Endothelial-to-hematopoietic Transition Research Articles

RECONSTITUTION OF T LYMPHOPOIESIS FROM PLURIPOTENT STEM CELLS BY DEFINED TRANSCRIPTION FACTORS

Pluripotent stem cells (PSC)-generated induced PTP could only reconstitute functional T lymphopoiesis in vivo when pre-embedded in congenic fetal thymic lobes, indicative of a cell-intrinsic flaw of homing capacity in vivo.In this study, we identified certain transcription factors that were absently expressed in induced hemogenic endothelial cells but abundantly expressed in their natural counterparts and further screened their combinatory roles in specifying T lymphopoiesis by tandem-TF-knock-in strategy. Coordinated inducible expression of TF1 and TF2 during endothelial to hematopoietic transition (EHT) and subsequent blood cell maturation stages in vitro preferentially resulted in full T lymphopoiesis in vivo after transplanting the derived ETP-abundant hematopoietic progenitors into conditioned NOD scid gamma mice via intravenous delivery. Remarkably, even a single induced hemogenic endothelial cells (iHEC) was able to generate T cells (iT cells) in vitro and reconstitute T lymphopoiesis in vivo. Following the same protocol, another TF-cocktails led to induced hematopoietic progenitors preferentially giving rise to B lymphopoiesis including B1 cells in vivo, strongly supporting the concept that certain transcription factors can specify lymphoid-lineage potentials at the early developmental stages before the emergence of hematopoietic stem cells. Physiologically, the mature iT cells distributed into lymphoid and other solid organs and exhibited abundant TCRαβ repertoire. Functionally, these regenerated iT cells successfully rejected allogeneic skin grafts and formed immune memory response. Translationally, the tumor-antigen-specific TCR-iT cells derived from gene-edited PSC successfully repressed tumor growth in vivo. This study provides insight into reconstituting T lymphopoiesis using PSC source.

GPR56 AND GPR97 PLAY REDUNDANT ROLES IN REGULATING HEMATOPOIESIS

Hematopoietic stem cells (HSCs) are responsible for the lifelong production and maintenance of adult blood cell types. They arise from the aorta at the embryonic day (E) 10.5 from a subset of endothelial cells that undergo to endothelial-to-hematopoietic transition (EHT). Our lab discovered that a G-coupled receptor protein, Gpr56, is required for the generation of HSCs in zebrafish. However, its role in mammalian hematopoiesis remains controversial. We investigate the functional role of Gpr56 in vitro using our mouse Gata2Venus (G2V) embryonic stem cell (ESC) line which allows us to highly enrich for hematopoietic progenitor cells (HPCs). G2V.G56-/-. We tested the Gpr56 loss of function by generating a full knock out (KO) Gpr56 mouse G2V (G2V.G56-/-) ESC line. The in vitro examination of phenotypic hematopoietic cell production by FACS and colony forming unit-culture (CFU-C) assay reported an increase in hematopoietic cells (HC) and HPCs production from G2V.G56-/- ESC as compared to G2V ESC. Further analyses performed on cell-sorted HPC population showed that another GPR protein, Gpr97 (located in the same locus), is highly expressed when Gpr56 is deleted. This led us to generate a novel G2V Gpr56/Gpr97 double knockout (G2V.G56/97-/-) ESC line. The in vitro differentiation of the G2V.G56/97-/- revealed a significant decrease in HPCs, supporting the idea that a compensatory mechanism by Gpr97 is taking place when Gpr56 is absent. Preliminary in vivo data using a Gpr56 conditional KO mouse model corroborates our hypothesis. The data demonstrate that the Gpr97 performs a redundant function upon deletion of Gpr56 in the HS/PC population and the deletion of both Gpr56 and Gpr97 is sufficient to impair the production of HS/PC and HC in vitro. We conclude that GPR signalling through Gpr56 and/or Gpr97 is required for HPC production.

