Hemogenic Endothelial Cells Research Articles

Tracing haematopoietic stem cell formation at single-cell resolution.

Haematopoietic stem cells (HSCs) are derived early from embryonic precursors, such as haemogenic endothelial cells and pre-haematopoietic stem cells (pre-HSCs), the molecular identity of which still remains elusive. Here we use potent surface markers to capture the nascent pre-HSCs at high purity, as rigorously validated by single-cell-initiated serial transplantation. Then we apply single-cell RNA sequencing to analyse endothelial cells, CD45(-) and CD45(+) pre-HSCs in the aorta-gonad-mesonephros region, and HSCs in fetal liver. Pre-HSCs show unique features in transcriptional machinery, arterial signature, metabolism state, signalling pathway, and transcription factor network. Functionally, activation of mechanistic targets of rapamycin (mTOR) is shown to be indispensable for the emergence of HSCs but not haematopoietic progenitors. Transcriptome data-based functional analysis reveals remarkable heterogeneity in cell-cycle status of pre-HSCs. Finally, the core molecular signature of pre-HSCs is identified. Collectively, our work paves the way for dissection of complex molecular mechanisms regulating stepwise generation of HSCs in vivo, informing future efforts to engineer HSCs for clinical applications.

A Multi-Lineage Screen Reveals mTORC1 Inhibition Enhances Human Pluripotent Stem Cell Mesendoderm and Blood Progenitor Production.

SummaryHuman pluripotent stem cells (hPSCs) exist in heterogeneous micro-environments with multiple subpopulations, convoluting fate-regulation analysis. We patterned hPSCs into engineered micro-environments and screened responses to 400 small-molecule kinase inhibitors, measuring yield and purity outputs of undifferentiated, neuroectoderm, mesendoderm, and extra-embryonic populations. Enrichment analysis revealed mammalian target of rapamycin (mTOR) inhibition as a strong inducer of mesendoderm. Dose responses of mTOR inhibitors such as rapamycin synergized with Bone Morphogenetic protein 4 (BMP4) and activin A to enhance the yield and purity of BRACHYURY-expressing cells. Mechanistically, small interfering RNA knockdown of RAPTOR, a component of mTOR complex 1, phenocopied the mesendoderm-enhancing effects of rapamycin. Functional analysis during mesoderm and endoderm differentiation revealed that mTOR inhibition increased the output of hemogenic endothelial cells 3-fold, with a concomitant enhancement of blood colony-forming cells. These data demonstrate the power of our multi-lineage screening approach and identify mTOR signaling as a node in hPSC differentiation to mesendoderm and its derivatives.

Efficient production of platelets from mouse embryonic stem cells by enforced expression of Gata2 in late hemogenic endothelial cells

Platelets are essential for blood circulation and coagulation. Previous study indicated that overexpression of Gata2 in differentiated mouse embryonic stem cells (ESCs) resulted in robust induction of megakaryocytes (Mks). To evaluate platelet production capacity of the Gata2-induced ESC-derived Mks, we generated iGata2-ESC line carrying the doxycycline-inducible Gata2 expression cassette. When doxycycline was added to day 5 hemogenic endothelial cells in the in vitro differentiation culture of iGata2-ESCs, c-Kit−Tie2−CD41+ Mks were predominantly generated. These iGata2-ESC-derived Mks efficiently produced CD41+CD42b+CD61+ platelets and adhered to fibrinogen-coated glass coverslips in response to thrombin stimulation. Transmission electron microscopy analysis demonstrated that the iGata2-ESC-derived platelets were discoid-shaped with α-granules and an open canalicular system, but were larger than peripheral blood platelets in size. These results demonstrated that an enforced expression of Gata2 in late HECs of differentiated ESCs efficiently promotes megakaryopoiesis followed by platelet production. This study provides valuable information for ex vivo platelet production from human pluripotent stem cells in future.

Molecular Analysis of Neutrophil Differentiation from Human Induced Pluripotent Stem Cells Delineates the Kinetics of Key Regulators of Hematopoiesis.

