A multistep computational approach reveals a neuro-mesenchymal cell population in the embryonic hematopoietic stem cell niche.

The first hematopoietic stem and progenitor cells (HSPCs) emerge in the Aorta-Gonad-Mesonephros (AGM) region of the mid-gestation mouse embryo. However, the precise nature of their supportive mesenchymal microenvironment remains largely unexplored. Here, we profiled transcriptomes of laser micro-dissected aortic tissues at three developmental stages and individual AGM cells. Computational analyses allowed the identification of several cell subpopulations within the E11.5 AGM mesenchyme, with the presence of a yet unidentified subpopulation characterized by the dual expression of genes implicated in adhesive or neuronal functions. We confirmed the identity of this cell subset as a neuro-mesenchymal population, through morphological and lineage tracing assays. Loss of function in the zebrafish confirmed that Decorin, a characteristic extracellular matrix component of the neuro-mesenchyme, is essential for HSPC development. We further demonstrated that this cell population is not merely derived from the neural crest, and hence, is a bona fide novel subpopulation of the AGM mesenchyme.

Dorsal aorta polarization and haematopoietic stem cell emergence.

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.

Portal fibroblasts with mesenchymal stem cell features form a reservoir of proliferative myofibroblasts in liver fibrosis.

BACKGROUND AND AIMS: In liver fibrosis, myofibroblasts derive from HSCs and as yet undefined mesenchymal cells. We aimed to identify portal mesenchymal progenitors of myofibroblasts. APPROACH AND RESULTS: Portal mesenchymal cells were isolated from mouse bilio-vascular tree and analyzed by single-cell RNA-sequencing. Thereby, we uncovered the landscape of portal mesenchymal cells in homeostatic mouse liver. Trajectory analysis enabled inferring a small cell population further defined by surface markers used to isolate it. This population consisted of portal fibroblasts with mesenchymal stem cell features (PMSCs), i.e., high clonogenicity and trilineage differentiation potential, that generated proliferative myofibroblasts, contrasting with nonproliferative HSC-derived myofibroblasts (-MF). Using bulk RNA-sequencing, we built oligogene signatures of the two cell populations that remained discriminant across myofibroblastic differentiation. SLIT2, a prototypical gene of PMSC/PMSC-MF signature, mediated profibrotic and angiogenic effects of these cells, which conditioned medium promoted HSC survival and endothelial cell tubulogenesis. Using PMSC/PMSC-MF 7-gene signature and slit guidance ligand 2 fluorescent in situ hybridization, we showed that PMSCs display a perivascular portal distribution in homeostatic liver and largely expand with fibrosis progression, contributing to the myofibroblast populations that form fibrotic septa, preferentially along neovessels, in murine and human liver disorders, irrespective of etiology. We also unraveled a 6-gene expression signature of HSCs/HSC-MFs that did not vary in these disorders, consistent with their low proliferation rate. CONCLUSIONS: PMSCs form a small reservoir of expansive myofibroblasts, which, in interaction with neovessels and HSC-MFs that mainly arise through differentiation from a preexisting pool, underlie the formation of fibrotic septa in all types of liver diseases.

The quail genome: insights into social behaviour, seasonal biology and infectious disease response.

BACKGROUND: The Japanese quail (Coturnix japonica) is a popular domestic poultry species and an increasingly significant model species in avian developmental, behavioural and disease research. RESULTS: We have produced a high-quality quail genome sequence, spanning 0.93 Gb assigned to 33 chromosomes. In terms of contiguity, assembly statistics, gene content and chromosomal organisation, the quail genome shows high similarity to the chicken genome. We demonstrate the utility of this genome through three diverse applications. First, we identify selection signatures and candidate genes associated with social behaviour in the quail genome, an important agricultural and domestication trait. Second, we investigate the effects and interaction of photoperiod and temperature on the transcriptome of the quail medial basal hypothalamus, revealing key mechanisms of photoperiodism. Finally, we investigate the response of quail to H5N1 influenza infection. In quail lung, many critical immune genes and pathways were downregulated after H5N1 infection, and this may be key to the susceptibility of quail to H5N1. CONCLUSIONS: We have produced a high-quality genome of the quail which will facilitate further studies into diverse research questions using the quail as a model avian species.

An in vitro model of hemogenic endothelium commitment and hematopoietic production.