MEIS1 EXPRESSION IN MOUSE EMBRYONIC HEMATOPOIESIS

Hematopoietic stem cells (HSCs) originate during embryogenesis and colonize the bone marrow, after expansion in the fetal liver, to sustain adult hematopoiesis. In vivo imaging studies have supported the emergence of HSCs form the ventral endothelium of the dorsal aorta in the aorta-gonado-mesonephros (AGM) region through a process termed endothelial-to-hematopoietic transition (EHT). Generation of HSC is a dynamic process requiring temporal and cell-specific changes within a subset of cells, the hemogenic endothelium, to drive a new cell fate. Although several pathways have been shown to play a critical role in this transition, the molecular programming driving EHT remains elusive. In this study, we aim to understand how the transcription factor Meis1 affects the emergence of HSC by taking advantage of various mouse models. Using a GFP reporter mouse, we mapped Meis1 expression in the developing dorsal aorta and showed that Meis1 expression enriches for the hemogenic endothelium compared to vascular endothelium. We combined these data with observations of endothelial-specific deletion of Meis1 in vivo, using VE-cadherin (VEC)-Cre recombinase, to gain a better understanding of the requirements for Meis1 during this dynamic developmental process. Conditional Meis1-deletion impaired the hematopoietic potential of the AGM endothelium based on specific gene expression and significantly reduce the emergence of cells with hematopoietic surface markers. Our ability to derived HSC in vitro is in its infancy and was largely influenced by studies of normal developmental processes. Therefore, understanding the regulatory network driving EHT remains essential to develop better strategies for HSC specification.

EXPLORING TRANSCRIPTOME AND FUNCTIONAL HETEROGENEITY WITHIN THE COHORT OF EMERGING HEMATOPOIETIC CELLS

Hematopoietic stem cells (HSC) are responsible for life-long maintenance and regeneration of the adult vertebrate blood system. The first HSCs arise from intra-aortic hematopoietic cluster cells (IAHC) in the mouse aorta at embryonic day (E)10.5 through a transdifferentiation process called endothelial-to-hematopoietic-transition (EHT). Previously we showed that the heptad factor Gata2, one of a group of 7 (heptad) transcription factors pivotal to definitive hematopoiesis, is required for HSC generation and survival. Gata2-/- embryos suffer lethality at E10.5 and Gata2+/- HSCs are qualitatively defective. Utilizing our Gata2Venus reporter mouse for time-lapse imaging of EHT in vivo, we found rapid pulsatile Gata2 expression level changes in single transitioning cells, implicating transcriptional instability during establishment of hematopoietic fate. Flow cytometric analysis of IAHCs revealed cells with varying levels of Venus (Vmedium, Vhigh). Hematopoietic progenitors (HPC) were highly enriched and HSCs exclusively found in the Vmed fraction. From these results, we hypothesized that fate choice is based on stochastic expression (levels and combinations) of the pivotal heptad factors. Here we test this by single cell transcriptomics and functional assays. Single-cell RNA sequencing of index-sorted Vmed cells shows surprising heterogeneity in heptad gene expression and has facilitated further HSC enrichment, defining intensity levels of known markers and providing additional markers. These new markers were functionally validated and their expression visualized by immunohistochemistry. Single-cell HPC assays also allowed for enrichment of immature HPCs. Further sorting and sequencing reiterations will refine the HSC transcriptomic profile and provide information on whether transcriptome heterogeneity correlates with and plays a role in generating both HSCs and HPCs.

Overexpression of GATA2 Enhances Development and Maintenance of Human Embryonic Stem Cell-Derived Hematopoietic Stem Cell-like Progenitors.

SummaryGATA2 is essential for the endothelial-to-hematopoietic transition (EHT) and generation of hematopoietic stem cells (HSCs). It is poorly understood how GATA2 controls the development of human pluripotent stem cell (hPSC)-derived HS-like cells. Here, using human embryonic stem cells (hESCs) in which GATA2 overexpression was induced by doxycycline (Dox), we elucidated the dual functions of GATA2 in definitive hematopoiesis before and after the emergence of CD34+CD45+CD90+CD38– HS-like cells. Specifically, GATA2 promoted expansion of hemogenic precursors via the EHT and then helped to maintain HS-like cells in a quiescent state by regulating cell cycle. RNA sequencing showed that hPSC-derived HS-like cells were very similar to human fetal liver-derived HSCs. Our findings will help to elucidate the mechanism that controls the early stages of human definitive hematopoiesis and may help to develop a strategy to generate hPSC-derived HSCs.