In vitro generation of mature neutrophils from human induced pluripotent stem cells (iPSCs) requires hematopoietic progenitor development followed by myeloid differentiation. The purpose of our studies was to extensively characterize this process, focusing on the critical window of development between hemogenic endothelium, hematopoietic stem/progenitor cells (HSPCs), and myeloid commitment, to identify associated regulators and markers that might enable the stem cell field to improve the efficiency and efficacy of iPSC hematopoiesis. We utilized a four-stage differentiation protocol involving: embryoid body (EB) formation (stage-1); EB culture with hematopoietic cytokines (stage-2); HSPC expansion (stage-3); and neutrophil maturation (stage-4). CD34(+) CD45(-) putative hemogenic endothelial cells were observed in stage-3 cultures, and expressed VEGFR-2/Flk-1/KDR and VE-cadherin endothelial markers, GATA-2, AML1/RUNX1, and SCL/TAL1 transcription factors, and endothelial/HSPC-associated microRNAs miR-24, miR-125a-3p, miR-126/126*, and miR-155. Upon further culture, CD34(+) CD45(-) cells generated CD34(+) CD45(+) HSPCs that produced hematopoietic CFUs. Mid-stage-3 CD34(+) CD45(+) HSPCs exhibited increased expression of GATA-2, AML1/RUNX1, SCL/TAL1, C/EBPα, and PU.1 transcription factors, but exhibited decreased expression of HSPC-associated microRNAs, and failed to engraft in immune-deficient mice. Mid-stage-3 CD34(-) CD45(+) cells maintained PU.1 expression and exhibited increased expression of hematopoiesis-associated miR-142-3p/5p and a trend towards increased miR-223 expression, indicating myeloid commitment. By late Stage-4, increased CD15, CD16b, and C/EBPɛ expression were observed, with 25%-65% of cells exhibiting morphology and functions of mature neutrophils. These studies demonstrate that hematopoiesis and neutrophil differentiation from human iPSCs recapitulates many features of embryonic hematopoiesis and neutrophil production in marrow, but reveals unexpected molecular signatures that may serve as a guide for enhancing iPSC hematopoiesis. Stem Cells 2016;34:1513-1526.

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.

Hematopoietic stem cells develop in the absence of endothelial cadherin 5 expression

AbstractRare endothelial cells in the aorta-gonad-mesonephros (AGM) transition into hematopoietic stem cells (HSCs) during embryonic development. Lineage tracing experiments indicate that HSCs emerge from cadherin 5 (Cdh5; vascular endothelial–cadherin)+ endothelial precursors, and isolated populations of Cdh5+ cells from mouse embryos and embryonic stem cells can be differentiated into hematopoietic cells. Cdh5 has also been widely implicated as a marker of AGM-derived hemogenic endothelial cells. Because Cdh5−/− mice embryos die before the first HSCs emerge, it is unknown whether Cdh5 has a direct role in HSC emergence. Our previous genetic screen yielded malbec (mlbbw306), a zebrafish mutant for cdh5, with normal embryonic and definitive blood. Using time-lapse confocal imaging, parabiotic surgical pairing of zebrafish embryos, and blastula transplantation assays, we show that HSCs emerge, migrate, engraft, and differentiate in the absence of cdh5 expression. By tracing Cdh5−/−green fluorescent protein (GFP)+/+ cells in chimeric mice, we demonstrated that Cdh5−/−GFP+/+ HSCs emerging from embryonic day 10.5 and 11.5 (E10.5 and E11.5) AGM or derived from E13.5 fetal liver not only differentiate into hematopoietic colonies but also engraft and reconstitute multilineage adult blood. We also developed a conditional mouse Cdh5 knockout (Cdh5flox/flox:Scl-Cre-ERT) and demonstrated that multipotent hematopoietic colonies form despite the absence of Cdh5. These data establish that Cdh5, a marker of hemogenic endothelium in the AGM, is dispensable for the transition of hemogenic endothelium to HSCs.

