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Hematopoietic stem cells (HSCs) develop from hemogenic endothelial cells (HECs) during mouse embryogenesis. Understanding the signaling molecules required for HSC development is crucial for the in vitro derivation of HSCs. We previously induced HSCs from embryonic HECs, isolated at embryonic day 10.5 (E10.5), in serum-free culture conditions with stem cell factor, thrombopoietin, and an endothelial feeder layer. Here, we aimed to elucidate signal requirements for inducing HSCs from earlier-stage HECs. Single-cell RNA sequencing (RNA-seq) analysis detected bone morphogenetic protein (BMP) signaling activation in E9.5 HECs. Adding BMP4 to the culture conditions led to the induction of HSCs from E9.5 HECs. Furthermore, isolating BMP4 receptor-expressing HECs from E9.5 embryos enriched progenitors with HSC-forming ability. This study identified BMP4 as an essential factor promoting the differentiation of early HECs into HSCs, opening up new possibilities for the in vitro derivation of HSCs., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Differentiation of induced pluripotent stem cells (iPSC) into hematopoietic lineages offers great therapeutic potential. During embryogenesis, hemogenic endothelium (HE) gives rise to hematopoietic stem and progenitor cells through the endothelial- to-hematopoietic transition (EHT). Understanding this process using iPSC is key to generating functional hematopoietic stem cells (HSC), a currently unmet challenge. In this study, we examined the role of the transcriptional factor GFI1B and its co-factor LSD1/KDM1A in EHT. To this end, we employed patient-derived iPSC lines with a dominant-negative dysfunctional GFI1B Q287* and irreversible pharmacological LSD1/KDM1A inhibition in healthy iPSC lines. The formation of HE remained unaffected; however, hematopoietic output was severely reduced in both conditions. Single-cell RNA sequencing (scRNAseq) performed on the CD144+/CD31+ population derived from healthy iPSC revealed similar expression dynamics of genes associated with in vivo EHT. Interestingly, LSD1/KDM1A inhibition in healthy lines before EHT resulted in a complete absence of hematopoietic output. However, uncommitted HE cells did not display GFI1B expression, suggesting a timed transcriptional program. To test this hypothesis, we ectopically expressed GFI1B in uncommitted HE cells, leading to downregulation of endothelial genes and upregulation of hematopoietic genes, including GATA2, KIT, RUNX1, and SPI1. Thus, we demonstrate that LSD1/KDM1A and GFI1B can function at distinct temporal points in different cellular subsets during EHT. Although GFI1B is not detected in uncommitted HE cells, its ectopic expression allows for partial hematopoietic specification. These data indicate that precisely timed expression of specific transcriptional regulators during EHT is crucial to the eventual outcome of EHT.

During human embryogenesis, distinct waves of hematopoiesis give rise to various blood cell types, originating from hemogenic endothelial (HE) cells. As HE cells reside in hypoxic conditions in the embryo, we investigated the role of hypoxia in human endothelial to hematopoietic transition and subsequent hematopoiesis. Using single-cell RNA sequencing, we describe hypoxia-related transcriptional changes in different HE-derived blood lineages, which reveal that erythroid cells are particularly susceptible to oxidative stress, due to decreased NRF2 activity in hypoxia. In contrast, nonerythroid CD45 + cells exhibit increased proliferative rates in hypoxic conditions and enhanced resilience to oxidative stress. We find that even in normoxia, erythroid cells present a clear predisposition to oxidative stress, with low glutathione levels and high lipid peroxidation, in contrast to CD45 + cells. Intriguingly, reactive oxygen species are produced at different sites in GPA + and CD45 + cells, revealing differences in oxidative phosphorylation and the use of canonical versus noncanonical tricarboxylic acid cycle in these lineages. Our findings elucidate how hypoxia and oxidative stress distinctly affect HE-derived hematopoietic lineages, uncovering critical transcriptional and metabolic pathways that influence blood cell development., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Undergoing endothelial-to-hematopoietic transition, a small fraction of embryonic aortic endothelial cells specializes into hemogenic endothelial cells (HECs) and eventually gives rise to hematopoietic stem cells (HSCs). Previously, we found that the activity of ribosome biogenesis (RiBi) is highly enriched in the HSC-primed HECs compared with adjacent arterial endothelial cells; however, whether RiBi is required in HECs for the generation of HSCs remains to be determined. Here, we have found that robust RiBi is markedly augmented during the endothelial-to-hematopoietic transition in mouse. Pharmacological inhibition of RiBi completely impeded the generation of HSCs in explant cultures. Moreover, disrupting RiBi selectively interrupted the HSC generation potential of HECs rather than T1 pre-HSCs, which was in line with its influence on cell cycle activity. Further investigation revealed that, upon HEC specification, the master transcription factor Runx1 dramatically bound to the loci of genes involved in RiBi, thereby facilitating this biological process. Taken together, our study provides functional evidence showing the indispensable role of RiBi in generating HSCs from HECs, providing previously unreported insights that may contribute to the improvement of HSC regeneration strategies., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

A diverse array of protocols have been established for the directed differentiation of human pluripotent stem cells (hPSCs) into a variety of cell types, including blood cells, for modeling development and disease, and for the development of cell-based therapeutics. These protocols recapitulate various signaling requirements essential for the establishment of the hematopoietic systems during embryonic development. However, in many instances, the functional properties of those progenitors, and their relevance to human development, remains unclear. The human embryo, much like other vertebrate model organisms, generates hematopoietic cells via successive anatomical location- and time-specific waves, each yielding cells with distinct functional and molecular characteristics. Each of these progenitor "waves" is characterized at the time of emergence of the direct hematopoietic progenitor in the vasculature, the hemogenic endothelial cell (HEC). Critically, despite decades of study in model organisms, the origins of each of these HEC populations remain unclear. Fortunately, through the directed differentiation of hPSCs, recent insights have been made into the earliest origins of each HEC population, revealing that each arises from transcriptionally and phenotypically distinct subsets of nascent mesoderm. Here, we outline the protocols to generate each mesodermal and HEC population via the formation of embryoid bodies and subsequent stage-specific signal manipulation. Through implementation of these discrete signal manipulations, it is possible to obtain human HEC populations that are exclusively extraembryonic-like or exclusively intraembryonic-like, enabling comparative developmental biology studies or specific translational applications., Competing Interests: Conflict of Interest Disclosure CMS and AD are inventors of patents (“Methods to Obtain Retinoic Acid-Dependent Hematopoietic Progenitors from Human Pluripotent Stem Cells,” International Publication No. WO2020154412 A1, inventors: CMS and AD; “Methods for isolating hemogenic endothelial cells,” International Publication No. WO2023247722 A1, inventor: AD) pertaining to the application of the methodologies described in this manuscript. CMS is a scientific founder and Scientific Advisory Board member of Clade Therapeutics. The remaining authors declare no competing interests., (Copyright © 2024 International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)