S863 ONE-YEAR EFFICACY OF RAVULIZUMAB (ALXN1210) IN ADULT PATIENTS WITH PAROXYSMAL NOCTURNAL HEMOGLOBINURIA NAIVE TO COMPLEMENT INHIBITORS

Background: GATA2 is the most frequently mutated gene in childhood AML. These patients have germline mutations in one allele of GATA2 and have a high predisposition for MDS/AML. Also, in mouse, Gata2 is required for embryonic HSC generation and survival and complete deletion of Gata2 is lethal at embryonic day (E)10, while Gata2 haploinsufficiency results in a severe reduction in the formation of HSCs. The first HSCs are formed at E10.5 through a process called endothelial-to-hematopoietic transition (EHT) from specialized hemogenic endothelial cells of the dorsal aorta in the aorta-gonad-mesonephros (AGM) region. Aims: As all patients suffering from GATA2 haploinsufficiency syndromes have innate GATA2 mutations, our hypothesis is that embryonic haploinsufficiency of GATA2 is responsible for the phenotype of the patients. Therefore, we want to understand how Gata2 haploinsufficiency affects the generation of HSCs through EHT. Methods: We sorted phenotypic HSPCs (CD31+cKit+ cells) from WT and Gata2+/- E11 AGMs and performed transcriptome analysis. Also, with whole-mount antibody staining performed on the WT and Gata2+/- AGMs, we analysed CD31+cKit+ cell populations. Results: Surprisingly, we found that the hematopoietic transcriptional program is not abrogated in Gata2+/- mouse E11 CD31+cKit+ cells. However, Gata2+/- HSPCs still express the endothelial program indicating that the hematopoietic cells are stuck in the endothelium and cannot complete EHT. We found that Gfi1b is downregulated in Gata2+/- HSPCs. As Gfi1b is known to be required for downregulation of the endothelial program for EHT during primitive hematopoiesis, we hypothesize that Gata2 directly regulates Gfi1b and that this regulation is required for normal EHT. Summary/Conclusion: In conclusion, we showed Gata2+/- HSPCs are transcriptionally different from WT HSPCs. We are currently studying if a specific HSPC subtype can overcome Gata2 haploinsufficiency and will still undergo EHT as this may have implications for the hematopoietic cells that eventually populate the hematopoietic tissues.

S862 THE ROLE OF GATA2 IN HSC GENERATION THROUGH ENDOTHELIAL‐TO‐HEMATOPOIETIC TRANSITION.

Background:GATA2 is the most frequently mutated gene in childhood AML. These patients have germline mutations in one allele of GATA2 and have a high predisposition for MDS/AML. Also, in mouse, Gata2 is required for embryonic HSC generation and survival and complete deletion of Gata2 is lethal at embryonic day (E)10, while Gata2 haploinsufficiency results in a severe reduction in the formation of HSCs. The first HSCs are formed at E10.5 through a process called endothelial‐to‐hematopoietic transition (EHT) from specialized hemogenic endothelial cells of the dorsal aorta in the aorta‐gonad‐mesonephros (AGM) region.Aims:As all patients suffering from GATA2 haploinsufficiency syndromes have innate GATA2 mutations, our hypothesis is that embryonic haploinsufficiency of GATA2 is responsible for the phenotype of the patients. Therefore, we want to understand how Gata2 haploinsufficiency affects the generation of HSCs through EHT.Methods:We sorted phenotypic HSPCs (CD31+cKit+ cells) from WT and Gata2 +/‐ E11 AGMs and performed transcriptome analysis. Also, with whole‐mount antibody staining performed on the WT and Gata2 +/‐ AGMs, we analysed CD31+cKit+ cell populations.Results:Surprisingly, we found that the hematopoietic transcriptional program is not abrogated in Gata2 +/‐ mouse E11 CD31+cKit+ cells. However, Gata2 +/‐ HSPCs still express the endothelial program indicating that the hematopoietic cells are stuck in the endothelium and cannot complete EHT. We found that Gfi1b is downregulated in Gata2 +/‐ HSPCs. As Gfi1b is known to be required for downregulation of the endothelial program for EHT during primitive hematopoiesis, we hypothesize that Gata2 directly regulates Gfi1b and that this regulation is required for normal EHT.Summary/Conclusion:In conclusion, we showed Gata2 +/‐ HSPCs are transcriptionally different from WT HSPCs. We are currently studying if a specific HSPC subtype can overcome Gata2 haploinsufficiency and will still undergo EHT as this may have implications for the hematopoietic cells that eventually populate the hematopoietic tissues.

Endothelial-to-haematopoietic transition: an update on the process of making blood.

