The Experts below are selected from a list of 906 Experts worldwide ranked by ideXlab platform
Georges Lacaud - One of the best experts on this subject based on the ideXlab platform.
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early human Hemogenic Endothelium generates primitive and definitive hematopoiesis in vitro
Stem cell reports, 2018Co-Authors: Eva Garciaalegria, Georges Lacaud, Sara Menegatti, Muhammad Z H Fadlullah, Pablo Menendez, Valerie KouskoffAbstract:Summary The differentiation of human embryonic stem cells (hESCs) to hematopoietic lineages initiates with the specification of Hemogenic Endothelium, a transient specialized endothelial precursor of all blood cells. This in vitro system provides an invaluable model to dissect the emergence of hematopoiesis in humans. However, the study of hematopoiesis specification is hampered by a lack of consensus in the timing of Hemogenic Endothelium analysis and the full hematopoietic potential of this population. Here, our data reveal a sharp decline in the Hemogenic potential of Endothelium populations isolated over the course of hESC differentiation. Furthermore, by tracking the dynamic expression of CD31 and CD235a at the onset of hematopoiesis, we identified three populations of hematopoietic progenitors, representing primitive and definitive subsets that all emerge from the earliest specified Hemogenic Endothelium. Our data establish that Hemogenic Endothelium populations endowed with primitive and definitive hematopoietic potential are specified simultaneously from the mesoderm in differentiating hESCs.
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hoxb4 promotes Hemogenic Endothelium formation without perturbing endothelial cell development
Stem cell reports, 2018Co-Authors: Nadine Teichweyde, Georges Lacaud, Valerie Kouskoff, Lara Kasperidus, Sebastian Carotta, Peter A Horn, Stefan Heinrichs, Hannes KlumpAbstract:Summary Generation of hematopoietic stem cells (HSCs) from pluripotent stem cells, in vitro , holds great promise for regenerative therapies. Primarily, this has been achieved in mouse cells by overexpression of the homeotic selector protein HOXB4. The exact cellular stage at which HOXB4 promotes hematopoietic development, in vitro , is not yet known. However, its identification is a prerequisite to unambiguously identify the molecular circuits controlling hematopoiesis, since the activity of HOX proteins is highly cell and context dependent. To identify that stage, we retrovirally expressed HOXB4 in differentiating mouse embryonic stem cells (ESCs). Through the use of Runx1 (−/−) ESCs containing a doxycycline-inducible Runx1 coding sequence, we uncovered that HOXB4 promoted the formation of Hemogenic Endothelium cells without altering endothelial cell development. Whole-transcriptome analysis revealed that its expression mediated the upregulation of transcription of core transcription factors necessary for hematopoiesis, culminating in the formation of blood progenitors upon initiation of Runx1 expression.
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regulation of runx1 dosage is crucial for efficient blood formation from Hemogenic Endothelium
Development, 2018Co-Authors: Michael Liealing, Valerie Kouskoff, Elli Marinopoulou, Andrew J Lilly, Mairi Challinor, Rahima Patel, Christophe Lancrin, Georges LacaudAbstract:ABSTRACT During ontogeny, hematopoietic stem and progenitor cells arise from Hemogenic Endothelium through an endothelial-to-hematopoietic transition that is strictly dependent on the transcription factor RUNX1. Although it is well established that RUNX1 is essential for the onset of hematopoiesis, little is known about the role of RUNX1 dosage specifically in Hemogenic Endothelium and during the endothelial-to-hematopoietic transition. Here, we used the mouse embryonic stem cell differentiation system to determine if and how RUNX1 dosage affects Hemogenic Endothelium differentiation. The use of inducible Runx1 expression combined with alterations in the expression of the RUNX1 co-factor CBFβ allowed us to evaluate a wide range of RUNX1 levels. We demonstrate that low RUNX1 levels are sufficient and necessary to initiate an effective endothelial-to-hematopoietic transition. Subsequently, RUNX1 is also required to complete the endothelial-to-hematopoietic transition and to generate functional hematopoietic precursors. In contrast, elevated levels of RUNX1 are able to drive an accelerated endothelial-to-hematopoietic transition, but the resulting cells are unable to generate mature hematopoietic cells. Together, our results suggest that RUNX1 dosage plays a pivotal role in Hemogenic Endothelium maturation and the establishment of the hematopoietic system.