Vascular Niche-Derived Angiocrine Factors Specify and Maintain Hematopoietic Stem Cells

Organ-specific endothelial cells (ECs) are both conduits for delivery of nutrients and also establish an instructive vascular niche. The vascular niche produces paracrine factors, (i.e., angiocrine factors), that balance self-renewal and differentiation of hematopoietic stem/progenitor cells (HSPCs) (1,2). Activation of Akt-mTOR pathway in sinusoidal ECs (SECs) stimulates physiological expression of angiocrine factors, including Kit-ligand, Notch-ligands, Wnts, FGFs, BMPs and TGFb, that expand long-term repopulating HSPCs. Activation of MAPkinase in ECs upregulates expression of GM-CSF, M-CSF, IL6, IL7, SDF-1 and G-CSF (..others) to accelerate HSPC multi-lineage differentiation. We developed an ex vivo vascular niche in which HSPC/EC co-cultures are maintained and expanded in serum-free conditions. This vascular niche platform produces physiologic levels of angiocrine factors that balance expansion/differentiation of human cord blood, mobilized peripheral blood, and steady state bone marrow HSPCs that maintain their ability to reconstitute hematopoiesis in vivo. In contrast to our vascular platform, co-culture with bone marrow-derived mesenchymal does not support long-term expansion of HSPCs. In collaboration with Drs. Kiem and Gori at Hutchinson Cancer Center, we have shown that ECs expand repopulating nonhuman primate marrow-derived HSPCs. Transplantation of the vascular-niche expanded gene-modified HSPCs reconstituted long-term multi-lineage hematopoiesis in autologous transplantation setting in nonhuman primates. Importantly, intravenous co-infusion of the vascular niche with HSPCs did not cause infusional toxicity. Vascular niche-expanded HSPCs supported robust hematopoietic recovery underscoring the essential function of vascular niche-signals in hematopoietic reconstitution without provoking fibrosis (3). The ECs also supplies key signals that induce emergence of HSPCs from hemogenic ECs. To prove this point, we transduced adult human or mouse ECs with Runx1/Spi1/Gfi1/FosB transcription factors along with vascular niche-induction allowing for conversion of these ECs into stable and long-term engraftable HSPCs, including functional immune cells (4). Importantly, transition through a pluripotent state results in poorly engraftable hematopoietic cells that are unstable and upon exposure to pathophysiological stressors differentiate aberrantly into other cell-types. Remarkably, signals from vascular niche support specification of repopulating multipotent-HSPCs from both human and nonhuman primate pluripotent stem cells (5). In summary, we developed and characterized a vascular niche platform that provides physiologically relevant levels of key angiocrine factors that stimulate safe clinical-scale expansion of authentic adult, cord blood, and primitive HSPCs under GMP-grade culture conditions. We are currently translating the vascular niche platform to the clinical setting, to evaluate the potential of co-transplantation of HSPCs with vascular niche cells to reconstruct injured EC niches thereby accelerating short- and long-term hematopoietic recovery. This first-in-man clinical application will set the stage for repopulation with true hematopoietic stem cells, thereby enabling use of a vascular niche for treatment of a wide range of acquired, inherited, and malignant hematopoietic diseases.1. Butler JM …… Rafii S. Endothelial cells are essential for the self-renewal and repopulation of Notch-dependent hematopoietic stem cells. Cell Stem Cell, 3:251-64, 2010.2. Nolan D........Rafii S. Molecular and cellular signatures of tissue-specific vascular heterogeneity in organ maintenance and regeneration. Developmental Cell, 26(2):204-19, 2013.3. Ding BS …..Rafii S. Divergent angiocrine signals from vascular niche balance liver regeneration and fibrosis.Nature 505(7481):97-102, 2014.4. Sandler VM, Lis R ...... Butler JM, Scandura JM, Rafii S. Reprogramming of Human Endothelium Into Engraftable Hematopoietic Progenitors by Vascular Niche Induction.Nature, 511(7509):312-8, 2014.5. Gori J., Butler JM, .....Rafii S, Kiem HP. Vascular niche promotes hematopoietic multipotent progenitor formation from pluripotent stem cells. Journal of Clinical Investigation, 125(3): 1243-54, 2015. DisclosuresRafii:Angiocrine Bioscience: Consultancy, Equity Ownership.