The first definitive blood cells during embryogenesis are derived from endothelial cells in a highly conserved process known as endothelial-to-haematopoietic transition (EHT). This conversion involves activation of a haematopoietic transcriptional programme in a subset of endothelial cells in the major vasculature of the embryo, followed by major morphological changes that result in transitioning cells rounding up, breaking the tight junctions to neighbouring endothelial cells and adopting a haematopoietic fate. The whole process is co-ordinated by a complex interplay of key transcription factors and signalling pathways, with additional input from surrounding tissues. Diverse model systems, including mouse, chick and zebrafish embryos as well as differentiation of pluripotent cells in vitro, have contributed to the elucidation of the details of the EHT, which was greatly accelerated in recent years by sophisticated live imaging techniques and advances in transcriptional profiling, such as single-cell RNA-Seq. A detailed knowledge of these developmental events is required in order to be able to apply it to the generation of haematopoietic stem cells from pluripotent stem cells in vitro — an achievement which is of obvious clinical importance. The aim of this review is to summarise the latest findings and describe how these may have contributed towards achieving this goal.

Quantification of Mouse Hematopoietic Progenitors' Formation Using Time-lapse Microscopy and Image Analysis.

In vitro differentiation of mouse embryonic stem cells (mESCs) towards blood cells constitutes a well-established system to study the endothelial-to-hematopoietic transition (EHT) at the onset of blood development. Assessing the emergence of small non-adherent round blood cells in the culture without disturbing it is essential to evaluate the progression of EHT and also to test conditions potentially enhancing or repressing this process. Here, we describe how to quantify the formation of mouse hematopoietic progenitors during EHT in normal conditions or following over-expression of eight essential transcription factors using time-lapse microscopy and image analysis.

MEIS2 regulates endothelial to hematopoietic transition of human embryonic stem cells by targeting TAL1

BackgroundDespite considerable progress in the development of methods for hematopoietic differentiation, efficient generation of transplantable hematopoietic stem cells (HSCs) and other genuine functional blood cells from human embryonic stem cells (hESCs) is still unsuccessful. Therefore, a better understanding of the molecular mechanism underlying hematopoietic differentiation of hESCs is highly demanded.MethodsIn this study, by using whole-genome gene profiling, we identified Myeloid Ectopic Viral Integration Site 2 homolog (MEIS2) as a potential regulator of hESC early hematopoietic differentiation. We deleted MEIS2 gene in hESCs using the CRISPR/CAS9 technology and induced them to hematopoietic differentiation, megakaryocytic differentiation.ResultsIn this study, we found that MEIS2 deletion impairs early hematopoietic differentiation from hESCs. Furthermore, MEIS2 deletion suppresses hemogenic endothelial specification and endothelial to hematopoietic transition (EHT), leading to the impairment of hematopoietic differentiation. Mechanistically, TAL1 acts as a downstream gene mediating the function of MEIS2 during early hematopoiesis. Interestingly, unlike MEIS1, MEIS2 deletion exerts minimal effects on megakaryocytic differentiation and platelet generation from hESCs.ConclusionsOur findings advance the understanding of human hematopoietic development and may provide new insights for large-scale generation of functional blood cells for clinical applications.

Cooperative Transcription Factor Induction Mediates Hemogenic Reprogramming

SUMMARYDuring development, hematopoietic stem and progenitor cells (HSPCs) arise from specialized endothelial cells by a process termed endothelial-to-hematopoietic transition (EHT). The genetic program driving human HSPC emergence remains largely unknown. We previously reported that the generation of hemogenic precursor cells from mouse fibroblasts recapitulates developmental hematopoiesis. Here, we demonstrate that human fibroblasts can be reprogrammed into hemogenic cells by the same transcription factors. Induced cells display dynamic EHT transcriptional programs, generate hematopoietic progeny, possess HSPC cell surface phenotype, and repopulate immunodeficient mice for 3 months. Mechanistically, GATA2 and GFI1B interact and co-occupy a cohort of targets. This cooperative binding is reflected by engagement of open enhancers and promoters, initiating silencing of fibroblast genes and activating the hemogenic program. However, GATA2 displays dominant and independent targeting activity during the early phases of reprogramming. These findings shed light on the processes controlling human HSC specification and support generation of reprogrammed HSCs for clinical applications.