Adult-type hematopoietic stem and progenitor cells are formed during ontogeny from a specialized subset of endothelium, termed the hemogenic endothelium, via an endothelial-to-hematopoietic transition (EHT) that occurs in the embryonic aorta and the associated arteries. Despite efforts to generate models, little is known about the mechanisms that drive endothelial cells to the hemogenic fate and about the subsequent molecular control of the EHT. Here, we have designed a stromal line-free controlled culture system utilizing the embryonic pre-somitic mesoderm to obtain large numbers of endothelial cells that subsequently commit into hemogenic endothelium before undergoing EHT. Monitoring the culture for up to 12 days using key molecular markers reveals stepwise commitment into the blood-forming system that is reminiscent of the cellular and molecular changes occurring during hematopoietic development at the level of the aorta. Long-term single-cell imaging allows tracking of the EHT of newly formed blood cells from the layer of hemogenic endothelial cells. By modifying the culture conditions, it is also possible to modulate the endothelial cell commitment or the EHT or to produce smooth muscle cells at the expense of endothelial cells, demonstrating the versatility of the cell culture system. This method will improve our understanding of the precise cellular changes associated with hemogenic endothelium commitment and EHT and, by unfolding these earliest steps of the hematopoietic program, will pave the way for future ex vivo production of blood cells.

The crosstalk between hematopoietic stem cells and their niches.

PURPOSE OF REVIEW: Hematopoietic stem cells (HSCs) reside in specific microenvironments also called niches that regulate HSC functions. Understanding the molecular and cellular mechanisms involved in the crosstalk between HSCs and niche cells is a major issue in stem cell biology and regenerative medicine. The purpose of this review is to discuss recent advances in this field with particular emphasis on the transcriptional landscape of HSC niche cells and the roles of extracellular vesicles (EVs) in the dialog between HSCs and their microenvironments. RECENT FINDINGS: The development of high-throughput technologies combined with computational methods has considerably improved our knowledge on the molecular identity of HSC niche cells. Accumulating evidence strongly suggest that the dialog between HSCs and their niches is bidirectional and that EVs play an important role in this process. SUMMARY: These advances bring a unique conceptual and methodological framework for understanding the molecular complexity of the HSC niche and identifying novel HSC regulators. They are also promising for exploring the reciprocal influence of HSCs on niche cells and delivering specific molecules to HSCs in regenerative medicine.

The European Hematology Association Roadmap for European Hematology Research: a consensus document.

The European Hematology Association (EHA) Roadmap for European Hematology Research highlights major achievements in diagnosis and treatment of blood disorders and identifies the greatest unmet clinical and scientific needs in those areas to enable better funded, more focused European hematology research. Initiated by the EHA, around 300 experts contributed to the consensus document, which will help European policy makers, research funders, research organizations, researchers, and patient groups make better informed decisions on hematology research. It also aims to raise public awareness of the burden of blood disorders on European society, which purely in economic terms is estimated at €23 billion per year, a level of cost that is not matched in current European hematology research funding. In recent decades, hematology research has improved our fundamental understanding of the biology of blood disorders, and has improved diagnostics and treatments, sometimes in revolutionary ways. This progress highlights the potential of focused basic research programs such as this EHA Roadmap.The EHA Roadmap identifies nine 'sections' in hematology: normal hematopoiesis, malignant lymphoid and myeloid diseases, anemias and related diseases, platelet disorders, blood coagulation and hemostatic disorders, transfusion medicine, infections in hematology, and hematopoietic stem cell transplantation. These sections span 60 smaller groups of diseases or disorders.The EHA Roadmap identifies priorities and needs across the field of hematology, including those to develop targeted therapies based on genomic profiling and chemical biology, to eradicate minimal residual malignant disease, and to develop cellular immunotherapies, combination treatments, gene therapies, hematopoietic stem cell treatments, and treatments that are better tolerated by elderly patients.

Dorso-ventral contributions in the formation of the embryonic aorta and the control of aortic hematopoiesis.

The embryonic dorsal aorta plays a pivotal role in the production of the first hematopoietic stem cells (HSCs), the founders of the adult hematopoietic system. HSC production is polarized by being restricted to the aortic floor where a specialized subset of endothelial cells (ECs) endowed with hemogenic properties undergo an endothelial-to-hematopoietic production resulting in the formation of the intra-aortic hematopoietic clusters. This production is tightly time- and space-controlled with the transcription factor Runx1 playing a key role in this process and the surrounding tissues controlling the aortic shape and fate. In this paper, we shall review (a) how hemogenic ECs differentiate from the mesoderm, (b) how the different aortic components assemble coordinately to establish the dorso-ventral polarity, and (c) how this results in the initiation of Runx1 expression in hemogenic ECs and the initiation of the hematopoietic program. These observations should elucidate the first steps in HSC commitment and help in developing techniques to manipulate adult HSCs.