Tropomyosins coat actin filaments to impact actin-related signaling and cell morphogenesis. Genome-wide association studies have linked Tropomyosin 1 (TPM1) with human blood trait variation. TPM1 has been shown to regulate blood cell formation in vitro, but it remains unclear how or when TPM1 affects hematopoiesis. Using gene-edited induced pluripotent stem cell (iPSC) model systems, we found that TPM1 knockout augmented developmental cell state transitions and key signaling pathways, including tumor necrosis factor alpha (TNF-α) signaling, to promote hemogenic endothelial (HE) cell specification and hematopoietic progenitor cell (HPC) production. Single-cell analyses revealed decreased TPM1 expression during human HE specification, suggesting that TPM1 regulated in vivo hematopoiesis via similar mechanisms. Analyses of a TPM1 gene trap mouse model showed that TPM1 deficiency enhanced HE formation during embryogenesis, without increasing the number of hematopoietic stem cells. These findings illuminate novel effects of TPM1 on developmental hematopoiesis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Arterial endothelial cells (AECs) are the founder cells for intraembryonic haematopoiesis. Here, we report a method for the efficient generation of human haemogenic DLL4+ AECs from pluripotent stem cells (PSC). Time-series single-cell RNA-sequencing reveals the dynamic evolution of haematopoiesis and lymphopoiesis, generating cell types with counterparts present in early human embryos, including stages marked by the pre-haematopoietic stem cell genes MECOM/EVI1, MLLT3 and SPINK2. DLL4+ AECs robustly support lymphoid differentiation, without the requirement for exogenous NOTCH ligands. Using this system, we find IL7 acts as a morphogenic factor determining the fate choice between the T and innate lymphoid lineages and also plays a role in regulating the relative expression level of RAG1. Moreover, we document a developmental pathway by which human RAG1+ lymphoid precursors give rise to the natural killer cell lineage. Our study describes an efficient method for producing lymphoid progenitors, providing insights into their endothelial and haematopoietic ontogeny, and establishing a platform to investigate the development of the human blood system., (© 2024. The Author(s).)

COVID-19 and infectious diseases have been included in strategic development goals (SDG) of United Nations (UN). The SARS-CoV-2 pandemic has unveiled complex pathophysiological mechanisms underpinning COVID-19, notably inducing a systemic acquired vascular hemopathy characterized by endothelial dysfunction and intussusceptive angiogenesis, a rapid vascular remodeling process identified as a hallmark in severe COVID-19 cases affecting pulmonary and cardiac tissues. Stem cell migration have been proposed as significant regulators of this neoangiogenic process. In a monocentric cross-sectional study, through spectral flow cytometry analysis of peripheral blood mononuclear cells, we identified a distinct stem cell subpopulation mobilized in critical COVID-19. Indeed, by an unsupervised analysis generating a UMAP representation we highlighted eleven different clusters in critical and non-critical COVID-19 patients. Only one cluster was significantly associated to critical COVID-19 compared to non-critical patients. This cluster expressed the markers: CD45dim, CD34+, CD117+, CD147+, and CD143+, and were negative for CD133. Higher level of expression of hemangioblast markers CD143 were found in critical COVID-19 patients. This population, indicative of hemangioblast-like cells, suggests a key role in COVID-19-related neoangiogenesis, potentially driving the severe vascular complications observed. Our findings underscore the need for further investigation into the contributions of adult stem cells in COVID-19 pathology, offering new insights into therapeutic targets and interventions., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Hematopoietic stem cells (HSCs) develop from hemogenic endothelial cells (HECs) in vivo during mouse embryogenesis. When cultured in vitro, cells from the embryo phenotypically defined as pre-HSC-I and pre-HSC-II have the potential to differentiate into HSCs. However, minimal factors required for HSC induction from HECs have not yet been determined. In this study, we demonstrated that stem cell factor (SCF) and thrombopoietin (TPO) induced engrafting HSCs from embryonic day (E) 11.5 pre-HSC-I in a serum-free and feeder-free culture condition. In contrast, E10.5 pre-HSC-I and HECs required an endothelial cell layer in addition to SCF and TPO to differentiate into HSCs. A single-cell RNA sequencing analysis of E10.5 to 11.5 dorsal aortae with surrounding tissues and fetal livers detected TPO expression confined in hepatoblasts, while SCF was expressed in various tissues, including endothelial cells and hepatoblasts. Our results suggest a transition of signal requirement during HSC development from HECs. The differentiation of E10.5 HECs to E11.5 pre-HSC-I in the aorta-gonad-mesonephros region depends on SCF and endothelial cell-derived factors. Subsequently, SCF and TPO drive the differentiation of E11.5 pre-HSC-I to pre-HSC-II/HSCs in the fetal liver. The culture system established in this study provides a beneficial tool for exploring the molecular mechanisms underlying the development of HSCs from HECs., Competing Interests: Competing interests statement:The authors declare no competing interest.

Hematopoietic stem cells emerge in the embryo from an aortic-derived tissue called the hemogenic endothelium (HE). The HE appears to give birth to cells of different nature and fate but the molecular principles underlying this complexity are largely unknown. Here we show, in the zebrafish embryo, that two cell types emerge from the aortic floor with radically different morphodynamics. With the support of live imaging, we bring evidence suggesting that the mechanics underlying the two emergence types rely, or not, on apicobasal polarity establishment. While the first type is characterized by reinforcement of apicobasal polarity and maintenance of the apical/luminal membrane until release, the second type emerges via a dynamic process reminiscent of trans-endothelial migration. Interfering with Runx1 function suggests that the balance between the two emergence types depends on tuning apicobasal polarity at the level of the HE. In support of this and unexpectedly, we show that Pard3ba - one of the four Pard3 proteins expressed in the zebrafish - is sensitive to interference with Runx1 activity, in aortic endothelial cells. This supports the idea of a signaling cross talk controlling cell polarity and its associated features, between aortic and hemogenic cells. In addition, using new transgenic fish lines that express Junctional Adhesion Molecules and functional interference, we bring evidence for the essential role of ArhGEF11/PDZ-RhoGEF in controlling the HE-endothelial cell dynamic interface, including cell-cell intercalation, which is ultimately required for emergence completion. Overall, we highlight critical cellular and dynamic events of the endothelial-to-hematopoietic transition that support emergence complexity, with a potential impact on cell fate., Competing Interests: LT, SM, CV, AS No competing interests declared, (© 2023, Torcq et al.)

During embryonic development, blood cells emerge from specialized endothelial cells, named haemogenic endothelial cells (HECs). As HECs are rare and only transiently found in early developing embryos, it remains difficult to distinguish them from endothelial cells. Here we performed transcriptomic analysis of 28- to 32-day human embryos and observed that the expression of Fc receptor CD32 (FCGR2B) is highly enriched in the endothelial cell population that contains HECs. Functional analyses using human embryonic and human pluripotent stem cell-derived endothelial cells revealed that robust multilineage haematopoietic potential is harboured within CD32 + endothelial cells and showed that 90% of CD32 + endothelial cells are bona fide HECs. Remarkably, these analyses indicated that HECs progress through different states, culminating in FCGR2B expression, at which point cells are irreversibly committed to a haematopoietic fate. These findings provide a precise method for isolating HECs from human embryos and human pluripotent stem cell cultures, thus allowing the efficient generation of haematopoietic cells in vitro., (© 2024. The Author(s).)

