ABSTRACT
Thymus is necessary for lifelong immunological tolerance and immunity. It displays a distinctive epithelial complexity and undergoes age-dependent atrophy. Nonetheless, it also retains regenerative capacity, which, if harnessed appropriately, might permit rejuvenation of adaptive immunity. By characterizing cortical and medullary compartments in the human thymus at single-cell resolution, in this study we have defined specific epithelial populations, including those that share properties with bona fide stem cells (SCs) of lifelong regenerating epidermis. Thymic epithelial SCs display a distinctive transcriptional profile and phenotypic traits, including pleiotropic multilineage potency, to give rise to several cell types that were not previously considered to have shared origin. Using here identified SC markers, we have defined their cortical and medullary niches and shown that, in vitro, the cells display long-term clonal expansion and self-organizing capacity. These data substantively broaden our knowledge of SC biology and set a stage for tackling thymic atrophy and related disorders.
Subject(s)
Stem Cells , Thymus Gland , Humans , Cell Differentiation , Stem Cells/metabolism , Thymus Gland/metabolism , Cells, Cultured , Epithelial Cells/metabolism , Atrophy/metabolismABSTRACT
The bone-marrow (BM) niche is the spatial environment composed by a network of multiple stromal components regulating adult hematopoiesis. We use multi-omics and computational tools to analyze multiple BM environmental compartments and decipher their mutual interactions in the context of acute myeloid leukemia (AML) xenografts. Under homeostatic conditions, we find a considerable overlap between niche populations identified using current markers. Our analysis defines eight functional clusters of genes informing on the cellular identity and function of the different subpopulations and pointing at specific stromal interrelationships. We describe how these transcriptomic profiles change during human AML development and, by using a proximity-based molecular approach, we identify early disease onset deregulated genes in the mesenchymal compartment. Finally, we analyze the BM proteomic secretome in the presence of AML and integrate it with the transcriptome to predict signaling nodes involved in niche alteration in AML.
Subject(s)
Bone Marrow Cells/metabolism , Leukemia, Myeloid, Acute/genetics , Proteomics/methods , Animals , Humans , Mice , Tumor MicroenvironmentABSTRACT
Myelodysplastic syndrome (MDS) are clonal stem cell diseases characterized mainly by ineffective hematopoiesis. Here, we present an approach that enables robust long-term engraftment of primary MDS stem cells (MDS-SCs) in mice by implantation of human mesenchymal cell-seeded scaffolds. Critically for modelling MDS, where patient sample material is limiting, mononuclear bone marrow cells containing as few as 104 CD34+ cells can be engrafted and expanded by this approach with the maintenance of the genetic make-up seen in the patients. Non-invasive high-resolution ultrasound imaging shows that these scaffolds are fully perfused. Our data shows that human microenvironment but not mouse is essential to MDS-SCs homing and engraftment. Notably, the alternative niche provided by healthy donor MSCs enhanced engraftment of MDS-SCs. This study characterizes a new tool to model MDS human disease with the level of engraftment previously unattainable in mice, and offers insights into human-specific determinants of MDS-SC microenvironment.
Subject(s)
Mesenchymal Stem Cells , Myelodysplastic Syndromes , Animals , Bone Marrow Cells , Hematopoiesis , Humans , Mice , Stem CellsABSTRACT
Acute myeloid leukemia (AML) is a heterogeneous disease, yet clinically most patients present with pancytopenia resulting from bone marrow failure, predisposing them to life-threatening infections and bleeding. The mechanisms by which AML mediates hematopoietic suppression is not well known. Indeed, much effort has so far been focused on how AML remodels the bone marrow niche to make it a more permissive environment, with less focus on how the remodeled niche affects normal hematopoietic cells. In this perspective, we present evidence of the key role of the bone marrow niche in suppressing hematopoietic stem cells (HSCs) during leukemic progression and provide perspectives on how future research on this topic may be exploited to provide treatments for one of the key complications of AML.
Subject(s)
Hematopoietic Stem Cells/cytology , Leukemia, Myeloid, Acute/pathology , Neoplastic Stem Cells/cytology , Stem Cell Niche , Tumor Microenvironment , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Cell Communication , Cell Hypoxia , Deferoxamine/pharmacology , Disease Progression , Endothelial Cells/cytology , Endothelial Cells/drug effects , Exosomes/physiology , Hematopoiesis , Hematopoietic Stem Cells/drug effects , Homeostasis , Humans , Leukemia, Myeloid, Acute/drug therapy , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/drug effects , Neoplastic Stem Cells/drug effects , Recurrence , Tumor Cells, CulturedABSTRACT
Adult haematopoietic stem cells (HSCs) mainly reside in the bone marrow, where stromal and haematopoietic cells regulate their function. The steady state HSC niche has been extensively studied. In this Review, we focus on how bone marrow microenvironment components respond to different insults including inflammation, malignant haematopoiesis and chemotherapy. We highlight common and unique patterns among multiple cell types and their environment and discuss current limitations in our understanding of this complex and dynamic tissue.