Sf3b1 Regulation of Jak/Stat Signaling Is Essential for Hematopoietic Stem Cell Formation

Hematopoietic stem cells (HSCs) maintain the hematopoietic system throughout the lifetime of an organism. During embryonic development, HSCs emerge through an endothelial-to-hematopoietic transition (EHT) from specialized hemogenic endothelial (HE) cells in the dorsal aorta. HSC fate specification depends on gene expression, which is the culmination of coordinated transcription, RNA splicing, and translation. Although transcriptional regulation of HSC fate choice is well studied, the regulatory role of RNA splicing in this process is poorly understood. Using zebrafish loss-of-function mutants for the spliceosomal component splicing factor 3b, subunit 1 (sf3b1), we identified that impaired splicing hindered HSC production. Surprisingly, we found that this constitutive splicing factor selectively regulates the fate of hemogenic endothelium while leaving the identity of closely-related non-hemogenic endothelium unperturbed. To identify Sf3b1-regulated transcripts important in EHT, we performed RNA-sequencing on purified kdrl:gfp+ endothelial cells from sf3b1 mutant and wild-type siblings at 24 hpf. Approximately 900 genes were mis-spliced, 144 of which were differentially expressed. Ingenuity Pathway Analysis identified Janus Kinase (Jak)/Signaling Transducer and Activator of Transcription (Stat) signaling, in particular Stat3, as one of the top perturbed pathways in the mis-spliced gene set. Stat3 is a transcription factor activated in response to several cytokine and inflammatory signals. To determine if altered splicing of stat3 was critical for HSC formation, we injected antisense splice-blocking morpholinos (MO) targeting the Sf3b1-sensitive stat3 exon19 into wild-type and sf3b1 heterozygous embryos, which normally generate equivalent levels of HSCs. We observed an impairment of HSC production in stat3 morpholino-injected sf3b1 heterozygotes, but not wild-type siblings, indicating a synthetic lethal interaction between sf3b1 and stat3. We also found that overexpression of a constitutively active form of Stat3 significantly suppressed the HSC defects in sf3b1 homozygous mutants. Together, these data indicate that Sf3b1-mediated splicing regulation of the Jak/Stat pathway is critical for HSC emergence. DisclosuresNo relevant conflicts of interest to declare.

Inflammasome-Mediated Regulation of Hematopoiesis in the Vertebrate Embryo

Hematopoietic stem and progenitor cells (HSPCs) arise in the embryonic dorsal aorta (DA) through a Runx1-dependent process of endothelial-to-hematopoietic transition (EHT). Subsequently, HSPCs colonize secondary niches to proliferate, differentiate and maintain lifelong hematopoiesis. We previously reported that elevated glucose metabolism and sterile inflammatory signaling each stimulate zebrafish HSPC production from hemogenic endothelium. Consistent with the hypothesis that transient metabolic activation induces an inflammatory response to influence HSPC formation, we found that glucose stimulation from 12-36 hours post fertilization (hpf) increased expression of pro-inflammatory cytokines and receptors by quantitative PCR. In particular, morpholino-mediated knockdown of il1b blunted the inductive effects of glucose metabolism on runx1 expression in the DA and numbers of CD41+ HSPCs in the caudal hematopoietic tissue (CHT). In contrast, overexpression of mature il1b increased HSPC numbers as assessed by flow cytometry and runx1/cmyb in situ hybridization. As IL-1β is cleaved and activated by the inflammasome, which is a well-known sensor of metabolic stimuli, we treated embryos with validated inflammasome activators, such as nigericin, and found that they also increased runx1/cmyb expression and Flk1+cMyb+ HSPCs. Inflammasome stimulation required NF-κB activity, and upregulated canonical IL-1β targets, including IL-6 and IL-8, which have been previously shown by our laboratory to play significant roles in embryonic HSPC formation. Importantly, loss of the inflammasome adapter pycard or effector caspa reduced HSPC numbers, and prevented expansion of the HSPC pool in response to glucose stimulation. In further support of a role for sterile inflammatory activity in regulating HSPC number downstream of metabolic activation, we found that inflammasome-mediated IL-1β action bypassed the suppressive effect of antioxidant treatment on runx1/cmyb expression in the DA. Conversely, inflammasome inhibition partially blocked effects of metabolism-associated HIF1α activation on runx1/cmyb expression, indicating that the inflammasome acts downstream of reactive-oxygen-species (ROS) generation and HIF1α activation to promote HSPC production and/or expansion from hemogenic endothelium. Inflammasome components are highly expressed in myeloid cells; targeted knockdown and cell ablation studies indicate that macrophages are necessary to mediate the effects of inflammasome stimulation on HSPC numbers in the CHT at 72hpf. Interestingly, prolonged inflammasome stimulation also expands myeloid and lymphoid progenitors, as cmyb, rag1 and mpo expression were each elevated at 120hpf; increased numbers of Rag2+ and Mpo+ cells were confirmed by flow cytometry. To determine whether the effects of inflammasome activation were conserved in higher vertebrates, we induced inflammasome activation during differentiation of human induced pluripotent stem cells (iPSC) into hematopoietic progenitors. In this system, nigericin treatment of iPSC-derived hemogenic endothelial cells yielded an increased frequency of functional colony-forming units by day 7 of EHT culture. Taken together, inflammasome-mediated IL-1β action appears to serve as an integrator of metabolic activity, downstream of ROS/ HIF1α, to promote HSPC formation and development of myeloid and lymphoid lineages in vivo and in vitro. These studies identify the inflammasome as a promising target to promote human HSPC production from iPSCs for therapeutic purposes. DisclosuresNo relevant conflicts of interest to declare.

