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.

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 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.

Extracellular vesicles of stromal origin target and support hematopoietic stem and progenitor cells.

Extracellular vesicles (EVs) have been recently reported as crucial mediators in cell-to-cell communication in development and disease. In this study, we investigate whether mesenchymal stromal cells that constitute a supportive microenvironment for hematopoietic stem and progenitor cells (HSPCs) released EVs that could affect the gene expression and function of HSPCs. By taking advantage of two fetal liver-derived stromal lines with widely differing abilities to maintain HSPCs ex vivo, we demonstrate that stromal EVs play a critical role in the regulation of HSPCs. Both supportive and nonsupportive stromal lines secreted EVs, but only those delivered by the supportive line were taken up by HSPCs ex vivo and in vivo. These EVs harbored a specific molecular signature, modulated the gene expression in HSPCs after uptake, and maintained the survival and clonogenic potential of HSPCs, presumably by preventing apoptosis. In conclusion, our study reveals that EVs are an important component of the HSPC niche, which may have major applications in regenerative medicine.

Molecular signature of erythroblast enucleation in human embryonic stem cells.

While enucleation is a critical step in the terminal differentiation of human red blood cells, the molecular mechanisms underlying this unique process remain unclear. To investigate erythroblast enucleation, we studied the erythroid differentiation of human embryonic stem cells (hESCs), which provide a unique model for deeper understanding of the development and differentiation of multiple cell types. First, using a two-step protocol, we demonstrated that terminal erythroid differentiation from hESCs is directly dependent on the age of the embryoid bodies. Second, by choosing hESCs in two extreme conditions of erythroid culture, we obtained an original differentiation model which allows one to study the mechanisms underlying the enucleation of erythroid cells by analyzing the gene and miRNA (miR) expression profiles of cells from these two culture conditions. Third, using an integrated analysis of mRNA and miR expression profiles, we identified five miRs potentially involved in erythroblast enucleation. Finally, by selective knockdown of these five miRs we found miR-30a to be a regulator of erythroblast enucleation in hESCs.

Transcriptomic analysis of mouse limb tendon cells during development.

The molecular signals driving tendon development are not fully identified. We have undertaken a transcriptome analysis of mouse limb tendon cells that were isolated at different stages of development based on scleraxis (Scx) expression. Microarray comparisons allowed us to establish a list of genes regulated in tendon cells during mouse limb development. Bioinformatics analysis of the tendon transcriptome showed that the two most strongly modified signalling pathways were TGF-ß and MAPK. TGF-ß/SMAD2/3 gain- and loss-of-function experiments in mouse limb explants and mesenchymal stem cells showed that TGF-ß signalling was sufficient and required via SMAD2/3 to drive mouse mesodermal stem cells towards the tendon lineage ex vivo and in vitro. TGF-ß was also sufficient for tendon gene expression in late limb explants during tendon differentiation. FGF does not have a tenogenic effect and the inhibition of the ERK MAPK signalling pathway was sufficient to activate Scx in mouse limb mesodermal progenitors and mesenchymal stem cells.

A systems biology approach for defining the molecular framework of the hematopoietic stem cell niche.

Despite progress in identifying the cellular composition of hematopoietic stem/progenitor cell (HSPC) niches, little is known about the molecular requirements of HSPC support. To address this issue, we used a panel of six recognized HSPC-supportive stromal lines and less-supportive counterparts originating from embryonic and adult hematopoietic sites. Through comprehensive transcriptomic meta-analyses, we identified 481 mRNAs and 17 microRNAs organized in a modular network implicated in paracrine signaling. Further inclusion of 18 additional cell strains demonstrated that this mRNA subset was predictive of HSPC support. Our gene set contains most known HSPC regulators as well as a number of unexpected ones, such as Pax9 and Ccdc80, as validated by functional studies in zebrafish embryos. In sum, our approach has identified the core molecular network required for HSPC support. These cues, along with a searchable web resource, will inform ongoing efforts to instruct HSPC ex vivo amplification and formation from pluripotent precursors.

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.

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.

The roles of BMP and IL-3 signaling pathways in the control of hematopoietic stem cells in the mouse embryo.