Subject(s)
Cell Differentiation/physiology , Hematopoiesis/physiology , Hematopoietic Stem Cells/metabolism , Stem Cell Niche/physiology , Animals , Bone Marrow/metabolism , Environment , HumansABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
Hematopoietic stem cells (HSCs) develop in the embryonic aorta-gonad-mesonephros (AGM) region and subsequently relocate to fetal liver. Runx1 transcription factor is essential for HSC development, but is largely dispensable for adult HSCs. Here, we studied tamoxifen-inducible Runx1 inactivation in vivo. Induction at pre-liver stages (up to embryonic day 10.5) reduced erythromyeloid progenitor numbers, but surprisingly did not block the appearance of Runx1-null HSCs in liver. By contrast, ex vivo analysis showed an absolute Runx1 dependency of HSC development in the AGM region. We found that, contrary to current beliefs, significant Cre-inducing tamoxifen activity persists in mouse blood for at least 72 hr after injection. This deferred recombination can hit healthy HSCs, which escaped early Runx1 ablation and result in appearance of Runx1-null HSCs in liver. Such extended recombination activity in vivo is a potential source of misinterpretation, particularly in analysis of dynamic developmental processes during embryogenesis.
Subject(s)
Aorta/embryology , Core Binding Factor Alpha 2 Subunit/genetics , Hematopoietic Stem Cells/cytology , Liver/embryology , Mesonephros/embryology , Animals , Aorta/cytology , Core Binding Factor Alpha 2 Subunit/metabolism , Female , Gene Deletion , Hematopoiesis , Hematopoietic Stem Cells/metabolism , Liver/cytology , Mesonephros/cytology , Mice, Inbred C57BL , Mice, TransgenicABSTRACT
During development, hematopoietic stem cells (HSCs) emerge in the aorta-gonad-mesonephros (AGM) region through a process of multi-step maturation and expansion. While proliferation of adult HSCs is implicated in the balance between self-renewal and differentiation, very little is known about the proliferation status of nascent HSCs in the AGM region. Using Fucci reporter mice that enable in vivo visualization of cell-cycle status, we detect increased proliferation during pre-HSC expansion followed by a slowing down of cycling once cells start to acquire a definitive HSC state, similar to fetal liver HSCs. We observe time-specific changes in intra-aortic hematopoietic clusters corresponding to HSC maturation stages. The proliferative architecture of the clusters is maintained in an orderly anatomical manner with slowly cycling cells at the base and more actively proliferating cells at the more apical part of the cluster, which correlates with c-KIT expression levels, thus providing an anatomical basis for the role of SCF in HSC maturation.
Subject(s)
Aorta/metabolism , Hematopoietic Stem Cells/metabolism , Animals , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Proliferation , Cells, Cultured , Core Binding Factor Alpha 2 Subunit/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Embryo, Mammalian/metabolism , Genes, Reporter , Gonads/metabolism , Hematopoietic Stem Cells/cytology , Leukocyte Common Antigens/metabolism , Mesonephros/metabolism , Mice , Mice, Inbred C57BL , Microscopy, Fluorescence , Platelet Membrane Glycoprotein IIb/metabolism , Proto-Oncogene Proteins c-kit/metabolismABSTRACT
The first definitive hematopoietic stem cells (dHSCs) in the mouse emerge in the dorsal aorta of the embryonic day (E) 10.5 to 11 aorta-gonad-mesonephros (AGM) region. Notch signaling is essential for early HSC development but is dispensable for the maintenance of adult bone marrow HSCs. How Notch signaling regulates HSC formation in the embryo is poorly understood. We demonstrate here that Notch signaling is active in E10.5 HSC precursors and involves both Notch1 and Notch2 receptors, but is gradually downregulated while they progress toward dHSCs at E11.5. This downregulation is accompanied by gradual functional loss of Notch dependency. Thus, as early as at final steps in the AGM region, HSCs begin acquiring the Notch independency characteristic of adult bone marrow HSCs as part of the maturation program. Our data indicate that fine stage-dependent tuning of Notch signaling may be required for the generation of definitive HSCs from pluripotent cells.