Background: Hemangioblasts are mesoderm-derived multipotent stem cells for differentiation of all hematopoietic and endothelial cells in the circulation system. However, the underlying molecular mechanism is poorly understood., Methods: CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (type II CRISPR RNA-guided endonuclease) editing was used to develop aggf1 -/- and emp2 -/- knockout zebra fish. Whole-mount in situ hybridization and transgenic Tg( gata1 -EGFP [enhanced green fluorescent protein]), Tg( mpx -EGFP), Tg( rag2 -DsRed [discosoma sp. red fluorescent protein]), Tg( cd41 -EGFP), Tg( kdrl -EGFP), and Tg( aggf1 -/- ; kdrl -EGFP) zebra fish were used to examine specification of hemangioblasts and hematopoietic stem and progenitor cells (HSPCs), hematopoiesis, and vascular development. Quantitative real-time polymerase chain reaction and Western blot analyses were used for expression analysis of genes and proteins., Results: Knockout of aggf1 impaired specification of hemangioblasts and HSPCs, hematopoiesis, and vascular development in zebra fish. Expression of npas4l / cloche -the presumed earliest marker for hemangioblast specification-was significantly reduced in aggf1 -/- embryos and increased by overexpression of aggf1 in embryos. Overexpression of npas4l rescued the impaired specification of hemangioblasts and HSPCs and development of hematopoiesis and intersegmental vessels in aggf1 -/- embryos, placing aggf1 upstream of npas4l in hemangioblast specification. To identify the underlying molecular mechanism, we identified emp2 as a key aggf1 downstream gene. Similar to aggf1 , emp2 knockout impaired the specification of hemangioblasts and HSPCs, hematopoiesis, and angiogenesis by increasing the phosphorylation of ERK1/2 (extracellular signal-regulated protein kinase 1/2). Mechanistic studies showed that aggf1 knockdown and knockout significantly decreased the phosphorylated levels of mTOR (mammalian target of rapamycin) and p70 S6K (ribosomal protein S6 kinase), resulting in reduced protein synthesis of Emp2 (epithelial membrane protein 2), whereas mTOR activator MHY1485 (4,6-dimorpholino-N-(4-nitrophenyl)-1,3,5-triazin-2-amine) rescued the impaired specification of hemangioblasts and HSPCs and development of hematopoiesis and intersegmental vessels and reduced Emp2 expression induced by aggf1 knockdown., Conclusions: These results indicate that aggf1 acts at the top of npas4l and becomes the earliest marker during specification of hemangioblasts. Our data identify a novel signaling axis of Aggf1 (angiogenic factor with G-patch and FHA domain 1)-mTOR-S6K-ERK1/2 for specification of hemangioblasts and HSPCs, primitive and definitive hematopoiesis, and vascular development. Our findings provide important insights into specification of hemangioblasts and HSPCs essential for the development of the circulation system., Competing Interests: Disclosures None.

Hemogenic endothelial cells (HECs) are specialized cells that undergo endothelial-to-hematopoietic transition (EHT) to give rise to the earliest precursors of hematopoietic progenitors that will eventually sustain hematopoiesis throughout the lifetime of an organism. Although HECs are thought to be primarily limited to the aorta-gonad-mesonephros (AGM) during early development, EHT has been described in various other hematopoietic organs and embryonic vessels. Though not defined as a hematopoietic organ, the lung houses many resident hematopoietic cells, aids in platelet biogenesis, and is a reservoir for hematopoietic stem and progenitor cells (HSPCs). However, lung HECs have never been described. Here, we demonstrate that the fetal lung is a potential source of HECs that have the functional capacity to undergo EHT to produce de novo HSPCs and their resultant progeny. Explant cultures of murine and human fetal lungs display adherent endothelial cells transitioning into floating hematopoietic cells, accompanied by the gradual loss of an endothelial signature. Flow cytometric and functional assessment of fetal-lung explants showed the production of multipotent HSPCs that expressed the EHT and pre-HSPC markers EPCR, CD41, CD43, and CD44. scRNA-seq and small molecule modulation demonstrated that fetal lung HECs rely on canonical signaling pathways to undergo EHT, including TGFβ/BMP, Notch, and YAP. Collectively, these data support the possibility that post-AGM development, functional HECs are present in the fetal lung, establishing this location as a potential extramedullary site of de novo hematopoiesis., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

In vertebrates, the earliest hematopoietic stem and progenitor cells (HSPCs) are derived from a subset of specialized endothelial cells, hemogenic endothelial cells, in the aorta-gonad-mesonephros region through endothelial-to-hematopoietic transition. HSPC generation is efficiently and accurately regulated by a variety of factors and signals; however, the precise control of these signals remains incompletely understood. Post-transcriptional regulation is crucial for gene expression, as the transcripts are usually bound by RNA-binding proteins (RBPs) to regulate RNA metabolism. Here, we report that the RBP protein Csde1-mediated translational control is essential for HSPC generation during zebrafish early development. Genetic mutants and morphants demonstrated that depletion of csde1 impaired HSPC production in zebrafish embryos. Mechanistically, Csde1 regulates HSPC generation through modulating Wnt/β-catenin signaling activity. We demonstrate that Csde1 binds to ctnnb1 mRNAs (encoding β-catenin, an effector of Wnt signaling) and regulates translation but not stability of ctnnb1 mRNA, which further enhances β-catenin protein level and Wnt signal transduction activities. Together, we identify Csde1 as an important post-transcriptional regulator and provide new insights into how Wnt/β-catenin signaling is precisely regulated at the post-transcriptional level., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Prior to the generation of hematopoietic stem cells (HSCs) from the hemogenic endothelial cells (HECs) mainly in the dorsal aorta in midgestational mouse embryos, multiple hematopoietic progenitors including erythro-myeloid progenitors and lymphoid progenitors are generated from yolk sac HECs. These HSC-independent hematopoietic progenitors have recently been identified as major contributors to functional blood cell production until birth. However, little is known about yolk sac HECs. Here, combining integrative analyses of multiple single-cell RNA-sequencing datasets and functional assays, we reveal that Neurl3-EGFP, in addition to marking the continuum throughout the ontogeny of HSCs from HECs, can also serve as a single enrichment marker for yolk sac HECs. Moreover, while yolk sac HECs have much weaker arterial characteristics than either arterial endothelial cells in the yolk sac or HECs within the embryo proper, the lymphoid potential of yolk sac HECs is largely confined to the arterial-biased subpopulation featured by the Unc5b expression. Interestingly, the B lymphoid potential of hematopoietic progenitors, but not for myeloid potentials, is exclusively detected in Neurl3-negative subpopulations in midgestational embryos. Taken together, these findings enhance our understanding of blood birth from yolk sac HECs and provide theoretical basis and candidate reporters for monitoring step-wise hematopoietic differentiation., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Neovascularization is a key therapeutic target for cancer treatment. However, anti-angiogenic therapies have shown modest success, as tumors develop rapid resistance to treatment owing to activation of redundant pathways that aid vascularization. We hypothesized that simultaneously targeting different pathways of neovascularization will circumvent the current issue of drug resistance and offer enhanced therapeutic benefits. To test this hypothesis, we made use of two distinct models of tumor-neovascularization, which exhibit equally dense microvasculature but show disparate sensitivity to anti-SDF-1 treatment. Lewis lung carcinoma (LLC) is primarily a vasculogenic-tumor that is associated with HSC functioning as a hemangioblast to generate circulating Endothelial Progenitor Cells contributing to formation of new blood vessels, and responds to anti-SDF-1 treatment. B16F0 melanoma is an angiogenic-tumor that derives new blood vessels from existing vasculature and is resistant to anti-SDF-1 therapy. In this study, we observed increased expression of the angiogenic-factor, Robo1 predominantly expressed on the blood vessels of B16F0 tumor. Blockade of Robo1 by the decoy receptor, RoboN, resulted in reduced microvascular-density and tumor-growth. However, this was associated with mobilization of BM-cells into the B16F0 tumor, thus switching the mode of neovascularization from angiogenic to vasculogenic. The use of a combinatorial treatment of RoboN and the monoclonal anti-SDF-1 antibody effectively attenuated tumor-growth and inhibited both angiogenic and BM-derived microvessels., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Pharyngeal arch artery (PAA) progenitors undergo proliferative expansion and angioblast differentiation to build vessels connecting the heart with the dorsal aortae. However, it remains unclear whether and how these two processes are orchestrated. Here we demonstrate that Tmem88 is required to fine-tune PAA progenitor proliferation and differentiation. Loss of zebrafish tmem88a/b leads to an excessive expansion and a failure of differentiation of PAA progenitors. Moreover, tmem88a/b deficiency enhances cyclin D1 expression in PAA progenitors via aberrant Wnt signal activation. Mechanistically, cyclin D1-CDK4/6 promotes progenitor proliferation through accelerating the G1/S transition while suppressing angioblast differentiation by phosphorylating Nkx2.5/Smad3. Ectodermal Wnt2bb signaling is confined by Tmem88 in PAA progenitors to ensure a balance between proliferation and differentiation. Therefore, the proliferation and angioblast differentiation of PAA progenitors manifest an inverse relationship and are delicately regulated by cell cycle machinery downstream of the Tmem88-Wnt pathway., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Hemogenic endothelium (HE) plays a pivotal and inevitable role in haematopoiesis and can generate all blood and endothelial lineage cells in the aorta-gonad-mesonephros of mouse embryos. Whether definitive HE can prospectively isolate pure HE from human pluripotent stem cells that can spontaneously differentiate into heterogeneous cells remains unknown. Here, we identified and validated a CD34 dim subpopulation with hemogenic potential. We also purified CD34 cells with a CXCR4 - CD73 - phenotype as a definitive HE population that generated haematopoietic stem cells and lymphocytes. The frequency of CXCR4 - CD73 - CD34 dim was evidently increased by bone morphogenetic protein 4, and purified HE cells differentiated into haematopoietic cells with myeloid and T lymphoid lineages including Vδ2+ subset of γ/δ T cells. We developed a simple method to purify HE cells that were enriched in CD34 dim cells. We uncovered an initial step in differentiating haematopoietic lineage cells that could be applied to basic and translational investigations into regenerative medicine., (© 2022 The Authors. Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.)