Modeling Fanconi Anemia Using Human Induced Pluripotent Stem Cells By Reversible Complementation

Modeling of Fanconi anemia (FA) using human induced pluripotent stem cells (iPSCs) has been hindered by the requirement for an intact FA DNA repair pathway for effective reprogramming and maintenance of pluripotency in patient-derived iPSCs. Temporary complementation of FA pathway defects could permit effective reprogramming, with removal of complementation upon directed differentiation to hematopoietic stem and progenitor cells (HSPCs) to model the hematopoietic phenotypes of FA. In this study, we used three FA patient-derived iPSC lines engineered with doxycycline inducible complementation of disease-causing FANCA mutations. We maintained complementation with doxycycline exposure in the pluripotent state and during morphogen directed differentiation of hemogenic endothelium (HE) within embryoid bodies, which possesses the capacity to differentiate to HSPCs. Upon initiation of the endothelial-to-hematopoietic transition (EHT) in purified iPSC-derived HE, doxycycline was either removed or maintained in culture to either inactivate or sustain FANCA expression. Morphologic EHT and emergence of CD34+CD45+ immunophenotypic human HSPCs were not affected by the status of FANCA expression, which allowed for the isolation of otherwise isogenic FANCA-expressing and FANCA-deficient HSPCs for disease modeling. FANCA-deficient HSPCs were unable to form FANCD2/γH2AX foci relative to FANCA-expressing HSPCs in response to genotoxic stress, confirming impairment of the function of the FA pathway. We found that uncomplimented, FANCA-deficient HSPCs showed impaired cell cycle progression, increased apoptosis, and markedly decreased colony forming activity in methylcellulose compared to complemented, FANCA-expressing HSPCs. Unexpectedly, we also found that FANCA-deficient HSPCs showed accelerated erythroid differentiation compared to cells with an intact FA pathway. RNA sequencing analysis showed enrichment of signatures of normal HSPCs in complemented HSPCs, while uncomplimented HSPCs showed signatures of more mature hematopoietic cells and cells undergoing erythroid differentiation. Together, these findings demonstrate the utility of reversible complementation in modeling FA with patient-derived iPSCs, and provide an inexhaustible source of otherwise isongenic complimented and uncomplimented human FA HSPCs for use in the investigation of new therapies. DisclosuresNo relevant conflicts of interest to declare.

Key genes and integrated modules in hematopoietic differentiation of human embryonic stem cells: a comprehensive bioinformatic analysis

