The N-Glycome regulates the endothelial-to-hematopoietic transition

AbstractHematopoietic stem and progenitor cells (HSPCs) that establish and maintain the blood system in adult vertebrates arise from the transdifferentiation of hemogenic endothelial cells (hemECs) during embryogenesis. This endothelial-to-hematopoietic transition (EHT) is tightly regulated, but the mechanisms are poorly understood. Here, we show that microRNA (miR)-223-mediated regulation of N-glycan biosynthesis in endothelial cells (ECs) regulates EHT. Single cell RNA-sequencing revealed that miR-223 is enriched in hemECs and in oligopotent nascent HSPCs. miR-223 restricts the EHT of lymphoid/myeloid lineages by suppressing the expression of mannosyltransferase alg2 and sialyltransferase st3gal2, two enzymes involved in N-linked protein glycosylation. High-throughput glycomics of ECs lacking miR-223 showed a decrease of high mannose versus sialylated complex/hybrid sugars on N-glycoproteins involved in EHT such as the metalloprotease Adam10. Endothelial-specific expression of an N-glycan Adam10 mutant or of the N-glycoenzymes phenocopied the aberrant HSPC production of miR-223 mutants. Thus, the N-glycome plays a previously unappreciated role as an intrinsic regulator of EHT, with specific mannose and sialic acid modifications serving as key endothelial determinants of their hematopoietic fate.One Sentence SummaryThe N-glycan “sugar code” governs the hematopoietic fate of endothelial cells and regulates blood stem cell production in vivo.

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The N-glycome regulates the endothelial-to-hematopoietic transition

Science ◽

10.1126/science.aaz2121 ◽

2020 ◽

Vol 370 (6521) ◽

pp. 1186-1191

Author(s):

Dionna M. Kasper ◽

Jared Hintzen ◽

Yinyu Wu ◽

Joey J. Ghersi ◽

Hanna K. Mandl ◽

...

Keyword(s):

Endothelial Cells ◽

Progenitor Cells ◽

Specific Expression ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

High Mannose

Definitive hematopoietic stem and progenitor cells (HSPCs) arise from the transdifferentiation of hemogenic endothelial cells (hemECs). The mechanisms of this endothelial-to-hematopoietic transition (EHT) are poorly understood. We show that microRNA-223 (miR-223)–mediated regulation of N-glycan biosynthesis in endothelial cells (ECs) regulates EHT. miR-223 is enriched in hemECs and in oligopotent nascent HSPCs. miR-223 restricts the EHT of lymphoid-myeloid lineages by suppressing the mannosyltransferase alg2 and sialyltransferase st3gal2, two enzymes involved in protein N-glycosylation. ECs that lack miR-223 showed a decrease of high mannose versus sialylated sugars on N-glycoproteins such as the metalloprotease Adam10. EC-specific expression of an N-glycan Adam10 mutant or of the N-glycoenzymes phenocopied miR-223 mutant defects. Thus, the N-glycome is an intrinsic regulator of EHT, serving as a key determinant of the hematopoietic fate.

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The role of the PZP domain of AF10 in acute leukemia driven by AF10 translocations

Nature Communications ◽

10.1038/s41467-021-24418-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Brianna J. Klein ◽

Anagha Deshpande ◽

Khan L. Cox ◽

Fan Xuan ◽

Mohamad Zandian ◽

...

Keyword(s):

Critical Role ◽

Cellular Transformation ◽

Acute Leukemias ◽

Hematopoietic Stem ◽

Nucleosome Core ◽

Stem And Progenitor Cells ◽

Transforming Activity

AbstractChromosomal translocations of the AF10 (or MLLT10) gene are frequently found in acute leukemias. Here, we show that the PZP domain of AF10 (AF10PZP), which is consistently impaired or deleted in leukemogenic AF10 translocations, plays a critical role in blocking malignant transformation. Incorporation of functional AF10PZP into the leukemogenic CALM-AF10 fusion prevents the transforming activity of the fusion in bone marrow-derived hematopoietic stem and progenitor cells in vitro and in vivo and abrogates CALM-AF10-mediated leukemogenesis in vivo. Crystallographic, biochemical and mutagenesis studies reveal that AF10PZP binds to the nucleosome core particle through multivalent contacts with the histone H3 tail and DNA and associates with chromatin in cells, colocalizing with active methylation marks and discriminating against the repressive H3K27me3 mark. AF10PZP promotes nuclear localization of CALM-AF10 and is required for association with chromatin. Our data indicate that the disruption of AF10PZP function in the CALM-AF10 fusion directly leads to transformation, whereas the inclusion of AF10PZP downregulates Hoxa genes and reverses cellular transformation. Our findings highlight the molecular mechanism by which AF10 targets chromatin and suggest a model for the AF10PZP-dependent CALM-AF10-mediated leukemogenesis.