Recent studies have highlighted the crucial role of the aorta microenvironment in the generation of the first haematopoietic stem cells (HSCs) from specialized haemogenic endothelial cells (HECs). Despite more than two decades of investigations, we require a better understanding of the cellular and molecular events driving aorta formation and polarization, which will be pivotal to establish the mechanisms that operate during HEC specification and HSC competency. Here, we outline the early mechanisms involved in vertebrate aorta formation by comparing four different species: zebrafish, chicken, mouse and human. We highlight how this process, which is tightly controlled in time and space, requires a coordinated specification of several cell types, in particular endothelial cells originating from distinct mesodermal tissues. We also discuss how molecular signals originating from the aorta environment result in its polarization, creating a unique entity for HSC generation., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

The E26 transformation-specific or E-twenty-six (ETS) genes encode a superfamily of transcription factors involved in diverse biological processes. Here, we report the identification and characterization of a previously unidentified member of the ETS transcription factors, Spi2, that is found exclusively in the ray-finned fish kingdom. We show that the expression of spi2 is restricted to hemogenic endothelial cells (HECs) and to hematopoietic stem and progenitor cells (HSPCs) in zebrafish. Using bacteria artificial chromosome transgenesis, we generate a spi2 reporter line, TgBAC(spi2:P2a-GFP), which manifests the GFP pattern recapitulating the endogenous spi2 expression. Genetic ablation of spi2 has little effect on HEC formation and the endothelial-to-hematopoietic transition, but results in compromised proliferation of HSPCs in the caudal hematopoietic tissue (CHT) during early development and in severe myeloid lineage defect in adulthood. Epistatic analysis shows that spi2 acts downstream of runx1 in regulating HSPC development in the CHT. Our study identifies Spi2 as an essential regulator for definitive hematopoietic cell development and creates a TgBAC(spi2:P2a-GFP) reporter line for tracking HECs, HSPCs, myeloid cells and thrombocytes from early development to adulthood., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Haematopoietic stem and progenitor cells emerge from specialized haemogenic endothelial cells in select vascular beds during embryonic development. Specification and commitment to the blood lineage, however, occur before endothelial cells are endowed with haemogenic competence, at the time of mesoderm patterning and production of endothelial cell progenitors (angioblasts). Whilst early blood cell fate specification has long been recognized, very little is known about the mechanisms that induce endothelial cell diversification and progressive acquisition of a blood identity by a subset of these cells. Here, we review the endothelial origin of the haematopoietic system and the complex developmental journey of blood-fated angioblasts. We discuss how recent technological advances will be instrumental to examine the diversity of the embryonic anatomical niches, signaling pathways and downstream epigenetic and transcriptional processes controlling endothelial cell heterogeneity and blood cell fate specification. Ultimately, this will give essential insights into the ontogeny of the cells giving rise to haematopoietic stem cells, that may aid in the development of novel strategies for their in vitro production for clinical purposes., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

To achieve efficient, reproducible differentiation of human pluripotent stem cells (hPSCs) towards specific hematopoietic cell-types, a comprehensive understanding of the necessary cell signaling and developmental trajectories involved is required. Previous studies have identified the mesodermal progenitors of extra-embryonic-like and intra-embryonic-like hemogenic endothelium (HE), via stage-specific WNT and ACTIVIN/NODAL, with GYPA/GYPB (CD235a/b) expression serving as a positive selection marker for mesoderm harboring exclusively extra-embryonic-like hemogenic potential. However, a positive mesodermal cell-surface marker with exclusively intra-embryonic-like hemogenic potential has not been identified. Recently, we reported that early mesodermal expression of CDX4 critically regulates definitive HE specification, suggesting that CDX4 may act in a cell-autonomous manner during hematopoietic development. To identify CDX4+ mesoderm, we performed single cell (sc)RNAseq on hPSC-derived mesodermal cultures, revealing CDX4 hi expressing mesodermal populations were uniquely enriched in the non-classical MHC-Class-1 receptor CD1D. Flow cytometry demonstrated approximately 60% of KDR+CD34-CD235a- mesoderm was CD1d+, and CDX4 was robustly enriched within CD1d+ mesoderm. Critically, only CD1d+ mesoderm harbored CD34+ HOXA+ HE with multilineage erythroid-myeloid-lymphoid potential. Thus, CDX4+CD1d+ expression within early mesoderm demarcates an early progenitor of HE. These insights may be used for further study of human hematopoietic development and improve hematopoietic differentiation conditions for regenerative medicine applications., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Vascular establishment is one of the early events in embryogenesis. It is believed that vessel-initiating endothelial progenitors cluster to form the first primitive vessel. Understanding the molecular identity of these progenitors is crucial in order to elucidate lineage hierarchy. In this study, we identify protein C receptor (Procr) as an endothelial progenitor marker and investigate the role of Procr+ progenitors during embryonic vascular development. Using a ProcrmGFP-2A-lacZ reporter, we reveal a much earlier Procr expression (embryonic day 7.5) than previously acknowledged (embryonic day 13.5). Genetic fate-mapping experiments using ProcrCre and ProcrCreER demonstrate that Procr+ cells give rise to blood vessels throughout the entire embryo proper. Single-cell RNA-sequencing analyses place Procr+ cells at the start of endothelial commitment and maturation. Furthermore, targeted ablation of Procr+ cells results in failure of vessel formation and early embryonic lethality. Notably, genetic fate mapping and scRNA-seq pseudotime analysis support the view that Procr+ progenitors can give rise to hemogenic endothelium. In this study, we establish a Procr expression timeline and identify Procr+ vessel-initiating progenitors, and demonstrate their indispensable role in establishment of the vasculature during embryo development., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

The generation of human hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) represents a major goal in regenerative medicine and is believed would follow principles of early development. HSCs arise from a type of endothelial cell called a "hemogenic endothelium" (HE), and human HSCs are experimentally detected by transplantation into SCID or other immune-deficient mouse recipients, termed SCID-Repopulating Cells (SRC). Recently, SRCs were detected by forced expression of seven transcription factors (TF) (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1, and SPI1) in hPSC-derived HE, suggesting these factors are deficient in hPSC differentiation to HEs required to generate HSCs. Here we derived PECAM-1-, Flk-1-, and VE-cadherin-positive endothelial cells that also lack CD45 expression (PFV CD45- ) which are solely responsible for hematopoietic output from iPSC lines reprogrammed from AML patients. Using HEs derived from AML patient iPSCs devoid of somatic leukemic aberrations, we sought to generate putative SRCs by the forced expression of 7TFs to model autologous HSC transplantation. The expression of 7TFs in hPSC-derived HE cells from an enhanced hematopoietic progenitor capacity was present in vitro, but failed to acquire SRC activity in vivo. Our findings emphasize the benefits of forced TF expression, along with the continued challenges in developing HSCs for autologous-based therapies from hPSC sources.