BackgroundThe generation of hematopoietic stem cells (HSCs) and blood cells from human embryonic stem cells (hESCs) is a major goal for regenerative medicine; however, the differentiation mechanisms are largely undefined. Here, we aimed to identify the regulated genes and functional modules related to the early differentiation of the endothelial-to-hematopoietic transition (EHT) using comprehensive bioinformatics analyses.MethodsUndifferentiated hESCs (hESC-H9), CD34+ cells from 10-day differentiated hESC-H9 cells, and CD34+ cells from umbilical cord cells were isolated and collected. Cells from these three groups were subjected to RNA extraction and microarray analysis by which differentially expressed genes (DEGs) and time-series profiles were analyzed by significance analysis of microarray (SAM) and short time-series expression miner (STEM) algorithms. Gene enrichment analysis was performed by ClusterProfiler Package in Rstudio, while a protein-protein interaction (PPI) network was constructed by search tool for the retrieval of interacting genes (STRING) and visualized in Cytoscape. Hub genes were further identified with the MCODE algorithm in Cytoscape.ResultsIn the present study, we identified 11,262 DEGs and 16 time-series profiles that were enriched in biological processes of chromosome segregation, cell cycle, and leukocyte activation and differentiation, as well as hematopoiesis. Analysis using the MCODE algorithm further identified six integrated modules that might play an important role in the EHT process, including mitosis/cell cycle, mitochondrial process, splicing, ubiquitination, ribosome, and apoptosis.ConclusionsThe study identified potential genes and integrated functional modules associated with the hematopoietic and endothelial differentiation of human ESCs.

Anisotropic organization of circumferential actomyosin characterizes hematopoietic stem cells emergence in the zebrafish.

Hematopoiesis leads to the formation of blood and immune cells. Hematopoietic stem cells emerge during development, from vascular components, via a process called the endothelial-to-hematopoietic transition (EHT). Here, we reveal essential biomechanical features of the EHT, using the zebrafish embryo imaged at unprecedented spatio-temporal resolution and an algorithm to unwrap the aorta into 2D-cartography. We show that the transition involves anisotropic contraction along the antero-posterior axis, with heterogenous organization of contractile circumferential actomyosin. The biomechanics of the contraction is oscillatory, with unusually long periods in comparison to other apical constriction mechanisms described so far in morphogenesis, and is supported by the anisotropic reinforcement of junctional contacts. Finally, we show that abrogation of blood flow impairs the actin cytoskeleton, the morphodynamics of EHT cells, and the orientation of the emergence. Overall, our results underline the peculiarities of the EHT biomechanics and the influence of the mechanical forces exerted by blood flow.

3126 - Glycolysis is Essential for Metabolic Switch-Mediated Endothelial to Hematopoietic Transition

While the metabolic regulation of hematopoietic stem cell (HSC) quiescence, maintenance and differentiation has been extensively studied, it is still unclear how metabolic parameters contribute to the first emergence of HSCs from the hemogenic endothelium (HE). Using a human induced pluripotent stem (iPS) cell differentiation system, we studied the implication of major metabolic pathways in progenitors upstream of HSCs during the endothelial to hematopoietic transition (EHT) process. We show here that there is a gradual increase in the use of glycolysis during EHT, with the highest glucose uptake and glycolytic activity in immunophenotypic HSCs. Moreover, blocking glycolysis leads to a significant decrease in blood output, showing that this pathway is required during the EHT process. Surprisingly, this increase in glycolysis is accompanied with high levels of mitochondrial activity which, when chemically disrupted, also impairs hematopoietic differentiation. These results point towards a metabolic switch during EHT, with an integral increase in metabolic activity that is essential for HSC emergence.

3129 - Dynamic Regulation of Runx1 at the Onset of Definitive Hematopoiesis

Hematopoietic stem cells (HSCs) have been used in medicine for several decades. However, the difficulty of generating, maintaining or expanding them in culture has made it difficult to advance HSC-based therapies further. In the embryo, HSCs first arise from a subset of the dorsal aorta endothelium, the so-called hemogenic endothelium (HE), through a process known as endothelial-to-hematopoietic transition (EHT). The transcription factor Runx1 is critically required for EHT. To begin to elucidate the regulatory pathways converging on Runx1, and thus orchestrating hematopoietic specification and EHT, we have identified and characterized several hematopoietic Runx1 enhancers. Analysis of enhancer-reporter transgenic mouse lines showed that one of these, the +23 enhancer, is sufficient to mediate reporter gene expression to all cells undergoing EHT, including HE and hematopoietic stem and progenitor cells. Two others, in contrast, show a more restricted pattern, with one marking HE but not hematopoietic stem or progenitor cells, while the other marks hematopoietic progenitors but not the HSC lineage or HE. Mutation of candidate transcription factor motifs in these enhancers implicated distinct upstream factors that may contribute to their tissue specificity. Deletion of individual Runx1 enhancers from the endogenous locus in mESCs resulted in a moderate reduction of Runx1 expression in cell populations undergoing EHT. Our results are consistent with a shadow enhancer model where each element is acting in an additive manner. Ongoing work using next generation chromosome conformation capture (NG Capture-C) further dissects Runx1 cis-interactions and supports a model of dynamic enhancer-promoter interactions at the onset of hematopoiesis. Ultimately, a better understanding of factors and signals involved in establishing the HSC lineage in mouse ontogeny is expected to inform the development of culture protocols to generate these cells in vitro.