A Human Bone Marrow-Derived Stromal Cell Population with Hemogenic Potential

During ontogeny, definitive hematopoietic stem/progenitor cells (HSC) are thought to arise from vascular endothelial cells, through an endothelial-to-hematopoietic transition, a natural process that occurs in unique, specialized embryonic hemogenic endothelial cells. Developmental studies, and experiments using pluripotent stem cells in an effort to recapitulate this process and thereby gain a better understanding of the emergence of definitive hematopoiesis, have collectively led to the prevailing view that the hemogenic endothelium constitutes a transient population of cells within the embryo that rapidly disappears during development and is absent in the adult. Herein, we provide the first evidence that at early time points of gestation, prior to the establishment of hematopoiesis, a unique subpopulation of Stro-1+ cells present within the inner part of the developing human bone marrow co-expresses APLNR, a marker of angiogenic mesoderm. Moreover, these Stro-1+APLNR+ cells express multiple other markers described for hemogenic endothelium, and subsequently contribute to the vasculature, cartilage, and bone. Importantly, we also show that cells expressing these same markers of primitive mesoderm/hemogenic endothelium persist at low frequency within the adult marrow. These adult-derived cells can be extensively expanded in vitro without loss of potential, but lack hematopoietic colony-forming potential in vitro. However, upon transplantation into a fetal microenvironment, clonally-derived populations of these adult Stro1+ isolated stromal progenitors (SIPs) not only contribute to the vasculature and nascent BM niches, but also efficiently generate, at a clonal level, hematopoietic stem cells (HSC) that are capable of robust, multilineage hematopoietic reconstitution, with generation of both myeloid and lymphoid cells upon serial transplantation. In conclusion, our studies have thus uncovered the latent potential of a highly expandable population of seemingly vestigial adult human somatic cells, whose ontogenic history includes a phenotype identical to that described for hemogenic endothelium. We have also shown that, if provided with the appropriate/necessary inductive factors, these unique adult cells are capable of giving rise to hematopoietic cells that fulfill the gold standard criteria for bona fide HSC. Therefore, these cells could potentially be more amenable to reprogramming technologies, to produce HSC that could be used to treat/cure a broad variety of blood diseases. DisclosuresNo relevant conflicts of interest to declare.

Hematopoietic Stem Cells Develop in the Absence of Endothelial Cadherin 5 Expression

Rare endothelial cells in the aorta-gonad-mesonephros (AGM) transition into hematopoietic stem cells (HSCs) during embryonic development. Lineage tracing experiments indicate that HSCs emerge from Cadherin 5 (Cdh5, VE-cadherin)+ endothelial precursors, and isolated populations of Cdh5+ cells from mouse embryos and embryonic stem (ES) cells can be differentiated into hematopoietic cells. Cdh5 has also been widely implicated as a marker of AGM-derived hemogenic endothelial cells. Since Cdh5-/- mice embryos die before the first HSCs emerge, it is unknown if Cdh5 has a direct role in HSC emergence. Our previous genetic screen yielded malbec (mlbbw306), a zebrafish mutant for cdh5, with normal embryonic and definitive blood. Utilizing time-lapse imaging, parabiotic surgical pairing of zebrafish embryos, and blastula transplantation assays, we show that HSCs emerge, migrate, engraft, and differentiate in the absence of cdh5 expression. By tracing Cdh5-/- GFP+/+ cells inchimeric mice, we demonstrated that Cdh5-/- GFP+/+ HSCs emerging from E10.5 and E11.5 AGM or derived from E13.5 fetal liver not only differentiate into hematopoietic colonies but also engraft and reconstitute multi-lineage adult blood. These data establish that Cdh5, a marker of hemogenic endothelium in the AGM, is dispensable for the transition of hemogenic endothelium to HSCs. DisclosuresBauer:Biogen: Research Funding; Editas Medicine: Consultancy. Zon:FATE Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Scholar Rock: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder. Orkin:Editas Medicine: Membership on an entity's Board of Directors or advisory committees; Biogen: Research Funding; Pfizer: Research Funding; Sangamo Biosciences: Consultancy.

On signaling pathways: hematopoietic stem cell specification from hemogenic endothelium.

Hematopoietic stem cells (HSCs) are specified and generated during the embryonic development and have remarkable potential to replenish the full set of blood cell lineages. Researchers have long been interested in clarifying the molecular events involved in HSC specification. Many studies have reported the development of methods for generating functional hematopoietic cells from pluripotent stem cells (PSCs-embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs)) for decades. However, the generation of HSCs with robust long-term repopulation potential remains a swingeing challenge, of which a major factor contributing to this failure is the difficulty to define the intraembryonic signals related to the specification of HSCs. Since HSCs directly derive from hemogenic endothelium, in this review, we summarize both in vivo and in vitro studies on conserved signaling pathways that control the specification of HSCs from hemogenic endothelial cells.

