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.

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.

[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.

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.

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.

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.

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.

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.

Inferring Gene Networks in Bone Marrow Hematopoietic Stem Cell-Supporting Stromal Niche Populations.

The cardinal property of bone marrow (BM) stromal cells is their capacity to contribute to hematopoietic stem cell (HSC) niches by providing mediators assisting HSC functions. In this study we first contrasted transcriptomes of stromal cells at different developmental stages and then included large number of HSC-supportive and non-supportive samples. Application of a combination of algorithms, comprising one identifying reliable paths and potential causative relationships in complex systems, revealed gene networks characteristic of the BM stromal HSC-supportive capacity and of defined niche populations of perivascular cells, osteoblasts, and mesenchymal stromal cells. Inclusion of single-cell transcriptomes enabled establishing for the perivascular cell subset a partially oriented graph of direct gene-to-gene interactions. As proof of concept we showed that R-spondin-2, expressed by the perivascular subset, synergized with Kit ligand to amplify ex vivo hematopoietic precursors. This study by identifying classifiers and hubs constitutes a resource to unravel candidate BM stromal mediators.

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.

In vivo generation of haematopoietic stem/progenitor cells from bone marrow-derived haemogenic endothelium.

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.

Adaptive dynamics of hematopoietic stem cells and their supporting stroma: a model and mathematical analysis.

We propose a mathematical model to describe the evolution of hematopoietic stem cells (HSCs) and stromal cells in considering the bi-directional interaction between them. Cancerous cells are also taken into account in our model. HSCs are structured by a continuous phenotype characterising the population heterogeneity in a way relevant to the question at stake while stromal cells are structured by another continuous phenotype representing their capacity of support to HSCs. We then analyse the model in the framework of adaptive dynamics. More precisely, we study single Dirac mass steady states, their linear stability and we investigate the role of parameters in the model on the nature of the evolutionary stable distributions (ESDs) such as monomorphism, dimorphism and the uniqueness properties. We also study the dominant phenotypes by an asymptotic approach and we obtain the equation for dominant phenotypes. Numerical simulations are employed to illustrate our analytical results. In particular, we represent the case of the invasion of malignant cells as well as the case of co-existence of cancerous cells and healthy HSCs.

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.