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hemangioblast Hemogenic Endothelium and primitive versus definitive hematopoiesis
Experimental Hematology, 2017Co-Authors: Georges Lacaud, Valerie KouskoffAbstract:The types of progenitors generated during the successive stages of embryonic blood development are now fairly well characterized. The terminology used to describe these waves, however, can still be confusing. What is truly primitive? What is uniquely definitive? These questions become even more challenging to answer when blood progenitors are derived in vitro upon the differentiation of embryonic stem cells or induced pluripotent stem cells. Similarly, the cellular origin of these blood progenitors can be controversial. Are all blood cells, including the primitive wave, derived from Hemogenic Endothelium? Is the hemangioblast an in vitro artifact or is this mesoderm entity also present in the developing embryo? Here, we discuss the latest findings and propose some consensus relating to these controversial issues.
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runx1 positively regulates a cell adhesion and migration program in murine Hemogenic Endothelium prior to blood emergence
Blood, 2014Co-Authors: Michael Liealing, Valerie Kouskoff, Elli Marinopoulou, Rahima Patel, Monika Stefanska, Constanze Bonifer, Yaoyong Li, Crispin J Miller, Georges LacaudAbstract:During ontogeny, the transcription factor RUNX1 governs the emergence of definitive hematopoietic cells from specialized endothelial cells called Hemogenic Endothelium (HE). The ultimate consequence of this endothelial-to-hematopoietic transition is the concomitant activation of the hematopoietic
Thierry Jaffredo - One of the best experts on this subject based on the ideXlab platform.
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an in vitro model of Hemogenic Endothelium commitment and hematopoietic production
Development, 2016Co-Authors: Laurent Yvernogeau, Thierry Jaffredo, Sara Menegatti, Rodolphe Gautier, Hanane Khoury, Melanie Schmidt, Jean Francois GillesAbstract: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.
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decoding the Hemogenic Endothelium in mammals
Cell Stem Cell, 2009Co-Authors: Francoise Dieterlenlievre, Thierry JaffredoAbstract:A collection of recent Nature papers examines the relationship between endothelial precursors and hematopoietic cells. Two of these studies (Eilken et al., 2009; Lancrin et al., 2009) use time-lapse imaging with live markers and genetic analysis of differentiating ESCs to reveal that even non-aortic-derived endothelial cells are Hemogenic.
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the embryonic origins of hematopoietic stem cells a tale of hemangioblast and Hemogenic Endothelium
Apmis, 2005Co-Authors: Karine Bollerot, Claire Pouget, Thierry JaffredoAbstract:The developmental origin of hematopoietic stem cells has been for decades the subject of great interest. Once thought to emerge from the yolk sac, hematopoietic stem cells have now been shown to originate from the embryonic aorta. Increasing evidence suggests that hematopoietic stem cells are produced from an endothelial intermediate designated by the authors as hemangioblast or Hemogenic Endothelium. Recently, the allantois in the avian embryo and the placenta in the mouse embryo were shown to be a site of hematopoietic cell production/expansion and thus appear to play a critical role in the formation of the hematopoietic system. In this review we shall give an overview of the data obtained from human, mouse and avian models on the cellular origins of the hematopoietic system and discuss some aspects of the molecular mechanisms controlling hematopoietic cell production.
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from hemangioblast to hematopoietic stem cell an endothelial connection
Experimental Hematology, 2005Co-Authors: Thierry Jaffredo, Karine Bollerot, Wade T Nottingham, Claire Pouget, Kate Liddiard, Marella De BruijnAbstract:The developmental origin of hematopoietic stem cells has been the subject of much research. Now that the developmental link between the hematopoietic system and the vasculature has been well established, questions remain regarding the precise cellular origin of definitive hematopoietic cells and at what point they branch off from the endothelial lineage. Do they emerge directly from a hemangioblast-type cell, similar to what is proposed for primitive yolk sac hematopoiesis, or are they generated via an endothelial intermediate, the Hemogenic Endothelium? In this review, we will give an overview of the data obtained from the mouse and avian models on the cellular origins of the hematopoietic system.
Valerie Kouskoff - One of the best experts on this subject based on the ideXlab platform.