Definitive Hematopoietic Multipotent Progenitor Cells Are Transiently Generated From Hemogenic Endothelial Cells in Human Pluripotent Stem Cells.

Generation of fully functional hematopoietic multipotent progenitor (MPP) cells from human pluripotent stem cells (hPSCs) has a great therapeutic potential to provide an unlimited cell source for treatment of hematological disorders. We previously demonstrated that CD34(+) CD31(+) CD144(+) population derived from hPSCs contain hemato-endothelial progenitors (HEPs) that give rise to hematopoietic and endothelial cells. Here, we report a differentiation system to generate definitive hematopoietic MPP cells from HEPs via endothelial monolayer. In the presence of angiogenic factors, HEPs formed an endothelial monolayer, from which hematopoietic clusters emerged through the process of endothelial-to-hematopoietic transition (EHT). EHT was significantly enhanced by hematopoietic growth factors. The definitive MPP cells generated from endothelial monolayer were capable of forming multilineage hematopoietic colonies, giving rise to T lymphoid cells, and differentiating into enucleated erythrocytes. Emergence of hematopoietic cells from endothelial monolayer occurred transiently. Hematopoietic potential was lost during prolonged culture of HEPs in endothelial growth conditions. Our study demonstrated that CD34(+) CD31(+) CD144(+) HEPs gave rise to hematopoietic MPP cells via hemogenic endothelial cells that exist transiently. The established differentiation system provides a platform for future investigation of regulatory factors involved in de novo generation of hematopoietic MPP cells and their applications in transplantation.

Activin A in combination with OP9 cells facilitates development of Flk-1+ PDGFRα− and Flk-1+ PDGFRα+ hematopoietic mesodermal cells from murine embryonic stem cells

Lateral mesoderm-derived hemogenic endothelial cells are known to originate the definitive hematopoietic lineage in mouse embryogenesis. The developmental process of the definitive hematopoietic lineage can be recapitulated by inducing differentiation of mouse embryonic stem (ES) cells in a co-culture system with OP9 stromal cells. However, the signaling molecules that can modulate the development of the definitive hematopoietic lineage in the OP9 co-culture system have yet to be identified. Here we report that activin A enhanced the hematopoietic potential of endothelial cells derived from ES cells in the OP9 co-culture system. Activin A in combination with OP9 cells augmented development of Flk-1+ PDGFRα+ early mesodermal cells and Flk-1+ PDGFRα− lateral mesodermal cells from ES cells. These Flk-1+ mesodermal cells further differentiated into CD41+ endothelial cells, which preferentially possessed high hematopoietic potential. Furthermore, Flk-1+ PDGFRα+ cells but not Flk-1+ PDGFRα− cells produced hematopoietic progenitors with a bimodal pattern when cultured as an aggregate with OP9 cells. Our results suggest that activin A in combination with OP9 cells facilitates differentiation of ES cells to Flk-1+ mesodermal cells, which encompass various precursors that separately contribute to the development of hematopoietic lineages.

Localization and characterization of hematopoietic cells in the mouse embryonic head

The midgestation mouse head harbors hematopoietic stem cells (HSCs) at the same time HSCs are detected in the aorta, gonads, mesonephros region (AGM). Within the complex structure of the embryonic head, the localization and phenotype of HSCs and hematopoietic progenitor cells (HPCs) remains unknown. Since the Ly6a(Sca1)GFP transgene is a reporter for all AGM HSCs and some hemogenic endothelial cells (HEC), we examined these embryos for head HSC, HEC and HPC. Wholemount staining reveals some GFP+ cells in E10.5 head vasculature. By FACS analysis, 0.6% of E11.5 head cells are GFP+. To examine whether head HPC/HSC are enriched in the Ly6aGFP+ population, we sorted E11.5-E12.5 head cells and performed functional assays. Preliminary data show that head HSCs are GFP+. When we subdissected the head into forebrain (FB), midbrain (MB), and hindbrain with brachial arches (HBA) to localize HPCs, we found erythro-myeloid progenitor cells in GFP+ and GFP- fractions of all three brain regions at E10-11.5. Interestingly, in OP9 co-cutures, B cell potential was mostly observed in CD31+GFP+ of the three subregions. These results suggest that head HSCs (Ly6aGFP) are phenotypically and functionally similar to AGM HSCs. We are continuing to identify the precise head subregion(s) in which HSCs occur, examine the hemogenic potential of the head vasculature, and also the pathways affecting their development.