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Live imaging of Runx1 expression in the dorsal aorta tracks the emergence of blood progenitors from endothelial cells

Blood ◽

10.1182/blood-2010-01-264382 ◽

2010 ◽

Vol 116 (6) ◽

pp. 909-914 ◽

Cited By ~ 114

Author(s):

Enid Yi Ni Lam ◽

Christopher J. Hall ◽

Philip S. Crosier ◽

Kathryn E. Crosier ◽

Maria Vega Flores

Keyword(s):

Endothelial Cells ◽

Transcription Factor ◽

Fluorescent Protein ◽

Living Organism ◽

Time Lapse ◽

Dorsal Aorta ◽

Transgenic Zebrafish ◽

Hematopoietic Stem ◽

Green Fluorescent

Abstract Blood cells of an adult vertebrate are continuously generated by hematopoietic stem cells (HSCs) that originate during embryonic life within the aorta-gonad-mesonephros region. There is now compelling in vivo evidence that HSCs are generated from aortic endothelial cells and that this process is critically regulated by the transcription factor Runx1. By time-lapse microscopy of Runx1-enhanced green fluorescent protein transgenic zebrafish embryos, we were able to capture a subset of cells within the ventral endothelium of the dorsal aorta, as they acquire hemogenic properties and directly emerge as presumptive HSCs. These nascent hematopoietic cells assume a rounded morphology, transiently occupy the subaortic space, and eventually enter the circulation via the caudal vein. Cell tracing showed that these cells subsequently populated the sites of definitive hematopoiesis (thymus and kidney), consistent with an HSC identity. HSC numbers depended on activity of the transcription factor Runx1, on blood flow, and on proper development of the dorsal aorta (features in common with mammals). This study captures the earliest events of the transition of endothelial cells to a hemogenic endothelium and demonstrates that embryonic hematopoietic progenitors directly differentiate from endothelial cells within a living organism.

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Endothelial Cells Promote Expansion of Long-Term Engrafting Marrow Hematopoietic Stem and Progenitor Cells in Primates

Stem Cells Translational Medicine ◽

10.5966/sctm.2016-0240 ◽

2016 ◽

Vol 6 (3) ◽

pp. 864-876 ◽

Cited By ~ 18

Author(s):

Jennifer L. Gori ◽

Jason M. Butler ◽

Balvir Kunar ◽

Michael G. Poulos ◽

Michael Ginsberg ◽

...

Keyword(s):

Endothelial Cells ◽

Progenitor Cells ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells

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Elevated Mcl-1 perturbs lymphopoiesis, promotes transformation of hematopoietic stem/progenitor cells, and enhances drug resistance

Blood ◽

10.1182/blood-2010-04-281071 ◽

2010 ◽

Vol 116 (17) ◽

pp. 3197-3207 ◽

Cited By ~ 80

Author(s):

Kirsteen J. Campbell ◽

Mary L. Bath ◽

Marian L. Turner ◽

Cassandra J. Vandenberg ◽

Philippe Bouillet ◽

...

Keyword(s):

Progenitor Cells ◽

Progenitor Cell ◽

Fetal Liver ◽

Cytotoxic Agents ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

Fetal Liver Cells ◽

Follicular Lymphomas ◽

The Impact

Abstract Diverse human cancers with poor prognosis, including many lymphoid and myeloid malignancies, exhibit high levels of Mcl-1. To explore the impact of Mcl-1 overexpression on the hematopoietic compartment, we have generated vavP-Mcl-1 transgenic mice. Their lymphoid and myeloid cells displayed increased resistance to a variety of cytotoxic agents. Myelopoiesis was relatively normal, but lymphopoiesis was clearly perturbed, with excess mature B and T cells accumulating. Rather than the follicular lymphomas typical of vavP-BCL-2 mice, aging vavP-Mcl-1 mice were primarily susceptible to lymphomas having the phenotype of a stem/progenitor cell (11 of 30 tumors) or pre-B cell (12 of 30 tumors). Mcl-1 overexpression dramatically accelerated Myc-driven lymphomagenesis. Most vavP-Mcl-1/ Eμ-Myc mice died around birth, and transplantation of blood from bitransgenic E18 embryos into unirradiated mice resulted in stem/progenitor cell tumors. Furthermore, lethally irradiated mice transplanted with E13 fetal liver cells from Mcl-1/Myc bitransgenic mice uniformly died of stem/progenitor cell tumors. When treated in vivo with cyclophosphamide, tumors coexpressing Mcl-1 and Myc transgenes were significantly more resistant than conventional Eμ-Myc lymphomas. Collectively, these results demonstrate that Mcl-1 overexpression renders hematopoietic cells refractory to many cytotoxic insults, perturbs lymphopoiesis and promotes malignant transformation of hematopoietic stem and progenitor cells.