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early human Hemogenic Endothelium generates primitive and definitive hematopoiesis in vitro
Stem cell reports, 2018Co-Authors: Eva Garciaalegria, Georges Lacaud, Sara Menegatti, Muhammad Z H Fadlullah, Pablo Menendez, Valerie KouskoffAbstract:Summary The differentiation of human embryonic stem cells (hESCs) to hematopoietic lineages initiates with the specification of Hemogenic Endothelium, a transient specialized endothelial precursor of all blood cells. This in vitro system provides an invaluable model to dissect the emergence of hematopoiesis in humans. However, the study of hematopoiesis specification is hampered by a lack of consensus in the timing of Hemogenic Endothelium analysis and the full hematopoietic potential of this population. Here, our data reveal a sharp decline in the Hemogenic potential of Endothelium populations isolated over the course of hESC differentiation. Furthermore, by tracking the dynamic expression of CD31 and CD235a at the onset of hematopoiesis, we identified three populations of hematopoietic progenitors, representing primitive and definitive subsets that all emerge from the earliest specified Hemogenic Endothelium. Our data establish that Hemogenic Endothelium populations endowed with primitive and definitive hematopoietic potential are specified simultaneously from the mesoderm in differentiating hESCs.
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hoxb4 promotes Hemogenic Endothelium formation without perturbing endothelial cell development
Stem cell reports, 2018Co-Authors: Nadine Teichweyde, Georges Lacaud, Valerie Kouskoff, Lara Kasperidus, Sebastian Carotta, Peter A Horn, Stefan Heinrichs, Hannes KlumpAbstract:Summary Generation of hematopoietic stem cells (HSCs) from pluripotent stem cells, in vitro , holds great promise for regenerative therapies. Primarily, this has been achieved in mouse cells by overexpression of the homeotic selector protein HOXB4. The exact cellular stage at which HOXB4 promotes hematopoietic development, in vitro , is not yet known. However, its identification is a prerequisite to unambiguously identify the molecular circuits controlling hematopoiesis, since the activity of HOX proteins is highly cell and context dependent. To identify that stage, we retrovirally expressed HOXB4 in differentiating mouse embryonic stem cells (ESCs). Through the use of Runx1 (−/−) ESCs containing a doxycycline-inducible Runx1 coding sequence, we uncovered that HOXB4 promoted the formation of Hemogenic Endothelium cells without altering endothelial cell development. Whole-transcriptome analysis revealed that its expression mediated the upregulation of transcription of core transcription factors necessary for hematopoiesis, culminating in the formation of blood progenitors upon initiation of Runx1 expression.
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regulation of runx1 dosage is crucial for efficient blood formation from Hemogenic Endothelium
Development, 2018Co-Authors: Michael Liealing, Valerie Kouskoff, Elli Marinopoulou, Andrew J Lilly, Mairi Challinor, Rahima Patel, Christophe Lancrin, Georges LacaudAbstract:ABSTRACT During ontogeny, hematopoietic stem and progenitor cells arise from Hemogenic Endothelium through an endothelial-to-hematopoietic transition that is strictly dependent on the transcription factor RUNX1. Although it is well established that RUNX1 is essential for the onset of hematopoiesis, little is known about the role of RUNX1 dosage specifically in Hemogenic Endothelium and during the endothelial-to-hematopoietic transition. Here, we used the mouse embryonic stem cell differentiation system to determine if and how RUNX1 dosage affects Hemogenic Endothelium differentiation. The use of inducible Runx1 expression combined with alterations in the expression of the RUNX1 co-factor CBFβ allowed us to evaluate a wide range of RUNX1 levels. We demonstrate that low RUNX1 levels are sufficient and necessary to initiate an effective endothelial-to-hematopoietic transition. Subsequently, RUNX1 is also required to complete the endothelial-to-hematopoietic transition and to generate functional hematopoietic precursors. In contrast, elevated levels of RUNX1 are able to drive an accelerated endothelial-to-hematopoietic transition, but the resulting cells are unable to generate mature hematopoietic cells. Together, our results suggest that RUNX1 dosage plays a pivotal role in Hemogenic Endothelium maturation and the establishment of the hematopoietic system.