Megakaryocyte-platelet induction from mouse embryonic stem cells by enforced expression of Gata2 in late hemogenic endothelial cells

Megakaryocyte-platelet induction from mouse embryonic stem cells by enforced expression of Gata2 in late hemogenic endothelial cells

Hematopoietic stem cell production and regulation in the embryo aorta

Hematopoietic Stem Cells (HSCs) are responsible for the constant replenishment of all blood cells during the entire life of an organism. Initially generated during embryonic development, HSCs are first detected in the aorta. Then, HSCs transit through different anatomical sites where they will expand before colonizing the bone marrow where they reside in adult. Despite their discovery more than fifty years ago, there is still uncertainty regarding the mechanisms underlying HSC production and regulation. Over the past years, we have shown by using live imaging technology that hematopoietic cells, organized in clusters, are de novo generated from specialized hemogenic endothelial cells in the aorta of the mouse embryo. These clusters of cells (referred to as Intra-Aortic Hematopoietic Clusters or IAHCs) are present in most vertebrate species and contain the first few HSCs. However, some discrepancies remained concerning the spatial and temporal appearance and the numbers of IAHCs and HSCs. Therefore, the exact cell composition and function of IAHCs remain incomplete. We have recently shown that IAHCs contain HSC precursors (pre-HSCs) that progressively mature into HSCs in vivo (as shown by successful long-term multilineage reconstitution of primary neonates and secondary adult recipients). We are currently analyzing single IAHC cells and single IAHCs (isolated mechanically) by RNA-sequencing to reveal the level of heterogeneity in IAHCs and the molecular program occurring during pre-HSC maturation and HSC production. We are also interested in deciphering how the microenvironment surrounding the aorta regulates HSC generation. To answer this question we are currently using a genome-wide RNA tomography technique (tomo-seq, which combines RNA-Sequencing data and spatial information) to accurately determine spatial gene expression patterns along the aorta at different developmental stages. Our goal is to infer the spatial and temporal dynamics of gene expression and to identify key genes in the microenvironment that might be involved in HSC generation in both mouse and chicken embryos. The data obtained from chicken and mouse embryos will be compared to find conserved genes and pathways in both species (and therefore possibly also in human). Our approach will have the unique advantage to provide a global view of the gene expression patterns in the whole microenvironment (as it is organized in vivo around the aorta). Our overall goal is to advance our knowledge of the complex network of regulatory genes involved during HSC formation in the aorta, both in the pre-HSCs/HSCs and in the cells of the surrounding microenvironment. It should prove useful in the future for engineering appropriate in vitro culture conditions to produce autologous HSC grafts needed to treat inherited and acquired blood disorders.

Single-cell resolution of morphological changes in hemogenic endothelium.

Endothelial-to-hematopoietic transition (EHT) occurs within a population of hemogenic endothelial cells during embryogenesis, and leads to the formation of the adult hematopoietic system. Currently, the prospective identification of specific endothelial cells that will undergo EHT, and the cellular events enabling this transition, are not known. We set out to define precisely the morphological events of EHT, and to correlate cellular morphology with the expression of the transcription factors RUNX1 and SOX17. A novel strategy was developed to allow for correlation of immunofluorescence data with the ultrastructural resolution of scanning electron microscopy. The approach can identify single endothelial cells undergoing EHT, as identified by the ratio of RUNX1 to SOX17 immunofluorescence levels, and the morphological changes associated with the transition. Furthermore, this work details a new technical resource that is widely applicable for correlative analyses of single cells in their native tissue environments.

Repression of arterial genes in hemogenic endothelium is sufficient for haematopoietic fate acquisition.

