of key haematopoietic markers over UMAPs of haemogenic gastruloid timecourse (Author response image 12). We also show preliminary qRT-PCR data with increased Hlf expression upon extension of the protocol to 264h (Author response image 13), further confirming haematopoietic specification, including of candidate definitive progenitor cells, in the haemogenic gastruloid model.

Author response image 13.

Hlf expression is up-regulated in late stage haemogenic gastruloids. Quantitative RT-PCR analysis of Hlf expression in unfractionated haemogenic gastruloids cultured for 264h. From 168h onwards, haemogenic gastruloids were cultured in N2B27 in the presence of VEGF, SCF, FLT3L and TPO, all recombinant mouse cytokines, as described in the manuscript. Shown are mean±standard deviation of n=5 replicates from 2 mouse ES cell lines, respectively Flk1-GFP and Rosa26-BFP::Flk1-GFP, reported in the manuscript; 2-tailed unpaired t-test with Welch correction.

(5) Mapping of scRNA-Seq data onto the dataset by Thambyrajah et al. is not proof of the generation of AGM HE. The dataset they are mapping to only contains AGM cells, therefore cells do not have the option to map onto something that is not AGM. The authors should try mapping to other publicly available datasets also including YS cells.

We have done this and the data are presented in Author response image 5 and 6. As detailed in response to Reviewer 1, we have conducted projections of our single-cell RNA-seq data against two studies which (1) capture arterial and haemogenic specification in the para-splanchnopleura (pSP) and AGM region between E8.0 and E11 (Hou et al, PMID: 32203131) (Author response image 5), and (2) uniquely capture YS, AGM and FL progenitors and the AGM endothelial-to-haematopoietic transition (EHT) in the same scRNA-seq dataset (Zhu et al, PMID: 32392346) (Author response image 6). Specifically in answering the Reviewers’ point, we show that different subsets of haemogenic gastruloid cells sorted on haemogenic surface markers c-Kit, CD41 and CD45 cluster onto pre-HE and HE, intra-aortic clusters and FL progenitor compartments, and to YS EMP and erythroid and myeloid progenitors. This lends support to our claim that the haemogenic gastruloid system specifies both YS-like and AGM-like cells.

(6) Conclusions in Figure 3, named "hGx specify cells with preHSC characteristics" are not supported by the data presented here. Again, I am not convinced that hematopoietic cells can be efficiently generated in this system, and certainly not HSCs or pre-HSCs.

We have provided evidence, both in the manuscript and in this response to Reviewers, that there is haematopoietic specification, including of progenitor cells, in the haemogenic gastruloid system (Fig. 3 and Author response image 7,9). We have added data in this response that supports the specification of YS-like and AGM-like cells (Author response image 5-6, 8). Importantly, we have never claimed that haemogenic gastruloids generate HSC. We accept the Reviewer’s comment that we have not provided sufficient evidence for the specification of pre-HSC-like cells. We will re-phrase Fig. 3 conclusion as “Haemogenic gastruloids specify cells with characteristics of definitive haematopoietic progenitors”.

- FACS analysis in 3A is again very unconvincing. I do not think the population identified as c-Kit+ CD144+ is real. Also, why not try gating the other way around, as commonly done (e.g. VE-Cad+ Kit+ and then CD41/CD45)?

There is nothing unconventional about our gating strategy, which was done from a more populated gate onto the less abundant one to ensure that the results are numerically more robust. In the case of haemogenic gastruloids, unlike the AGM preparations the Reviewer may be referring to, CD41 and CD45+ cells are more abundant as there is no circulation of more differentiated haematopoietic cells away from the endothelial structures. This said, we did perform the gating as suggested (Author response image 14), indeed confirming that most VE-cad+ Kit+ cells are CD45+. Interestingly VE-cad+Kit- are predominantly CD41+, reinforcing the true haematopoietic nature of these cells.

Author response image 14.

Flow cytometry analysis of VE-cadherin+ cells in haemogenic gastruloids at 216h of the differentiation protocol, probing co-expression of CD45, CD41 and c-Kit.