3163 - Hypoxia Enhances Hematopoietic Development of Human Pluripotent Stem Cells via Sequential HIF1 Activation at Stages of Mesoderm and Endothelial to Hematopoietic Transition

Human early embryonic hematopoiesis occurs in a hypoxic microenvironment before establishment of the circulatory system. In vitro hematopoietic differentiation from human pluripotent stem cells (hPSCs) provides a unique approach to explore the mechanism of hypoxia on early hematopoiesis. Although hypoxia is often used in hPSC differentiation protocol, its actual role in hematopoiesis is largely unknown. Current methods for hematopoiesis from hPSCs rely on stromal cell coculture or embryoid body (EB) formation, which contain poorly defined components or have the solid sphere-like structure that makes against the infiltration of hypoxia. To better understand the effect of hypoxia on early hematopoiesis, we developed a chemically defined monolayer differentiation strategy. In this stepwise system, we monitored the sequential development of mesodermal progenitors (Brachyury+KDR-), hematopoietic-endothelial progenitors (KDR+CD34+) and CD43+CD41a+ hematopoietic cells. We found that hematopoiesis was significantly enhanced by hypoxia. The expression of HIF1 gene was upregulated in two waves, suggesting two distinct effects during hPSC differentiation. Around the first HIF1 peaked period, KDR+CD34+ cells were increased, but not Brachyury+KDR- cells, suggesting that hypoxia enhanced hematopoietic-endothelial lineage commitment from mesodermal progenitors. Around the second HIF1 peaked period, hypoxia promoted endothelial-to-hematopoietic transition (EHT) in a HIF1-dependent manner while the hemogenic endothelial cells (CD34+CD144+CD73-CD184-) was not enhanced by hypoxia. Interestingly, under hypoxic condition, arterial endothelial cells (CD34+CD144+CD73-CD184+) increased significantly, and arterial-specific gene DLL4, a Notch ligand, was also highly expressed. Further study indicated HIF1 suppression impaired DLL4 expression as well as the CD43+ hematopoietic cells generation, which was suppressed by Notch inhibitor. Collectively, we demonstrate for the first time that sequential HIF1 activation is able to significantly increase hematopoiesis by enhancing hematopoietic-endothelial lineage commitment and EHT, likely via Notch signals from arterial endothelial niche.

3200 - Identification of Extrinsic Regulators of Hematopoietic Stem Cell Formation in the Embryonic Aorta Microenvironment by Tomo-Sequencing

In all vertebrates, hematopoietic stem cell (HSC) emergence mainly occurs in the dorsal aorta of the embryo. It is now well accepted that HSCs arise from specialized endothelial cells of the aorta, named hemogenic endothelial cells, via an endothelial-to-hematopoietic transition (EHT) process. This process is tightly controlled in time and space by regulatory signals emitted by the surrounding microenvironment in all species (zebrafish, chicken, mouse and human). Nevertheless, a complete picture of the genes expressed in the surrounding tissues of the aorta that are involved during the highly regulated HSC emergence is still missing. o decipher the genes that are (differentially) expressed in specific regions of the aorta during HSC generation, we used a genome-wide RNA tomography (tomo-seq) approach (Junker 2014). Tomo-seq was applied on whole aortas (subdissected with the surrounding mesenchyme) and thick transversal embryo slices from zebrafish, chicken, mouse and human embryos. Using this approach, we provided a complete resource of the gene expression profiles around the dorsal aorta in all species. We then identified a series of genes specifically expressed either in the ventral or dorsal side of the aorta and validated their expression pattern using in situ hybridization and/or immunostainings. We further demonstrated their functional implication in HSC emergence and hematopoietic production by knocking-down these genes in zebrafish embryos. Finally, our comparative data analysis in between species unravelled conserved genes and molecular pathways involved in the regulation of the first HSC generated during embryonic development.