Identification of the pre-T-cell receptor alpha chain in nonmammalian vertebrates challenges the structure-function of the molecule.

In humans and mice, the early development of αß T cells is controlled by the pre-T-cell receptor α chain (pTα) that is covalently associated with the T-cell receptor ß (TCRß) chain to form the pre-T-cell receptor (pre-TCR) at the thymocyte surface. Pre-TCR functions in a ligand-independent manner through self-oligomerization mediated by pTα. Using in silico and gene synteny-based approaches, we identified the pTα gene (PTCRA) in four sauropsid (three birds and one reptile) genomes. We also identified 25 mammalian PTCRA sequences now covering all mammalian lineages. Gene synteny around PTCRA is remarkably conserved in mammals but differences upstream of PTCRA in sauropsids suggest chromosomal rearrangements. PTCRA organization is highly similar in sauropsids and mammals. However, comparative analyses of the pTα functional domains indicate that sauropsids, monotremes, marsupials, and lagomorphs display a short pTα cytoplasmic tail and lack most residues shown to be critical for human and murine pre-TCR self-oligomerization. Chicken PTCRA transcripts similar to those in mammals were detected in immature double-negative and double-positive thymocytes. These findings give clues about the evolution of this key molecule in amniotes and suggest that the ancestral function of pTα was exclusively to enable expression of the TCRß chain at the thymocyte surface and to allow binding of pre-TCR to the CD3 complex. Together, our data provide arguments for revisiting the current model of pTα signaling.

[Exploring together with Françoise Dieterlen the origin of hematopoietic stem cells]. / Plongée avec Françoise Dieterlen dans l'origine des cellules souches hématopoïétiques.

This article summarizes Françoise Dieterlen's major scientific discoveries about the hematopoietic and endothelial systems during her 40 years' career. Her most remarkable achievements include notably the demonstration of an intraembryonic source of hematopoietic stem cells, the characterization of the polarization of the aorta, the identification of a hemogenic endothelium as well as that of the allantois as an organ of hematopoietic amplification in the mouse embryo, and the demonstration of the existence of a hemogenic endothelium capable of generating hematopoietic stem cells in the bone marrow of the chicken and mouse embryo. While this last discovery was not made directly by Françoise Dieterlen, it was inspired by the many conversations I have had with her and the lessons she has taught me throughout my career. Her rich career will forever shape the field of hematopoietic development, in which she will remain a guiding figure.

Title: Plongée avec Françoise Dieterlen dans l'origine des cellules souches hématopoïétiques. Abstract: Cet article récapitule les principales découvertes scientifiques réalisées par Françoise Dieterlen sur le système hématopoïétique et endothélial au cours de sa carrière qui s'est déroulée sur plus de 40 années. Ses contributions, toutes majeures, portent notamment sur la démonstration d'une source intra-embryonnaire de cellules souches hématopoïetiques impliquant la polarisation de l'aorte et la formation d'un endothélium homogénique, la mise en évidence de l'allantoïde comme organe d'amplification hématopoïétique chez l'embryon de souris et la démonstration de l'existence d'un endothélium hémogénique capable de générer des cellules souches hématopoïétiques dans la moelle osseuse de l'embryon de poulet et de souris. Cette dernière découverte, bien que n'ayant pas été réalisée directement par Françoise Dieterlen, a été inspirée par les nombreuses discussions que j'ai pu avoir avec elle et les enseignements qu'elle m'a prodigués au début de ma carrière. Les avancées remarquables accomplies par Françoise Dieterlen dans le champ du développement hématopoïétique sont unanimement reconnues par tous les spécialistes pour qui elle reste à jamais l'une des fondatrices de ce domaine de recherche.

Efficient Vascularization of Kidney Organoids through Intracelomic Transplantation in Chicken Embryos.

Kidney organoids derived from human induced pluripotent stem cells contain nephron-like structures that resemble those in the adult kidney to a certain degree. Unfortunately, their clinical applicability is hampered by the lack of a functional vasculature and consequently limited maturation in vitro. The transplantation of kidney organoids in the celomic cavity of chicken embryos induces vascularization by perfused blood vessels, including the formation of glomerular capillaries, and enhances their maturation. This technique is very efficient, allowing for the transplantation and analysis of large numbers of organoids. This paper describes a detailed protocol for the intracelomic transplantation of kidney organoids in chicken embryos, followed by the injection of fluorescently labeled lectin to stain the perfused vasculature, and the collection of transplanted organoids for imaging analysis. This method can be used to induce and study organoid vascularization and maturation to find clues for enhancing these processes in vitro and improve disease modeling.