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hemangioblast Hemogenic Endothelium and primitive versus definitive hematopoiesis
Experimental Hematology, 2017Co-Authors: Georges Lacaud, Valerie KouskoffAbstract:The types of progenitors generated during the successive stages of embryonic blood development are now fairly well characterized. The terminology used to describe these waves, however, can still be confusing. What is truly primitive? What is uniquely definitive? These questions become even more challenging to answer when blood progenitors are derived in vitro upon the differentiation of embryonic stem cells or induced pluripotent stem cells. Similarly, the cellular origin of these blood progenitors can be controversial. Are all blood cells, including the primitive wave, derived from Hemogenic Endothelium? Is the hemangioblast an in vitro artifact or is this mesoderm entity also present in the developing embryo? Here, we discuss the latest findings and propose some consensus relating to these controversial issues.
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runx1 positively regulates a cell adhesion and migration program in murine Hemogenic Endothelium prior to blood emergence
Blood, 2014Co-Authors: Michael Liealing, Valerie Kouskoff, Elli Marinopoulou, Rahima Patel, Monika Stefanska, Constanze Bonifer, Yaoyong Li, Crispin J Miller, Georges LacaudAbstract:During ontogeny, the transcription factor RUNX1 governs the emergence of definitive hematopoietic cells from specialized endothelial cells called Hemogenic Endothelium (HE). The ultimate consequence of this endothelial-to-hematopoietic transition is the concomitant activation of the hematopoietic
Ben Van Handel - One of the best experts on this subject based on the ideXlab platform.
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abstract 356 scl represses cardiogenesis via distant enhancers during Hemogenic Endothelium specification
Circulation Research, 2013Co-Authors: Dan Duan, Roberto Ferrari, Amelie Montelhagen, Ben Van Handel, Rajkumar Sasidharan, Stuart H Orkin, Siavash K Kurdistani, Hanna K A MikkolaAbstract:Understanding the mechanisms directing mesoderm specification holds a great potential to advance the development of cell-based therapies for cardiovascular and blood disorders. The bHLH transcription factor Scl is known as the master regulator of the hematopoietic fate. We recently discovered that, in addition to its critical function in promoting the establishment of Hemogenic Endothelium during hematopoietic stem/progenitor cell (HS/PC) development, Scl is also required to repress cardiomyogenesis in Endothelium in hematopoietic tissues and endocardium in the heart. However, the mechanisms for the cardiac repression have remained unknown. Using ChIP-sequencing and microarray analysis of Flk+ mesoderm differentiated from mouse ES cells, we show that Scl both directly activates a broad gene regulatory network required for Hemogenic Endothelium and HS/PC development (e.g. Runx1, cMyb, Lyl1, Mef2C, Sox7 etc.), and directly represses transcriptional regulators required for cardiogenesis (e.g. Gata4, Gata6, Myocd, etc.) and mesoderm development (Eomes, Mixl1, Etv2, etc.). Repression of cardiac and mesodermal programs occurs during a short developmental window through Scl binding to distant enhancers, while binding to hematopoietic regulators extends throughout HS/PC and red blood cell development and encompasses both distant and proximal binding sites. We also discovered that, surprisingly, Scl complex partners Gata 1 and 2 are dispensable for hematopoietic vs. cardiac specification and Scl binding to majority of its target genes. Nevertheless, Gata factors co-operate with Scl to activate selected transcription factors that facilitate HS/PC emergence from Hemogenic Endothelium. These results denote Scl as a true master regulator of hematopoietic vs. cardiac fate choice and suggest a mechanism by which lineage-specific bHLH factors direct the divergence of competing fates.