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Age- and stage-specific regulation patterns in the hematopoietic stem cell hierarchy

Blood ◽

10.1182/blood.v98.10.2966 ◽

2001 ◽

Vol 98 (10) ◽

pp. 2966-2972 ◽

Cited By ~ 82

Author(s):

Hartmut Geiger ◽

Jarrod M. True ◽

Gerald de Haan ◽

Gary Van Zant

Keyword(s):

Linkage Analysis ◽

Molecular Mechanisms ◽

Hematopoietic Stem ◽

Cell Compartments ◽

Quantitative Trait Linkage Analysis ◽

Specific Manner ◽

Stem And Progenitor Cells ◽

Specific Regulation ◽

Stem Cell Hierarchy

Abstract The molecular mechanisms that regulate self-renewal and differentiation of very primitive hematopoietic stem and progenitor cells in vivo are still poorly understood. Despite the clinical relevance, even less is known about the mechanisms that regulate these cells in old animals. In a forward genetic approach, using quantitative trait linkage analysis in the mouse BXD recombinant inbred set, this study identified loci that regulate the genetic variation in the size of primitive hematopoietic cell compartments of young and old C57BL6 and DBA/2 animals. Linked loci were confirmed through the generation and analysis of congenic animals. In addition, a comparative linkage analysis revealed that the number of primitive hematopoietic cells and hematopoietic stem cells are regulated in a stage-specific and an age-specific manner.

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Dectin-1 Stimulation of Hematopoietic Stem and Progenitor Cells Occurs In Vivo and Promotes Differentiation Toward Trained Macrophages via an Indirect Cell-Autonomous Mechanism

mBio ◽

10.1128/mbio.00781-20 ◽

2020 ◽

Vol 11 (3) ◽

Cited By ~ 1

Author(s):

Cristina Bono ◽

Alba Martínez ◽

Javier Megías ◽

Daniel Gozalbo ◽

Alberto Yáñez ◽

...

Keyword(s):

Bone Marrow ◽

Candida Albicans ◽

Progenitor Cells ◽

Recipient Mouse ◽

Dependent Manner ◽

Toll Like Receptor ◽

Hematopoietic Stem ◽

Content Type ◽

Stem And Progenitor Cells

ABSTRACT Toll-like receptor (TLR) agonists drive hematopoietic stem and progenitor cells (HSPCs) to differentiate along the myeloid lineage. In this study, we used an HSPC transplantation model to investigate the possible direct interaction of β-glucan and its receptor (dectin-1) on HSPCs in vivo. Purified HSPCs from bone marrow of B6Ly5.1 mice (CD45.1 alloantigen) were transplanted into dectin-1−/− mice (CD45.2 alloantigen), which were then injected with β-glucan (depleted zymosan). As recipient mouse cells do not recognize the dectin-1 agonist injected, interference by soluble mediators secreted by recipient cells is negligible. Transplanted HSPCs differentiated into macrophages in response to depleted zymosan in the spleens and bone marrow of recipient mice. Functionally, macrophages derived from HSPCs exposed to depleted zymosan in vivo produced higher levels of inflammatory cytokines (tumor necrosis factor alpha [TNF-α] and interleukin 6 [IL-6]). These results demonstrate that trained immune responses, already described for monocytes and macrophages, also take place in HSPCs. Using a similar in vivo model of HSPC transplantation, we demonstrated that inactivated yeasts of Candida albicans induce differentiation of HSPCs through a dectin-1- and MyD88-dependent pathway. Soluble factors produced following exposure of HSPCs to dectin-1 agonists acted in a paracrine manner to induce myeloid differentiation and to influence the function of macrophages derived from dectin-1-unresponsive or β-glucan-unexposed HSPCs. Finally, we demonstrated that an in vitro transient exposure of HSPCs to live C. albicans cells, prior to differentiation, is sufficient to induce a trained phenotype of the macrophages they produce in a dectin-1- and Toll-like receptor 2 (TLR2)-dependent manner. IMPORTANCE Invasive candidiasis is an increasingly frequent cause of serious and often fatal infections. Understanding host defense is essential to design novel therapeutic strategies to boost immune protection against Candida albicans. In this article, we delve into two new concepts that have arisen over the last years: (i) the delivery of myelopoiesis-inducing signals by microbial components directly sensed by hematopoietic stem and progenitor cells (HSPCs) and (ii) the concept of “trained innate immunity” that may also apply to HSPCs. We demonstrate that dectin-1 ligation in vivo activates HSPCs and induces their differentiation to trained macrophages by a cell-autonomous indirect mechanism. This points to new mechanisms by which pathogen detection by HSPCs may modulate hematopoiesis in real time to generate myeloid cells better prepared to deal with the infection. Manipulation of this process may help to boost the innate immune response during candidiasis.