Generation of transgene-free hematopoietic stem cells from human induced pluripotent stem cells.

Hematopoietic stem cells (HSCs) are the rare cells responsible for the lifelong curative effects of hematopoietic cell (HC) transplantation. The demand for clinical-grade HSCs has increased significantly in recent decades, leading to major difficulties in treating patients. A promising but not yet achieved goal is the generation of HSCs from pluripotent stem cells. Here, we have obtained vector- and stroma-free transplantable HSCs by differentiating human induced pluripotent stem cells (hiPSCs) using an original one-step culture system. After injection into immunocompromised mice, cells derived from hiPSCs settle in the bone marrow and form a robust multilineage hematopoietic population that can be serially transplanted. Single-cell RNA sequencing shows that this repopulating activity is due to a hematopoietic population that is transcriptionally similar to human embryonic aorta-derived HSCs. Overall, our results demonstrate the generation of HSCs from hiPSCs and will help identify key regulators of HSC production during human ontogeny.

VE-cadherin expression allows identification of a new class of hematopoietic stem cells within human embryonic liver.

Edification of the human hematopoietic system during development is characterized by the production of waves of hematopoietic cells separated in time, formed in distinct embryonic sites (ie, yolk sac, truncal arteries including the aorta, and placenta). The embryonic liver is a major hematopoietic organ wherein hematopoietic stem cells (HSCs) expand, and the future, adult-type, hematopoietic cell hierarchy becomes established. We report herein the identification of a new, transient, and rare cell population in the human embryonic liver, which coexpresses VE-cadherin, an endothelial marker, CD45, a pan-hematopoietic marker, and CD34, a common endothelial and hematopoietic marker. This population displays an outstanding self-renewal, proliferation, and differentiation potential, as detected by in vitro and in vivo hematopoietic assays compared with its VE-cadherin negative counterpart. Based on VE-cadherin expression, our data demonstrate the existence of 2 phenotypically and functionally separable populations of multipotent HSCs in the human embryo, the VE-cadherin(+) one being more primitive than the VE-cadherin(-) one, and shed a new light on the hierarchical organization of the embryonic liver HSC compartment.

Endoglin expression level discriminates long-term hematopoietic from short-term clonogenic progenitor cells in the aorta.

CD105 is an auxiliary receptor for the transforming growth factor beta superfamily, highly expressed on proliferating endothelial cells and adult hematopoietic stem cells. Because CD105 mRNA expression was reported in the developing aortic region, we further characterized its expression profile in the aorta and examined the hematopoietic potential of CD105(+) cells. Aortic endothelial cells, intra-aortic hematopoietic cell clusters and the purified cell fraction enriched in progenitor/hematopoietic stem cell activity expressed CD105. Aortic hematopoietic short-term clonogenic progenitors were highly enriched in the CD105(intermediate) population whereas more immature long-term progenitors/hematopoietic stem cells are contained within the CD105(high) population. This places CD105 on the short list of molecules discriminating short-term versus long-term progenitors in the aorta. Furthermore, decreasing transforming growth factor beta signaling increases the number of clonogenic progenitors. This suggests that CD105 expression level defines a hierarchy among aortic hematopoietic cells allowing purification of clonogenic versus more immature hematopoietic progenitors, and that the transforming growth factor beta pathway plays a critical role in this process.

Single-cell transcriptomics reveals age-resistant maintenance of cell identities, stem cell compartments and differentiation trajectories in long-lived naked mole-rats skin.

Naked mole-rats (NMR) are subterranean rodents characterized by an unusual longevity coupled with an unexplained resistance to aging. In the present study, we performed extensive in situ analysis and single-cell RNA-sequencing comparing young and older animals. At variance with other species, NMR exhibited a striking stability of skin compartments and cell types, which remained stable over time without aging-associated changes. Remarkably, the number of stem cells was constant throughout aging. We found three classical cellular states defining a unique keratinocyte differentiation trajectory that were not altered after pseudo-temporal reconstruction. Epidermal gene expression did not change with aging either. Langerhans cell clusters were conserved, and only a higher basal stem cell expression of Igfbp3 was found in aged animals. In accordance, NMR skin healing closure was similar in young and older animals. Altogether, these results indicate that NMR skin is characterized by peculiar genetic and cellular features, different from those previously demonstrated for mice and humans. The remarkable stability of the aging NMR skin transcriptome likely reflects unaltered homeostasis and resilience.