- The authors must have tried really hard, but the lack of short- or long-engraftment in a number of immunodeficient mouse models (lines 305-313) really suggests that no blood progenitors are generated in their system. I am not familiar with the adrenal gland transplant system, but it seems like a very non-physiological system for trying to assess the maturation of putative pre-HSCs. The data supporting the engraftment of these mice, essentially seen only by PCR and in some cases with a very low threshold for detection, are very weak, and again unconvincing. It is stated that "BFP engraftment of the Spl and BM by flow cytometry was very low level albeit consistently above control (Fig. S4E)" (lines 337-338). I do not think that two dots in a dot plot can be presented as evidence of engraftment.

We have presented the data with full disclosure and do not deny that the engraftment achieved is low-level and short-term, indicating incomplete maturation of definitive haematopoietic progenitors in the current haemogenic gastruloid system. However, we call the Reviewer’s attention to the fact that detection of BFP+ cells by PCR and flow cytometry in the recipient animals at 4 weeks is consistent between the 2 methods (Author response image 7).

Furthermore, we have now also been able to detect low-level myelo-lymphoid engraftment in the bone marrow 8 weeks after adrenal implantation, again suggesting the presence of a small number of definitive haematopoietic progenitors that potentially mature from the 3 haemogenic gastruloids implanted (Author response image 9).

(7) Given the above, I find that the foundations needed for extracting meaningful data from the system when perturbed are very shaky at best. Nevertheless, the authors proceed to overexpress MNX1 by LV transduction, a system previously shown to transform fetal liver cells, mimicking the effect of the t(7;12) AML-associated translocation. Comments on this section:
- The increase in the size of the organoid when MNX1 is expressed is a very unspecific finding and not necessarily an indication of any hematopoietic effect of MNX1 OE.

We agree with the Reviewer on this point; it is nevertheless a reproducible observation which we thought relevant to describe for completeness and data reproducibility.

- The mild increase of cKit+ cells (Figure 4E) at the 144hr timepoint and the lack of any changes in CD41+ or CD45+ cells suggests that the increase in Kit+ cells % is not due to any hematopoietic effect of MNX1 OE. No hematopoietic GO categories are seen in RNA seq analysis, which supports this interpretation. Could it be that just endothelial cells are being generated?

The Reviewer is correct that the MNX1-overexpressing cells have a strong endothelial signature, which is present in the patients (Fig. 4A). We investigated a potential link with c-Kit by staining cells from the replating colonies during the process of in vitro transformation with CD31. We observed that 40-50% of c-Kit+ cells (20-30% total colony cells) co-expressed CD31(Author response image 15), at least at early plating. These cells co-exist with haematopoietic cells, namely Ter119+ cells, as expected from the YS-like erythroid and EMP-like affiliation of haematopoietic output from 144h-haemogenic gastruloids (Fig. 5F).

Author response image 15.

Endothelial affiliation of MNX1-oe replating cells from haemogenic gastruloid. A. Representative flow cytometry plot of plate 1 CFC from MNX1-overexpressing haemogenic gastruloids at 144h. B. Quantification of the proportion of CD31+c-Kit+ cells in plates 1 and 2 of MNX1-oe-driven in vitro transformation.

(8) There seems to be a relatively convincing increase in replating potential upon MNX1-OE, but this experiment has been poorly characterized. What type of colonies are generated? What exactly is the "proportion of colony forming cells" in Figures 5B-D? The colony increase is accompanied by an increase in Kit+ cells; however, the flow cytometry analysis has not been quantified.

Given the inability to replate control EV cells, there is not a population to compare with in terms of quantification. The level of c-Kit+ represented in Fig. 5E is achieved at plate 2 or 3 (depending on the experiment), both of which are significantly enriched for colony-forming cells relative to control (Fig. 5B, D).

(9) Do hGx cells engraft upon MNX1-OE? This experiment, which appears not to have been performed, is essential to conclude that leukemic transformation has occurred.

For the purpose of this study, we are satisfied with confirmation of in vitro transformation potential of MNX1 haemogenic gastruloids, which can be used for screening purposes. Although interesting, in vivo leukaemia engraftment from haemogenic gastruloids is beyond the scope of this study.