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Bone marrow regeneration requires mitochondrial transfer from donor Cx43-expressing hematopoietic progenitors to stroma

Blood ◽

10.1182/blood.2020005399 ◽

2020 ◽

Vol 136 (23) ◽

pp. 2607-2619 ◽

Cited By ~ 6

Author(s):

Karin Golan ◽

Abhishek K. Singh ◽

Orit Kollet ◽

Mayla Bertagna ◽

Mark J. Althoff ◽

...

Keyword(s):

Bone Marrow ◽

Connexin 43 ◽

Purinergic Receptor ◽

Cell Contact ◽

Hematopoietic Stem ◽

Mitochondrial Transfer ◽

Stem And Progenitor Cells ◽

Mitochondria Transfer ◽

Bone Marrow Regeneration

Abstract The fate of hematopoietic stem and progenitor cells (HSPC) is tightly regulated by their bone marrow (BM) microenvironment (ME). BM transplantation (BMT) frequently requires irradiation preconditioning to ablate endogenous hematopoietic cells. Whether the stromal ME is damaged and how it recovers after irradiation is unknown. We report that BM mesenchymal stromal cells (MSC) undergo massive damage to their mitochondrial function after irradiation. Donor healthy HSPC transfer functional mitochondria to the stromal ME, thus improving mitochondria activity in recipient MSC. Mitochondrial transfer to MSC is cell-contact dependent and mediated by HSPC connexin-43 (Cx43). Hematopoietic Cx43-deficient chimeric mice show reduced mitochondria transfer, which was rescued upon re-expression of Cx43 in HSPC or culture with isolated mitochondria from Cx43 deficient HSPCs. Increased intracellular adenosine triphosphate levels activate the purinergic receptor P2RX7 and lead to reduced activity of adenosine 5′-monophosphate–activated protein kinase (AMPK) in HSPC, dramatically increasing mitochondria transfer to BM MSC. Host stromal ME recovery and donor HSPC engraftment were augmented after mitochondria transfer. Deficiency of Cx43 delayed mesenchymal and osteogenic regeneration while in vivo AMPK inhibition increased stromal recovery. As a consequence, the hematopoietic compartment reconstitution was improved because of the recovery of the supportive stromal ME. Our findings demonstrate that healthy donor HSPC not only reconstitute the hematopoietic system after transplantation, but also support and induce the metabolic recovery of their irradiated, damaged ME via mitochondria transfer. Understanding the mechanisms regulating stromal recovery after myeloablative stress are of high clinical interest to optimize BMT procedures and underscore the importance of accessory, non-HSC to accelerate hematopoietic engraftment.

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Stromal Derived Factor-1–Induced Chemokinesis of Cord Blood CD34+ Cells (Long-Term Culture-Initiating Cells) Through Endothelial Cells Is Mediated by E-Selectin

Blood ◽

10.1182/blood.v94.12.4011 ◽

1999 ◽

Vol 94 (12) ◽

pp. 4011-4019 ◽

Cited By ~ 72

Author(s):

Afzal J. Naiyer ◽

Deog-Yeon Jo ◽

Jongcheol Ahn ◽

Robert Mohle ◽

Mario Peichev ◽

...