Vasculogenesis in kidney organoids upon transplantation.

Human induced pluripotent stem cell-derived kidney organoids have potential for disease modeling and to be developed into clinically transplantable auxiliary tissue. However, they lack a functional vasculature, and the sparse endogenous endothelial cells (ECs) are lost upon prolonged culture in vitro, limiting maturation and applicability. Here, we use intracoelomic transplantation in chicken embryos followed by single-cell RNA sequencing and advanced imaging platforms to induce and study vasculogenesis in kidney organoids. We show expansion of human organoid-derived ECs that reorganize into perfused capillaries and form a chimeric vascular network with host-derived blood vessels. Ligand-receptor analysis infers extensive potential interactions of human ECs with perivascular cells upon transplantation, enabling vessel wall stabilization. Perfused glomeruli display maturation and morphogenesis to capillary loop stage. Our findings demonstrate the beneficial effect of vascularization on not only epithelial cell types, but also the mesenchymal compartment, inducing the expansion of ´on target´ perivascular stromal cells, which in turn are required for further maturation and stabilization of the neo-vasculature. The here described vasculogenic capacity of kidney organoids will have to be deployed to achieve meaningful glomerular maturation and kidney morphogenesis in vitro.

Hematopoietic progenitors polarize in contact with bone marrow stromal cells in response to SDF1.

The fate of hematopoietic stem and progenitor cells (HSPCs) is regulated by their interaction with stromal cells in the bone marrow. However, the cellular mechanisms regulating HSPC interaction with these cells and their potential impact on HSPC polarity are still poorly understood. Here we evaluated the impact of cell-cell contacts with osteoblasts or endothelial cells on the polarity of HSPC. We found that an HSPC can form a discrete contact site that leads to the extensive polarization of its cytoskeleton architecture. Notably, the centrosome was located in proximity to the contact site. The capacity of HSPCs to polarize in contact with stromal cells of the bone marrow appeared to be specific, as it was not observed in primary lymphoid or myeloid cells or in HSPCs in contact with skin fibroblasts. The receptors ICAM, VCAM, and SDF1 were identified in the polarizing contact. Only SDF1 was independently capable of inducing the polarization of the centrosome-microtubule network.

Unexpected contribution of fibroblasts to muscle lineage as a mechanism for limb muscle patterning.

Positional information driving limb muscle patterning is contained in connective tissue fibroblasts but not in myogenic cells. Limb muscles originate from somites, while connective tissues originate from lateral plate mesoderm. With cell and genetic lineage tracing we challenge this model and identify an unexpected contribution of lateral plate-derived fibroblasts to the myogenic lineage, preferentially at the myotendinous junction. Analysis of single-cell RNA-sequencing data from whole limbs at successive developmental stages identifies a population displaying a dual muscle and connective tissue signature. BMP signalling is active in this dual population and at the tendon/muscle interface. In vivo and in vitro gain- and loss-of-function experiments show that BMP signalling regulates a fibroblast-to-myoblast conversion. These results suggest a scenario in which BMP signalling converts a subset of lateral plate mesoderm-derived cells to a myogenic fate in order to create a boundary of fibroblast-derived myonuclei at the myotendinous junction that controls limb muscle patterning.

OC-116, the chicken ortholog of mammalian MEPE found in eggshell, is also expressed in bone cells.

In chicken, ovocleidin 116 (OC-116) is found in the eggshell matrix and its encoding gene, OC-116, is expressed in uterine cells. In mammals, its orthologue MEPE encodes the matrix extracellular phosphoglycoprotein (MEPE), which has been shown to be involved in bone mineralization. Using RT-PCR and in situ hybridization on sections, we have checked whether OC-116 was also expressed in osteoblasts and osteocytes during bone development and mineralization in chicken embryos. We monitored OC-116 expression in the tibia and mandible of a growth series of chicken embryos from E3 to E19. Transcripts were identified in the osteoblasts as early as E5 in the tibia and E7 in the mandible, before matrix mineralization, then from these stages onwards in both the osteoblasts lining the mineralized bone matrix and the osteocytes. Therefore, early in chicken ontogeny and as soon as osteogenesis begins, OC-116 is involved. Its function, which remains still unknown, is maintained during further bone growth and mineralization, and later in adult, in which it is recruited for eggshell formation. We hypothesize that the ancestral OC-116/MEPE in a stem amniote was involved in these two functions and that the loss of eggshell in the mammalian lineage has probably favored the recruitment of some MEPE domains toward new functions in osteogenesis and mineralization, and in phosphatemia regulation.