Changes in cell fate and identity are essential for endothelial-to-haematopoietic transition (EHT), an embryonic process that generates the first adult populations of haematopoietic stem cells (HSCs) from hemogenic endothelial cells. Dissecting EHT regulation is a critical step towards the production of in vitro derived HSCs. Yet, we do not know how distinct endothelial and haematopoietic fates are parsed during the transition. Here we show that genes required for arterial identity function later to repress haematopoietic fate. Tissue-specific, temporally controlled, genetic loss of arterial genes (Sox17 and Notch1) during EHT results in increased production of haematopoietic cells due to loss of Sox17-mediated repression of haematopoietic transcription factors (Runx1 and Gata2). However, the increase in EHT can be abrogated by increased Notch signalling. These findings demonstrate that the endothelial haematopoietic fate switch is actively repressed in a population of endothelial cells, and that derepression of these programs augments haematopoietic output.

Origin of Hematopoietic Stem Cells in Bone Marrow--Endothelial to Hematopoietic Transition (EHT)?

In contrast to primitive hematopoiesis, during embryonic definitive hematopoiesis, it has been demonstrated that multilineage hematopoietic stem/progenitor cells (HSPCs) arise from hemogenic endothelium, and the endothelial to hematopoietic transition (EHT) exists within the yolk sac, placenta, AGM, mouse head vascular and extraembryonic vessels. However, whether hemogenic endothelial cells contribute to blood cell development at other sites of definitive hematopoiesis, including fetal liver and bone marrow, remains largely unknown. Recently, more and more researches showed that hematopoiesis within bone marrow had a close relationship with vascular endothelium development, too. This review summarizes the mechanism of EHT during embryo development, and discuss whether EHT exists in adult hematopoiesis.

Development of hematopoietic stem and progenitor cells from human pluripotent stem cells.

Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), provide a new cell source for regenerative medicine, disease modeling, drug discovery, and preclinical toxicity screening. Understanding of the onset and the sequential process of hematopoietic cells from differentiated hPSCs will enable the achievement of personalized medicine and provide an in vitro platform for studying of human hematopoietic development and disease. During embryogenesis, hemogenic endothelial cells, a specified subset of endothelial cells in embryonic endothelium, are the primary source of multipotent hematopoietic stem cells. In this review, we discuss current status in the generation of multipotent hematopoietic stem and progenitor cells from hPSCs via hemogenic endothelial cells. We also review the achievements in direct reprogramming from non-hematopoietic cells to hematopoietic stem and progenitor cells. Further characterization of hematopoietic differentiation in hPSCs will improve our understanding of blood development and expedite the development of hPSC-derived blood products for therapeutic purpose.

Bmi1 maintains the self-replenishing ability of B-1a cells in postnatal mice. (HEM1P.230)

Abstract B-1a cells are innate immune cells that reside in the peritoneal cavity. We have recently reported that some B-1 cells originate from hemogenic endothelial cells in the early mouse embryo, and are independent of HSC-derived hematopoiesis. B-1a cells are also considered to possess self-replenishing capacity. The mechanisms that permit B-1a cells to undergo self-replenishment in vivo independent of HSCs remain unclear. Bmi1 is a polycomb group protein that is known to be critical for self-renewal of adult BM HSCs. In Bmi1-/- postnatal BM, the number of HSCs was markedly reduced and long-term reconstituting ability was lost. We hypothesized that Bmi1 is also critical for B-1a cell self-replenishment ability. Bmi1 mRNA expression was higher in B-1a cells than other lymphoid subsets in adult wild type mice. In Bmi1-/- mice, absolute cell numbers in the spleen, BM, and peritoneal cavity were decreased but the percentage of each mature lymphoid population was not altered except for B-1a cells. The percentage of peritoneal B-1a cells was 4-7 fold less than wild type. Bmi1-/- B-1a cells displayed impaired self-replenishing ability upon transplantation into the peritoneal cavity of recipient mice. Ink4a-Arf is one of the known target genes of Bmi1, and its expression is increased in Bmi1-/- HSCs and B-1a cells. The percentage and numbers of B-1a cells were restored in Ink4a-/-Arf-/-Bmi1-/- mice. These data suggest that Bmi1 maintains the self-replenishing ability of B-1a cells.