Keyword(s):

Endothelial Cells ◽

Adhesion Molecules ◽

Constitutive Expression ◽

Transendothelial Migration ◽

Hematopoietic Stem ◽

Long Term Culture ◽

Migration System ◽

Cd34 Cells

Abstract Homing of hematopoietic stem cells to the bone marrow (BM) involves sequential interaction with adhesion molecules expressed on BM endothelium (BMEC) and chemokine stromal derived factor-1 (SDF-1). However, the mechanism whereby adhesion molecules regulate the SDF-1–induced transendothelial migration process is not known. E-selectin is an endothelial-specific selectin that is constitutively expressed by the BMEC in vivo. Hence, we hypothesized that E-selectin may mediate SDF-1–induced transendothelial migration of CD34+ cells. We show that CD34+ cells express both E-selectin ligand and fucosyltransferase-VII (FucT-VII). Soluble E-selectin–IgG chimera binds avidly to 75% ± 10% of CD34+ cells composed mostly of progenitors and cells with long-term culture-initiating cell (LTC-IC) potential. To assess the functional capacity of E-selectin to mediate CD34+ cell migration in a transendothelial migration system, CD34+ cells were placed on transwell plates coated with interleukin-1β–activated BMEC. In the absence of SDF-1, there was spontaneous migration of 7.0% ± 1.4% of CD34+ cells and 14.1% ± 2.2% of LTC-IC. SDF-1 induced migration of an additional 23.0% ± 4.4% of CD34+cells and 17.6% ± 3.6% of LTC-IC. Blocking MoAb to E-selectin inhibited SDF-1–induced migration of CD34+ cells by 42.0% ± 2.5% and LTC-IC by 90.9% ± 16.6%. To define the mechanism of constitutive expression of E-selectin by the BMEC in vivo, we have found that vascular endothelial growth factor (VEGF165) induces E-selectin expression by cultured endothelial cells. VEGF-stimulated endothelial cells support transendothelial migration of CD34+ cells that could be blocked by MoAb to E-selectin. These results suggest that trafficking of subsets of CD34+ cells with LTC-IC potential is determined in part by sequential interactions with E-selectin and SDF-1.

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Organ-Selective Homing Defines Engraftment Kinetics of Murine Hematopoietic Stem Cells and Is Compromised by Ex Vivo Expansion

Blood ◽

10.1182/blood.v93.5.1557 ◽

1999 ◽

Vol 93 (5) ◽

pp. 1557-1566 ◽

Cited By ~ 113

Author(s):

Stephen J. Szilvassy ◽

Michael J. Bass ◽

Gary Van Zant ◽

Barry Grimes

Keyword(s):

Ex Vivo ◽

Ex Vivo Expansion ◽

Hematopoietic Stem ◽

Murine Bone Marrow ◽

Hematopoietic Reconstitution ◽

Dramatic Decline ◽

Stem And Progenitor Cells ◽

Clonogenic Cells

Abstract Hematopoietic reconstitution of ablated recipients requires that intravenously (IV) transplanted stem and progenitor cells “home” to organs that support their proliferation and differentiation. To examine the possible relationship between homing properties and subsequent engraftment potential, murine bone marrow (BM) cells were labeled with fluorescent PKH26 dye and injected into lethally irradiated hosts. PKH26+ cells homing to marrow or spleen were then isolated by fluorescence-activated cell sorting and assayed for in vitro colony-forming cells (CFCs). Progenitors accumulated rapidly in the spleen, but declined to only 6% of input numbers after 24 hours. Although egress from this organ was accompanied by a simultaneous accumulation of CFCs in the BM (plateauing at 6% to 8% of input after 3 hours), spleen cells remained enriched in donor CFCs compared with marrow during this time. To determine whether this differential homing of clonogenic cells to the marrow and spleen influenced their contribution to short-term or long-term hematopoiesis in vivo, PKH26+ cells were sorted from each organ 3 hours after transplantation and injected into lethally irradiated Ly-5 congenic mice. Cells that had homed initially to the spleen regenerated circulating leukocytes (20% of normal counts) approximately 2 weeks faster than cells that had homed to the marrow, or PKH26-labeled cells that had not been selected by a prior homing step. Both primary (17 weeks) and secondary (10 weeks) recipients of “spleen-homed” cells also contained approximately 50% higher numbers of CFCs per femur than recipients of “BM-homed” cells. To examine whether progenitor homing was altered upon ex vivo expansion, highly enriched Sca-1+c-kit+Lin−cells were cultured for 9 days in serum-free medium containing interleukin (IL)-6, IL-11, granulocyte colony-stimulating factor, stem cell factor, flk-2/flt3 ligand, and thrombopoietin. Expanded cells were then stained with PKH26 and assayed as above. Strikingly, CFCs generated in vitro exhibited a 10-fold reduction in homing capacity compared with fresh progenitors. These studies demonstrate that clonogenic cells with differential homing properties contribute variably to early and late hematopoiesis in vivo. The dramatic decline in the homing capacity of progenitors generated in vitro underscores critical qualitative changes that may compromise their biologic function and potential clinical utility, despite their efficient numerical expansion.

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