During mouse ontogeny, the first adult-type hematopoietic stem cells (HSC) are autonomously generated at mid-gestation in the AGM (Aorta-Gonad-Mesonephros) region. Successively present in different anatomical sites where they will expand, HSCs will finally colonize the bone marrow (BM) where they will reside during the entire adult life. In the bone marrow, both HSC self-renewal and differentiation are controlled at cellular and molecular levels by interactions with the stromal microenvironment. So far, very little is known about the extracellular factors involved in the regulation of embryonic HSC emergence, survival and expansion. In the present review, we outline the BMP and IL-3 signaling pathways that are critical for the growth and potential of embryonic HSCs. We will also discuss how these pathways might be integrated with the ones of Notch and Mpl/thrombopoietin, also identified as important key regulators of AGM HSC activity.

Embryonic stromal clones reveal developmental regulators of definitive hematopoietic stem cells.

Hematopoietic stem cell (HSC) self-renewal and differentiation is regulated by cellular and molecular interactions with the surrounding microenvironment. During ontogeny, the aorta-gonad-mesonephros (AGM) region autonomously generates the first HSCs and serves as the first HSC-supportive microenvironment. Because the molecular identity of the AGM microenvironment is as yet unclear, we examined two closely related AGM stromal clones that differentially support HSCs. Expression analyses identified three putative HSC regulatory factors, beta-NGF (a neurotrophic factor), MIP-1gamma (a C-C chemokine family member) and Bmp4 (a TGF-beta family member). We show here that these three factors, when added to AGM explant cultures, enhance the in vivo repopulating ability of AGM HSCs. The effects of Bmp4 on AGM HSCs were further studied because this factor acts at the mesodermal and primitive erythropoietic stages in the mouse embryo. In this report, we show that enriched E11 AGM HSCs express Bmp receptors and can be inhibited in their activity by gremlin, a Bmp antagonist. Moreover, our results reveal a focal point of Bmp4 expression in the mesenchyme underlying HSC containing aortic clusters at E11. We suggest that Bmp4 plays a relatively late role in the regulation of HSCs as they emerge in the midgestation AGM.

Mesenchymal lineage potentials of aorta-gonad-mesonephros stromal clones.

BACKGROUND AND OBJECTIVES: The characterization of stem cell microenvironments throughout ontogeny is of fundamental interest in the field of stem cell biology. Within the adult blood system, hematopoietic stem cells (HSC) are supported in the osteoblastic and endothelial bone marrow microenvironments. During mouse mid-gestation, the first HSC emerge autonomously in the aorta-gonad-mesonephros (AGM) region. However, little is known about this microenvironment. To study the cellular complexity of the AGM hematopoietic microenvironment and its relationship to HSC, we examined the potential of AGM stromal clones to differentiate into several mesenchymal lineages. DESIGN AND METHODS: Stromal cell clones from the mid-gestation mouse were cultured in appropriate conditions known to support osteogenic, adipogenic, chondrogenic and endothelial differentiation. Potentials of the stromal cells were scored by morphological examination of the cultures, specific staining and gene expression profile. RESULTS: We show that most clones possess uni/bilineage osteogenic, adipogenic and/or endothelial potential. The differentiation potential of the stromal clones appears to relate to their site of origin but not to their ability to support hematopoiesis. Moreover, we show that AGM HSC activity is unaffected by the osteogenic differentiation of UG26.1B6 stromal cells. INTERPRETATION AND CONCLUSIONS: These results confirm the existence of mesenchymal stem/progenitor cells in the AGM region and suggest that the AGM hematopoietic microenvironment is highly complex, containing stromal cells with various mesenchymal lineage potentials.

An unexpected role for IL-3 in the embryonic development of hematopoietic stem cells.