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scl represses cardiomyogenesis in prospective Hemogenic Endothelium and endocardium
Cell, 2012Co-Authors: Ben Van Handel, Roberto Ferrari, Amelie Montelhagen, Rajkumar Sasidharan, Haruko Nakano, Cornelis J Boogerd, Johann Schredelseker, Yanling Wang, Sean HunterAbstract:Summary Endothelium in embryonic hematopoietic tissues generates hematopoietic stem/progenitor cells; however, it is unknown how its unique potential is specified. We show that transcription factor Scl/Tal1 is essential for both establishing the hematopoietic transcriptional program in Hemogenic Endothelium and preventing its misspecification to a cardiomyogenic fate. Scl −/− embryos activated a cardiac transcriptional program in yolk sac Endothelium, leading to the emergence of CD31 + Pdgfrα + cardiogenic precursors that generated spontaneously beating cardiomyocytes. Ectopic cardiogenesis was also observed in Scl −/− hearts, where the disorganized endocardium precociously differentiated into cardiomyocytes. Induction of mosaic deletion of Scl in Scl fl/fl Rosa26Cre-ER T2 embryos revealed a cell-intrinsic, temporal requirement for Scl to prevent cardiomyogenesis from Endothelium. Scl −/− Endothelium also upregulated the expression of Wnt antagonists, which promoted rapid cardiomyocyte differentiation of ectopic cardiogenic cells. These results reveal unexpected plasticity in embryonic Endothelium such that loss of a single master regulator can induce ectopic cardiomyogenesis from endothelial cells.
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latent cardiogenic potential in endocardium and Hemogenic Endothelium revealed in the absence of scl tal1
Blood, 2011Co-Authors: Amelie Montelhagen, Roberto Ferrari, Ben Van Handel, Rajkumar Sasidharan, Haruko Nakano, Kees Boogerd, Jian Zhou, Xinmin Li, Matteo Pellegrini, Stuart H OrkinAbstract:Abstract 2362 The Endothelium in embryonic and extraembryonic hematopoietic tissues has the capacity to generate hematopoietic stem and progenitor cells (HS/PC). However, it is unknown how this unique Endothelium is specified. Microarray analysis of endothelial cells from hematopoietic tissues of embryos deficient for the bHLH transcription factor Scl/tal1 revealed that Scl establishes a robust hematopoietic transcriptional program in the Endothelium. Surprisingly, lack of Scl also induced an unexpected fate switching of the prospective Hemogenic Endothelium to the cardiac lineage. Scl deficient embryos displayed a dramatic upregulation of cardiac transcription factors and structural proteins within the yolk sac vasculature, resulting in the generation of spontaneously beating cardiomyocytes. Ectopic cardiac potential in Scl deficient embryos arose from endothelial-derived CD31+Pdgfrα+ cardiogenic progenitor cells (CPCs), which were present in all sites of HS/PC generation. Analysis of Runx1-deficient embryos revealed, that although Runx1 acts downstream of Scl during the emergence of definitive HS/PCs, it is not required for the suppression of the cardiac fate in the Endothelium. The only wild type tissue that contained CD31+Pdgfrα+ putative CPCs was the heart, and this population was greatly expanded in Scl deficient embryos. Strikingly, endocardium in Scl−/− hearts also activated a robust cardiomyogenic transcriptional program and generated Troponin T+ cardiomyocytes both in vivo and in vitro. Although CD31+Pdgfrα+ CPCs from wild type hearts did not generate readily beating cells in culture, they produced cells expressing endothelial, smooth muscle and cardiomyocyte specific genes, implying multipotentiality of this novel CPC population. Furthermore, CD31+Pdgfrα+ CPCs were greatly reduced in Isl1−/− hearts, which fail to generate functional, multipotential CPCs. Lineage tracing using VE-cadherin Cre Rosa-YFP mouse strain demonstrated that, in addition to generating HS/PCs in hematopoietic tissues, endothelial cells are also the cell of origin for CD31+Pdgfrα+ CPCs in the heart. Together, these data suggest a broader role for embryonic Endothelium as a potential source of tissue-specific stem and progenitor cells and implicate Scl/tal1 as an important regulator of endothelial fate choice. Disclosures: No relevant conflicts of interest to declare.