Epigenetic modification is an important process during hematopoietic cell differentiation. Histone deacetylase (HDAC) inhibitors have previously been shown to enhance expansion of umbilical cord blood-derived hematopoietic stem cells (HSCs). However, the effect of HDAC inhibitors on pluripotent stem cells (PSCs) in this context is less understood. For years, investigators have considered PSC-derived natural killer (NK) and T-cell therapies. These "off-the-shelf" cellular therapies are now entering the clinic. However, the in vitro commitment of PSCs to the hematopoietic lineage is inefficient and represents a major bottleneck. We investigated whether HDAC inhibitors (HDACi) influence human PSC differentiation into CD34+CD45+ hematopoietic stem and progenitor cells (HSPCs), focusing on hemogenic endothelium (HE). Pluripotent stem cells cultured in the presence of HDACi showed a 2-5 times increase in HSPCs. Concurrent with this, HDACi-treated PSCs increased expression of 7 transcription factors (HOXA5, HOXA9, HOXA10, RUNX1, ERG, SPI1, and LCOR) recently shown to convert HE to HSPCs. ChIP-qPCR showed that SAHA upregulated acetylated-H3 at the promoter region of the above key genes. SAHA-treated human PSC-derived CD34+CD45+ cells showed primary engraftment in immunodeficient mice, but not serial transplantation. We further demonstrate that SAHA-derived HSPCs could differentiate into functional NK cells in vitro. The addition of SAHA is an easy and effective approach to overcoming the bottleneck in the transition from PSC to HSPCs for "off-the-shelf" cellular immunotherapy., (© The Author(s) 2022. Published by Oxford University Press.)

The earliest hematopoietic stem and progenitor cells (HSPCs) are generated from the ventral wall of the dorsal aorta, through endothelial-to-hematopoietic transition during vertebrate embryogenesis. Notch signaling is crucial for HSPC generation across vertebrates; however, the precise control of Notch during this process remains unclear. In the present study, we used multi-omics approaches together with functional assays to assess global DNA methylome dynamics during the endothelial cells to HSPCs transition in zebrafish, and determined that DNA methyltransferase 1 (Dnmt1) is essential for HSPC generation via repression of Notch signaling. Depletion of dnmt1 resulted in decreased DNA methylation levels and impaired HSPC production. Mechanistically, we found that loss of dnmt1 induced hypomethylation of Notch genes and consequently elevated Notch activity in hemogenic endothelial cells, thereby repressing the generation of HSPCs. This finding deepens our understanding of HSPC specification in vivo, which will provide helpful insights for designing new strategies for HSPC generation in vitro., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

The hematopoietic stem cells (HSCs) that produce blood for the lifetime of an animal arise from RUNX1+ hemogenic endothelial cells (HECs) in the embryonic vasculature through a process of endothelial-to-hematopoietic transition (EHT). Studies have identified inflammatory mediators and fluid shear forces as critical environmental stimuli for EHT, raising the question of how such diverse inputs are integrated to drive HEC specification. Endothelial cell MEKK3-KLF2/4 signaling can be activated by both fluid shear forces and inflammatory mediators, and it plays roles in cardiovascular development and disease that have been linked to both stimuli. Here we demonstrate that MEKK3 and KLF2/4 are required in endothelial cells for the specification of RUNX1+ HECs in both the yolk sac and dorsal aorta of the mouse embryo and for their transition to intraaortic hematopoietic cluster (IAHC) cells. The inflammatory mediators lipopolysaccharide and interferon-γ increase RUNX1+ HECs in an MEKK3-dependent manner. Maternal administration of catecholamines that stimulate embryo cardiac function and accelerate yolk sac vascular remodeling increases EHT by wild-type but not MEKK3-deficient endothelium. These findings identify MEKK-KLF2/4 signaling as an essential pathway for EHT and provide a molecular basis for the integration of diverse environmental inputs, such as inflammatory mediators and hemodynamic forces, during definitive hematopoiesis., (© 2022 by The American Society of Hematology.)

Objectives: During embryonic haematopoiesis, haematopoietic stem/progenitor cells (HSPCs) develop from hemogenic endothelial cells (HECs) though endothelial to haematopoietic transition (EHT). However, little is known about how EHT is regulated in human. Here, we report that GFI1 plays an essential role in enabling normal EHT during haematopoietic differentiation of human embryonic stem cells (hESCs)., Results: GFI1 deletion in hESCs leads to a complete EHT defect due to a closed chromatin state of hematopoietic genes in HECs. Mechanically, directly regulates important signaling pathways essential for the EHT such as PI3K signaling.etc., Conclutions: Together, our findings reveal an essential role of GFI1 mediated epigenetic mechanism underlying human EHT during hematopoiesis., (© 2022 The Authors. Cell Proliferation published by John Wiley & Sons Ltd.)

Hemogenic endothelial (HE) cells in the dorsal aorta undergo an endothelial-to-hematopoietic transition (EHT) to form multipotent progenitors, lympho-myeloid biased progenitors (LMPs), pre-hematopoietic stem cells (pre-HSCs) and adult-repopulating HSCs. These briefly accumulate in intra-arterial hematopoietic clusters (IAHCs) before being released into the circulation. It is generally assumed that the number of IAHC cells correlates with the number of HSCs. Here, we show that changes in the number of IAHC cells, LMPs and HSCs can be uncoupled. Mutations impairing MyD88-dependent toll-like receptor (TLR) signaling decreased the number of IAHC cells and LMPs, but increased the number of HSCs in the aorta-gonad-mesonephros region of mouse embryos. TLR4-deficient embryos generated normal numbers of HE cells, but IAHC cell proliferation decreased. Loss of MyD88-dependent TLR signaling in innate immune myeloid cells had no effect on IAHC cell numbers. Instead, TLR4 deletion in endothelial cells (ECs) recapitulated the phenotype observed with germline deletion, demonstrating that MyD88-dependent TLR signaling in ECs and/or in IAHCs regulates the numbers of LMPs and HSCs., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

During embryonic development, hematopoiesis occurs through primitive and definitive waves, giving rise to distinct blood lineages. Hematopoietic stem cells (HSCs) emerge from hemogenic endothelial (HE) cells, through endothelial-to-hematopoietic transition (EHT). In the adult, HSC quiescence, maintenance, and differentiation are closely linked to changes in metabolism. However, metabolic processes underlying the emergence of HSCs from HE cells remain unclear. Here, we show that the emergence of blood is regulated by multiple metabolic pathways that induce or modulate the differentiation toward specific hematopoietic lineages during human EHT. In both in vitro and in vivo settings, steering pyruvate use toward glycolysis or OXPHOS differentially skews the hematopoietic output of HE cells toward either an erythroid fate with primitive phenotype, or a definitive lymphoid fate, respectively. We demonstrate that glycolysis-mediated differentiation of HE toward primitive erythroid hematopoiesis is dependent on the epigenetic regulator LSD1. In contrast, OXPHOS-mediated differentiation of HE toward definitive hematopoiesis is dependent on cholesterol metabolism. Our findings reveal that during EHT, metabolism is a major regulator of primitive versus definitive hematopoietic differentiation., (© 2021 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