Subject(s)
Aorta/embryology , Embryo, Mammalian/cytology , Gonads/embryology , Hematopoietic Stem Cells/cytology , Mesonephros/embryology , Receptor, Notch2/metabolism , Stromal Cells/cytology , Animals , Aorta/metabolism , Cells, Cultured , Embryo, Mammalian/metabolism , Gonads/metabolism , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/metabolism , Mesonephros/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Signal Transduction , Stromal Cells/metabolismABSTRACT
During embryonic development, adult haematopoietic stem cells (HSCs) emerge preferentially in the ventral domain of the aorta in the aorta-gonad-mesonephros (AGM) region. Several signalling pathways such as Notch, Wnt, Shh and RA are implicated in this process, yet how these interact to regulate the emergence of HSCs has not previously been described in mammals. Using a combination of ex vivo and in vivo approaches, we report here that stage-specific reciprocal dorso-ventral inductive interactions and lateral input from the urogenital ridges are required to drive HSC development in the aorta. Our study strongly suggests that these inductive interactions in the AGM region are mediated by the interplay between spatially polarized signalling pathways. Specifically, Shh produced in the dorsal region of the AGM, stem cell factor in the ventral and lateral regions, and BMP inhibitory signals in the ventral tissue are integral parts of the regulatory system involved in the development of HSCs.
Subject(s)
Aorta/metabolism , Gonads/metabolism , Hematopoietic Stem Cells/metabolism , Mesonephros/metabolism , Signal Transduction , Animals , Aorta/embryology , Bone Morphogenetic Protein 4/genetics , Bone Morphogenetic Protein 4/metabolism , Female , Gonads/embryology , Hedgehog Proteins/genetics , Hedgehog Proteins/metabolism , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/cytology , Male , Mesonephros/embryology , Mice, Inbred C57BLABSTRACT
Definitive hematopoietic stem cells (HSCs) develop in the aorta gonad mesonephros (AGM) region in a stepwise manner. Type I pre-HSCs express CD41 but lack CD45 expression, which is subsequently upregulated in type II pre-HSCs prior to their maturation into definitive HSCs. Here, using ex vivo modeling of HSC development, we identify precursors of definitive HSCs in the trunk of the embryonic day 9.5 (E9.5) mouse embryo. These precursors, termed here pro-HSCs, are less mature than type I and II pre-HSCs. Although pro-HSCs are CD41(+), they lack the CD43 marker, which is gradually upregulated in the developing HSC lineage. We show that stem cell factor (SCF), but not interleukin-3 (IL-3), is a major effector of HSC maturation during E9-E10. This study extends further the previously established hierarchical organization of the developing HSC lineage and presents it as a differentially regulated four-step process and identifies additional targets that could facilitate the generation of transplantable HSCs from pluripotent cells for clinical needs.
Subject(s)
Hematopoiesis , Hematopoietic Stem Cells/cytology , Interleukin-3/metabolism , Leukosialin/genetics , Stem Cell Factor/metabolism , Animals , Aorta/embryology , Cell Lineage , Embryo, Mammalian/embryology , Embryo, Mammalian/metabolism , Gene Deletion , Hematopoietic Stem Cells/metabolism , Mesonephros/embryology , Mice , Mice, Inbred C57BLABSTRACT
Haematopoiesis in adult animals is maintained by haematopoietic stem cells (HSCs), which self-renew and can give rise to all blood cell lineages. The AGM region is an important intra-embryonic site of HSC development and a wealth of evidence indicates that HSCs emerge from the endothelium of the embryonic dorsal aorta and extra-embryonic large arteries. This, however, is a stepwise process that occurs through sequential upregulation of CD41 and CD45 followed by emergence of fully functional definitive HSCs. Although largely dispensable at later stages, the Runx1 transcription factor is crucially important during developmental maturation of HSCs; however, exact points of crucial involvement of Runx1 in this multi-step developmental maturation process remain unclear. Here, we have investigated requirements for Runx1 using a conditional reversible knockout strategy. We report that Runx1 deficiency does not preclude formation of VE-cad+CD45-CD41+ cells, which are phenotypically equivalent to precursors of definitive HSCs (pre-HSC Type I) but blocks transition to the subsequent CD45+ stage (pre-HSC Type II). These data emphasise that developmental progression of HSCs during a very short period of time is regulated by precise stage-specific molecular mechanisms.