Cytokines are important in adult hematopoiesis, yet their function in embryonic hematopoiesis has been largely unexplored. During development, hematopoietic stem cells (HSCs) are found in the aorta-gonad-mesonephros (AGM) region, yolk sac (YS), and placenta and require the Runx1 transcription factor for their normal generation. Since IL-3 is a Runx1 target and this cytokine acts on adult hematopoietic cells, we examined whether IL-3 affects HSCs in the mouse embryo. Using Runx1 haploinsufficient mice, we show that IL-3 amplifies HSCs from E11 AGM, YS, and placenta. Moreover, we show that IL-3 mutant embryos are deficient in HSCs and that IL-3 reveals the presence of HSCs in the AGM and YS prior to the stage at which HSCs are normally detected. Thus, our studies support an unexpected role for IL-3 during development and strongly suggest that IL-3 functions as a proliferation and/or survival factor for the earliest HSCs in the embryo.

Embryonic beginnings of adult hematopoietic stem cells.

Hematopoietic stem cells (HSC) are at the foundation of the adult hematopoietic system. HSC give rise to all blood cells through a complex series of proliferation and differentiation events that occur throughout the lifespan of the individual. Because of their clinical importance in transplantation protocols, recent research has focused on the developmental origins and potential of embryonic HSC. In both mammalian and non-mammalian vertebrate embryos, two independent anatomical sites have been found to generate hematopoietic cells. The yolk sac (or its equivalent in amphibians, the ventral blood islands) participates in a first transient wave of hematopoiesis by producing primitive erythrocytes. Importantly, adult-type HSCs emerge autonomously in a second wave of hematopoietic generation in an intraembryonic region surrounding the dorsal aorta, the aorta-gonads-mesonephros (AGM) region. In this review, we will discuss research advances in the field of developmental hematopoiesis, with a particular emphasis on the cellular origins of AGM HSC and their regulation by the embryonic hematopoietic microenvironment.

Identification and expression of Helios, a member of the Ikaros family, in the Mexican axolotl: implications for the embryonic origin of lymphocyte progenitors.

Transcription factors of the Ikaros gene family are critical for the differentiation of T and B lymphocytes from pluripotent hematopoietic stem cells. To study the first steps of lymphopoiesis in the Mexican axolotl, we have cloned the Helios ortholog in this urodele amphibian species. We demonstrated that the axolotl Helios contains a 144-bp deletion at the 5' end of the activation domain. Helios is expressed in both the thymus and spleen but not in the liver of the pre-adult axolotl. During ontogeny, Helios transcripts are detected from neurula stage, before the apparition of the first Ikaros transcripts and the colonization of lymphoid tissues. Interestingly, Helios and Ikaros mRNA are found predominantly in the ventral blood islands of late tail-bud embryos. These results suggest that in contrast to the Xenopus and amniote embryos where two sites of hematopoiesis have been characterized, the ventral blood islands could be the major site of hematopoiesis in the axolotl.

Structure, diversity and expression of the TCRdelta chains in the Mexican axolotl.

Mammals and birds have two major populations of T cells, based on the molecular composition and biological properties of their antigen receptors (TCR). alpha beta T cells recognize antigenic peptides linked to major histocompatibility complex (MHC) molecules, and gamma delta T cells recognize native peptide or non-peptide antigens independently of MHC. Very little is known about gamma delta T cells in ectothermic vertebrates. We have cloned and characterized the TCRdelta chains of an urodele amphibian, the Mexican axolotl (Ambystoma mexicanum). The Cdelta domain is structurally similar to its mammalian homologues and the transmembrane domain is very well conserved. Four of the six Valpha regions that can associate with Calpha (Valpha2, Valpha3, Valpha5 and Valpha6) can also associate with Cdelta, but no specific Vdelta regions were found. This suggests that the axolotl TRD locus is nested within the TRA locus, as in mammals, and that this organization has been present in all tetrapod vertebrates and in the common ancestor of Lissamphibians and mammals, for over 400 million years. Two Jdelta regions were identified, but no Ddelta segments were clearly recognized at the Vdelta-Jdelta junctions. This results in shorter and less variable CDR3 loops than in other vertebrates and the size range of the Vdelta-Jdelta junctions is similar to that of mammalian immunoglobulin light chains. Equivalent quantities of TRD mRNA were found in the lymphoid organs, and in the skin and the intestines of normal and thymectomized axolotls. The analysis of several Valpha/delta6-Cdelta and Vbeta7-Cbeta junctions showed that both the TCRdelta and the TCRbeta chains were limited in diversity in thymectomized axolotls.