In vitro generation and expansion of hematopoietic stem cells (HSCs) holds great promise for the treatment of any ailment that relies on bone marrow or blood transplantation. To achieve this, it is essential to resolve the molecular and cellular pathways that govern HSC formation in the embryo. HSCs first emerge in the aorta-gonad-mesonephros (AGM) region, where a rare subset of endothelial cells, hemogenic endothelium (HE), undergoes an endothelial-to-hematopoietic transition (EHT). Here, we present full-length single-cell RNA sequencing (scRNA-seq) of the EHT process with a focus on HE and dorsal aorta niche cells. By using Runx1b and Gfi1/1b transgenic reporter mouse models to isolate HE, we uncovered that the pre-HE to HE continuum is specifically marked by angiotensin-I converting enzyme (ACE) expression. We established that HE cells begin to enter the cell cycle near the time of EHT initiation when their morphology still resembles endothelial cells. We further demonstrated that RUNX1 AGM niche cells consist of vascular smooth muscle cells and PDGFRa+ mesenchymal cells and can functionally support hematopoiesis. Overall, our study provides new insights into HE differentiation toward HSC and the role of AGM RUNX1+ niche cells in this process. Our expansive scRNA-seq datasets represents a powerful resource to investigate these processes further., (© 2022 by The American Society of Hematology.)

Definitive long-term hematopoietic stem cells (LT-HSCs) arise during embryogenesis in a process termed endothelial-to-hematopoietic transition (EHT), in which specialized hemogenic endothelial cells (HECs) transform into hematopoietic cells. The transcription factor RUNX1 marks HECs and is essential for EHT. Ectopic RUNX1 expression in non-HECs is sufficient to convert them into HECs. However, the conversion efficiency depends on the developmental timing of expression. In this issue of Genes & Development , Howell and colleagues (pp. 1475-1489) leverage this observation to further understand how RUNX1 mediates EHT. They engineered mice that ectopically express RUNX1 in endothelial cells at different developmental time points and doses. They then performed chromatin accessibility and other analyses and correlate this with hemogenic potential. They found that RUNX1 collaborates with TGFβ signaling transcription factors to drive chromatin accessibility changes that specify HECs. They also highlight interesting parallels between EHT and endothelial-to-mesenchymal transition (EndoMT), which occurs during cardiac development. The results of Howell and colleagues provide new mechanistic insights into EHT and take us one step closer to generating patient-specific LT-HSCs from induced pluripotent stem cells., (© 2021 Samarakkody and Cantor; Published by Cold Spring Harbor Laboratory Press.)

Hematopoietic stem and progenitor cells (HSPCs) are generated de novo in the embryo from hemogenic endothelial cells (HECs) via an endothelial-to-hematopoietic transition (EHT) that requires the transcription factor RUNX1. Ectopic expression of RUNX1 alone can efficiently promote EHT and HSPC formation from embryonic endothelial cells (ECs), but less efficiently from fetal or adult ECs. Efficiency correlated with baseline accessibility of TGFβ-related genes associated with endothelial-to-mesenchymal transition (EndoMT) and participation of AP-1 and SMAD2/3 to initiate further chromatin remodeling along with RUNX1 at these sites. Activation of TGFβ signaling improved the efficiency with which RUNX1 specified fetal ECs as HECs. Thus, the ability of RUNX1 to promote EHT depends on its ability to recruit the TGFβ signaling effectors AP-1 and SMAD2/3, which in turn is determined by the changing chromatin landscape in embryonic versus fetal ECs. This work provides insight into regulation of EndoMT and EHT that will guide reprogramming efforts for clinical applications., (© 2021 Howell et al.; Published by Cold Spring Harbor Laboratory Press.)

During embryogenesis, waves of hematopoietic progenitors develop from hemogenic endothelium (HE) prior to the emergence of self-renewing hematopoietic stem cells (HSCs). Although previous studies have shown that yolk-sac-derived erythromyeloid progenitors and HSCs emerge from distinct populations of HE, it remains unknown whether the earliest lymphoid-competent progenitors, multipotent progenitors, and HSCs originate from common HE. In this study, we demonstrate by clonal assays and single-cell transcriptomics that rare HE with functional HSC potential in the early murine embryo are distinct from more abundant HE with multilineage hematopoietic potential that fail to generate HSCs. Specifically, HSC-competent HE are characterized by expression of CXCR4 surface marker and by higher expression of genes tied to arterial programs regulating HSC dormancy and self-renewal. Taken together, these findings suggest a revised model of developmental hematopoiesis in which the initial populations of multipotent progenitors and HSCs arise independently from HE with distinct phenotypic and transcriptional properties., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

During hematopoietic development, definitive hematopoietic cells are derived from hemogenic endothelial (HE) cells through a process known as endothelial to hematopoietic transition (EHT). During EHT, transitioning cells proliferate and undergo progressive changes in gene expression culminating in the new cell identity with corresponding changes in function, phenotype and morphology. However, the metabolic pathways fueling this transition remain unclear. We show here that glutamine is a crucial regulator of EHT and a rate limiting metabolite in the hematopoietic differentiation of HE cells. Intriguingly, different hematopoietic lineages require distinct derivatives of glutamine. While both derivatives, α-ketoglutarate and nucleotides, are required for early erythroid differentiation of HE during glutamine deprivation, lymphoid differentiation relies on α-ketoglutarate alone. Furthermore, treatment of HE cells with α-ketoglutarate in glutamine-free conditions pushes their differentiation towards lymphoid lineages both in vitro and in vivo, following transplantation into NSG mice. Thus, we report an essential role for glutamine metabolism during EHT, regulating both the emergence and the specification of hematopoietic cells through its various derivatives., (© 2021. The Author(s).)

Hematopoietic ontogeny consists of two broad programs: an initial hematopoietic stem cell (HSC)-independent program followed by HSC-dependent hematopoiesis that sequentially seed the fetal liver and generate blood cells. However, the transition from HSC-independent to HSC-derived hematopoiesis remains poorly characterized. To help resolve this question, we developed Mds1 CreERT2 mice, which inducibly express Cre-recombinase in emerging HSCs in the aorta and label long-term adult HSCs, but not HSC-independent yolk-sac-derived primitive or definitive erythromyeloid (EMP) hematopoiesis. Our lineage-tracing studies indicate that HSC-derived erythroid, myeloid, and lymphoid progeny significantly expand in the liver and blood stream between E14.5 and E16.5. Additionally, we find that HSCs contribute the majority of F4/80+ macrophages in adult spleen and marrow, in contrast to their limited contribution to macrophage populations in brain, liver, and lungs. The Mds1 CreERT2 mouse model will be useful to deconvolute the complexity of hematopoiesis as it unfolds in the embryo and functions postnatally., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021. Published by Elsevier Inc.)

Macrophages play pivotal roles in immunity, hematopoiesis, and tissue homeostasis. In mammals, macrophages have been shown to originate from yolk-sac-derived erythro-myeloid progenitors and aorta-gonad-mesonephros (AGM)-derived hematopoietic stem cells. However, whether macrophages can arise from other embryonic sites remains unclear. Here, using single-cell RNA sequencing, we profile the transcriptional landscape of mouse fetal placental hematopoiesis. We uncover and experimentally validate that a CD44 + subpopulation of placental endothelial cells (ECs) exhibits hemogenic potential. Importantly, lineage tracing using the newly generated Hoxa13 reporter line shows that Hoxa13-labeled ECs can produce placental macrophages, named Hofbauer cell (HBC)-like cells. Furthermore, we identify two subtypes of HBC-like cells, and cell-cell interaction analysis identifies their potential roles in angiogenesis and antigen presentation, separately. Our study provides a comprehensive understanding of placental hematopoiesis and highlights the placenta as a source of macrophages, which has important implications for both basic and translational research., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Across species, hematopoietic stem and progenitor cells (HSPCs) arise during embryogenesis from a specialized arterial population, termed hemogenic endothelium. Here, we describe a mechanistic role for the epigenetic regulator, Enhancer of zeste homolog-1 (Ezh1), in vertebrate HSPC production via regulation of hemogenic commitment. Loss of ezh1 in zebrafish embryos favored acquisition of hemogenic (gata2b) and HSPC (runx1) fate at the expense of the arterial program (ephrinb2a, dll4). In contrast, ezh1 overexpression blocked hematopoietic progression via maintenance of arterial gene expression. The related Polycomb group subunit, Ezh2, functioned in a non-redundant, sequential manner, whereby inhibition had no impact on arterial identity, but was capable of blocking ezh1-knockdown-associated HSPC expansion. Single-cell RNA sequencing across ezh1 genotypes revealed a dropout of ezh1 +/- cells among arterial endothelium associated with positive regulation of gene transcription. Exploitation of Ezh1/2 modulation has potential functional relevance for improving in vitro HSPC differentiation from induced pluripotent stem cell sources., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Current understanding of hematopoietic stem cell (HSC) development comes from mouse models is considered to be evolutionarily conserved in human. However, the cross-species comparison of the transcriptomic profiles of developmental HSCs at single-cell level is still lacking. Here, we performed integrative transcriptomic analysis of a series of key cell populations during HSC development in human and mouse, including HSC-primed hemogenic endothelial cells and pre-HSCs in mid-gestational aorta-gonad-mesonephros (AGM) region, and mature HSCs in fetal liver and adult bone marrow. We demonstrated the general similarity of transcriptomic characteristics between corresponding cell populations of the two species. Of note, one of the previously transcriptomically defined hematopoietic stem progenitor cell (HSPC) populations with certain arterial characteristics in AGM region of human embryos showed close transcriptomic similarity to pre-HSCs in mouse embryos. On the other hand, the other two HSPC populations in human AGM region displayed molecular similarity with fetal liver HSPCs, suggesting the maturation in AGM before HSCs colonizing the fetal liver in human, which was different to that in mouse. Finally, we re-clustered cells based on the integrated dataset and illustrated the evolutionarily conserved molecular signatures of major cell populations. Our results revealed transcriptomic conservation of critical cell populations and molecular characteristics during HSC development between human and mouse, providing a resource and theoretic basis for future studies on mammalian HSC development and regeneration by using mouse models., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Apelin receptor (APLNR/AGTRLl1/APJ) marks a transient cell population during the differentiation of hematopoietic stem and progenitor cells (HSPCs) from pluripotent stem cells, but its function during the production and maintenance of hematopoietic stem cells is not clear. We generated an Aplnr-tdTomato reporter mouse embryonic stem cell (mESC) line and showed that HSPCs are generated exclusively from mesodermal cells that express Aplnr-tdTomato. HSPC production from mESCs was impaired when Aplnr was deleted, implying that this pathway is required for their production. To address the role of APLNR signaling in HSPC maintenance, we added APELIN ligands to ex vivo AGM cultures. Activation of the APLNR pathway in this system impaired the generation of long-term reconstituting HSPCs and appeared to drive myeloid differentiation. Our data suggest that the APLNR signaling is required for the generation of cells that give rise to HSCs, but that its subsequent downregulation is required for their maintenance., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Extra-embryonic mesoderm (ExM)-composed of the earliest cells that traverse the primitive streak-gives rise to the endothelium as well as haematopoietic progenitors in the developing yolk sac. How a specific subset of ExM becomes committed to a haematopoietic fate remains unclear. Here we demonstrate using an embryonic stem cell model that transient expression of the T-box transcription factor Eomesodermin (Eomes) governs haemogenic competency of ExM. Eomes regulates the accessibility of enhancers that the transcription factor stem cell leukaemia (SCL) normally utilizes to specify primitive erythrocytes and is essential for the normal development of Runx1 + haemogenic endothelium. Single-cell RNA sequencing suggests that Eomes loss of function profoundly blocks the formation of blood progenitors but not specification of Flk-1 + haematoendothelial progenitors. Our findings place Eomes at the top of the transcriptional hierarchy regulating early blood formation and suggest that haemogenic competence is endowed earlier during embryonic development than was previously appreciated.

During embryogenesis, lymphoid tissue inducer (LTi) cells are essential for lymph node organogenesis. These cells are part of the innate lymphoid cell (ILC) family. Although their earliest embryonic hematopoietic origin is unclear, other innate immune cells have been shown to be derived from early hemogenic endothelium in the yolk sac as well as the aorta-gonad-mesonephros. A proper model to discriminate between these locations was unavailable. In this study, using a Cxcr4-CreERT2 lineage tracing model, we identify a major contribution from embryonic hemogenic endothelium, but not the yolk sac, toward LTi progenitors. Conversely, embryonic LTi cells are replaced by hematopoietic stem cell-derived cells in adults. We further show that, in the fetal liver, common lymphoid progenitors differentiate into highly dynamic alpha-lymphoid precursor cells that, at this embryonic stage, preferentially mature into LTi precursors and establish their functional LTi cell identity only after reaching the periphery., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Hox genes encode transcription factors that have been implicated in embryonic, adult and disease processes. The earliest developmental program known to be directed by Hox genes is the timing of ingression of presumptive axial mesoderm during gastrulation. We previously used morpholino (MO)-based knockdown to implicate the zebrafish hoxd4a gene in the specification of the hemangioblast, an event occurring at pre-gastrulation stages, well before the earliest known Hox gene function. The precise time at which hoxd4a function is required for this specification is not defined. We therefore fused the hoxd4a coding region to the human estrogen receptor (hER T2 ). Following co-injection of anti-hoxd4a MO with mRNA encoding the Hoxd4a-ER T2 fusion protein, hemangioblast specification was fully rescued when embryos were exposed to the estrogen analog 4-hydroxy-tamoxifen (4-OHT) at 4 hr post-fertilization (hpf), but only poorly at 6 hpf and not at all at 8 hpf, thereby defining a pre-gastrulation role for Hoxd4a, the earliest developmental function of a vertebrate Hox gene so far described. Both DNA binding and interaction with cofactor Pbx were further shown to be required for rescue of the morphant phenotype. Confirmation of the morphant phenotype was sought via the generation of hoxd4a null mutants using CRISPR/Cas9 technology. Null mutants of hoxd4a up to the third generation (F 3 ) failed to recapitulate the morphant phenotype, and were largely refractory to the effects of injected anti-hoxd4a MO suggesting the action of genetic compensation., (© 2020 Wiley Periodicals, Inc.)

ADP-ribosylation factor-like 4aa (Arl4aa) is a member of the ADP-ribosylation factor family. It is expressed in hematopoietic tissue during embryonic development, but its function was unknown. Zebrafish arl4aa is preferentially expressed in the ventral wall of the dorsal aorta (VDA) at 24 and 36 hpf and in caudal hematopoietic tissue at 48 hpf. Morpholino knockdown and transcription activator-like effector nuclease (TALEN) knockout of arl4aa significantly reduced expression of genes associated with definitive hematopoietic stem cells (HSCs). Golgi complex integrity in VDA was disrupted as shown by transmission electron microscopy and immunostaining of Golgi membrane Giantin. Mechanistically, arl4aa knockdown reduced Notch signaling in the VDA and its target gene expression. Protein expression of NICD was also reduced. Effects of arl4aa knockdown on definitive hematopoiesis could be restored by NICD expression. This study identified arl4aa as a factor regulating initiation of definitive HSCs by maintaining the integrity of Golgi complex and, secondarily, maturation of the Notch receptor., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Transcription factor (TF)-based reprogramming of somatic tissues holds great promise for regenerative medicine. Previously, we demonstrated that the TFs GATA2, GFI1B, and FOS convert mouse and human fibroblasts to hemogenic endothelial-like precursors that generate hematopoietic stem progenitor (HSPC)-like cells over time. This conversion is lacking in robustness both in yield and biological function. Herein, we show that inclusion of GFI1 to the reprogramming cocktail significantly expands the HSPC-like population. AFT024 coculture imparts functional potential to these cells and allows quantification of stem cell frequency. Altogether, we demonstrate an improved human hemogenic induction protocol that could provide a valuable human in vitro model of hematopoiesis for disease modeling and a platform for cell-based therapeutics. DATABASE: Gene expression data are available in the Gene Expression Omnibus (GEO) database under the accession number GSE130361., (© 2019 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Objectives: Cell-based therapy has emerged as a promising strategy for the treatment of patients with heart failure. Increasing evidence supports the hypothesis that paracrine mechanisms mediated by soluble factors released by the cells play a predominate role in reparative processes. The aim of our study was to analyze which cytokines are released by CD34 + enriched cell products intended for autologous transendocardial CD34 + cell transplantation in patients with cardiomyopathy., Material and Methods: The peripheral blood CD34 + cells from 12 patients were mobilized with granulocyte colony-stimulating factor, collected via apheresis and enriched by immunoselection., Results: In CD34 + enriched cell population, hematopoietic, but not mesenchymal or endothelial, progenitors were detected. Except for angiopoietin-1, other measured cytokines (FGF1, FGF2, VEGF, PDGF, IL-6, HGH, SDF-1α/CXCL12, NRG1) were not released by CD34 + cells. The average concentration of angiopoietin-1 released by 5×10 6 CD34 + cells grown in neutral DMEM medium was 213.6±130.0pg/mL (range: 74-448pg/mL). Angiopoietin-1 secretion correlated well with CD34 + cell's capacity for generating colonies derived from hematopoietic progenitors (Pearson's correlation=0.964; P<0.001)., Conclusion: Our study presents angiopoietin-1 as an interesting candidate and suggests future studies to explore how its release by CD34 + cells might impact the success of autologous CD34 + cell transplantation., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

It is well established that haematopoietic stem and progenitor cells (HSPCs) are generated from a transient subset of specialized endothelial cells termed haemogenic, present in the yolk sac, placenta and aorta, through an endothelial-to-haematopoietic transition (EHT). HSPC generation via EHT is thought to be restricted to the early stages of development. By using experimental embryology and genetic approaches in birds and mice, respectively, we document here the discovery of a bone marrow haemogenic endothelium in the late fetus/young adult. These cells are capable of de novo producing a cohort of HSPCs in situ that harbour a very specific molecular signature close to that of aortic endothelial cells undergoing EHT or their immediate progenies, i.e., recently emerged HSPCs. Taken together, our results reveal that HSPCs can be generated de novo past embryonic stages. Understanding the molecular events controlling this production will be critical for devising innovative therapies.

Tracing the emergence of the first hematopoietic stem cells (HSCs) in human embryos, particularly the scarce and transient precursors thereof, is so far challenging, largely due to the technical limitations and the material rarity. Here, using single-cell RNA sequencing, we constructed the first genome-scale gene expression landscape covering the entire course of endothelial-to-HSC transition during human embryogenesis. The transcriptomically defined HSC-primed hemogenic endothelial cells (HECs) were captured at Carnegie stage (CS) 12-14 in an unbiased way, showing an unambiguous feature of arterial endothelial cells (ECs) with the up-regulation of RUNX1, MYB and ANGPT1. Importantly, subcategorizing CD34 + CD45 - ECs into a CD44 + population strikingly enriched HECs by over 10-fold. We further mapped the developmental path from arterial ECs via HSC-primed HECs to hematopoietic stem progenitor cells, and revealed a distinct expression pattern of genes that were transiently over-represented upon the hemogenic fate choice of arterial ECs, including EMCN, PROCR and RUNX1T1. We also uncovered another temporally and molecularly distinct intra-embryonic HEC population, which was detected mainly at earlier CS 10 and lacked the arterial feature. Finally, we revealed the cellular components of the putative aortic niche and potential cellular interactions acting on the HSC-primed HECs. The cellular and molecular programs that underlie the generation of the first HSCs from HECs in human embryos, together with the ability to distinguish the HSC-primed HECs from others, will shed light on the strategies for the production of clinically useful HSCs from pluripotent stem cells.

In vertebrates, GATA2 is a master regulator of hematopoiesis and is expressed throughout embryo development and in adult life. Although the essential role of GATA2 in mouse hematopoiesis is well established, its involvement during early human hematopoietic development is not clear. By combining time-controlled overexpression of GATA2 with genetic knockout experiments, we found that GATA2, at the mesoderm specification stage, promotes the generation of hemogenic endothelial progenitors and their further differentiation to hematopoietic progenitor cells, and negatively regulates cardiac differentiation. Surprisingly, genome-wide transcriptional and chromatin immunoprecipitation analysis showed that GATA2 bound to regulatory regions, and repressed the expression of cardiac development-related genes. Moreover, genes important for hematopoietic differentiation were upregulated by GATA2 in a mostly indirect manner. Collectively, our data reveal a hitherto unrecognized role of GATA2 as a repressor of cardiac fates, and highlight the importance of coordinating the specification and repression of alternative cell fates., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Blood is an example of a highly regenerative tissue and its regeneration depends on the presence of stem cells residing in the bone marrow in humans. A better understanding of how these stem cells are programmed would benefit their use in clinical practice and shed light on the mechanisms by which the unique properties of stem cells are established. Our approach is to delineate the gene regulatory networks (GRNs) that specify these cells during their development in the embryo, and we use the amphibian experimental model because a wealth of evidence shows that the mechanisms used are conserved in mammals including humans. Blood stem cells are made during the intraembryonic wave of hematopoiesis during embryonic development where they emerge from endothelial precursors in the floor of the dorsal aorta (DA). These cells are derived from lateral plate mesoderm and so we have focused on the subset of cells in the lateral plate mesoderm fated to become blood and endothelium known as definitive hemangioblasts. We have found that their programming results from the activities of vascular endothelial growth factor A (VEGFA) and bone morphogenetic protein (BMP) signaling and the inhibition by miRNA of transforming growth factor beta signaling. VEGFA is first generated in the somites adjacent to the lateral plate mesoderm, and one of the responses of the lateral plate mesoderm is to activate endogenous VEGFA expression. BMP has multiple inputs into the programming of these cells via the activation of the transcription factor (TF), Gata2, and of the VEGFA receptor. These actions culminate in the expression of the leukemia-associated TF, Scl/Tal1, which is essential for blood fate specification. The activity of VEGFA in driving endothelial development resides in the small isoform, but the medium and large isoforms are required to initiate the blood stem cell program in the floor of the DA. The expression of the small isoform is dependent on the blood TF with leukemia connections, Tel1/Etv6, whereas the larger isoforms depend on another transcription-associated factor with leukemia connections, Eto2, raising the possibility that the regulation of VEGFA expression may be the mode of action of these leukemic factors. The action of Tel1/Etv6 in directly activating VEGFA expression in the somites was unexpected because this TF had only been reported to repress transcription. Using chromatin immunoprecipitation technology, we were able to show that Tel1/Etv6 does indeed work by repressing the expression of a VEGFA repressor, FoxC3, but it also acts directly to activate VEGFA expression, working together with Klf4. Finally, we have also looked at the mesodermal population that gives rise to the earlier waves of hematopoiesis, which do not generate a stem cell. We find significant differences including differential use of TFs of the E-Twenty-Six (ETS) family. In conclusion, we have elucidated the GRN responsible for preparing the lateral mesoderm for blood stem cell production.