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Latent Cardiogenic Potential in Endocardium and Hemogenic Endothelium Revealed in the Absence of Scl/tal1
Blood, 2011Co-Authors: Amelie Montel-hagen, Roberto Ferrari, Ben Van Handel, Rajkumar Sasidharan, Haruko Nakano, Kees Boogerd, Jian Zhou, Xinmin Li, Matteo PellegriniAbstract:Abstract 2362 The Endothelium in embryonic and extraembryonic hematopoietic tissues has the capacity to generate hematopoietic stem and progenitor cells (HS/PC). However, it is unknown how this unique Endothelium is specified. Microarray analysis of endothelial cells from hematopoietic tissues of embryos deficient for the bHLH transcription factor Scl/tal1 revealed that Scl establishes a robust hematopoietic transcriptional program in the Endothelium. Surprisingly, lack of Scl also induced an unexpected fate switching of the prospective Hemogenic Endothelium to the cardiac lineage. Scl deficient embryos displayed a dramatic upregulation of cardiac transcription factors and structural proteins within the yolk sac vasculature, resulting in the generation of spontaneously beating cardiomyocytes. Ectopic cardiac potential in Scl deficient embryos arose from endothelial-derived CD31+Pdgfrα+ cardiogenic progenitor cells (CPCs), which were present in all sites of HS/PC generation. Analysis of Runx1-deficient embryos revealed, that although Runx1 acts downstream of Scl during the emergence of definitive HS/PCs, it is not required for the suppression of the cardiac fate in the Endothelium. The only wild type tissue that contained CD31+Pdgfrα+ putative CPCs was the heart, and this population was greatly expanded in Scl deficient embryos. Strikingly, endocardium in Scl−/− hearts also activated a robust cardiomyogenic transcriptional program and generated Troponin T+ cardiomyocytes both in vivo and in vitro. Although CD31+Pdgfrα+ CPCs from wild type hearts did not generate readily beating cells in culture, they produced cells expressing endothelial, smooth muscle and cardiomyocyte specific genes, implying multipotentiality of this novel CPC population. Furthermore, CD31+Pdgfrα+ CPCs were greatly reduced in Isl1−/− hearts, which fail to generate functional, multipotential CPCs. Lineage tracing using VE-cadherin Cre Rosa-YFP mouse strain demonstrated that, in addition to generating HS/PCs in hematopoietic tissues, endothelial cells are also the cell of origin for CD31+Pdgfrα+ CPCs in the heart. Together, these data suggest a broader role for embryonic Endothelium as a potential source of tissue-specific stem and progenitor cells and implicate Scl/tal1 as an important regulator of endothelial fate choice. Disclosures: No relevant conflicts of interest to declare.
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blood flow is required for the release of hematopoietic stem and progenitor cells from Hemogenic Endothelium in the placenta
Blood, 2009Co-Authors: Katrin E Rhodes, Ben Van Handel, Yanling Wang, Michele Wang, Akanksha Chhabra, Hanna K A MikkolaAbstract:Abstract 698 Hematopoietic stem cells (HSCs) are required for continuous blood cell production throughout life. HSCs emerge only within a short developmental time window during embryogenesis. Mounting evidence posits that HSCs arise directly from Hemogenic endothelial cells during midgestation within the large arteries of the conceptus, which include the dorsal aorta, the umbilical and vitelline arteries and the chorioallantoic vessels of the placenta. However, the microenvironmental signals that mediate this temporally regulated process remain unclear. Here we examine, by using Ncx1 −/− embryos that lack heartbeat and circulation, how blood flow imparts instructive cues that ensure proper HSC development. Immunostaining revealed that CD41 + hematopoietic cells, although present, were markedly decreased in Ncx1 -/- placentas as compared to wild-type controls. Furthermore, mutant placentas evidenced large clusters of round CD31 + cells protruding into the lumens of the chorioallantoic vessels. Based on these data, we hypothesized that lack of blood flow may impede the generation of hematopoietic stem and progenitor cells (HS/PCs) and that the endothelial clusters represent Hemogenic intermediates. FACS analysis and colony forming assays confirmed a dramatic reduction in the number of clonogenic progenitors in the placenta and the embryo proper of Ncx mutants, while the yolk sac was unaffected. However, HS/PC generation in the placenta and embryo could be rescued by culturing explants on OP9 stroma before plating in colony forming assays, verifying intact hematopoietic potential. To determine if the rescue observed was due to expansion of existing progenitors or generation of new HS/PCs, we sorted CD41 med ckit + hematopoietic progenitors and CD31 + CD41 − endothelial cells from hematopoietic tissues and co-cultured them on stroma. These experiments demonstrated that endothelial cells from placenta, embryo proper and yolk sac can generate HS/PCs following stroma stimulation, confirming the presence of Hemogenic Endothelium in these organs. Immunostaining of Ncx −/− placentas revealed that although the development of the arterio-venous vascular network was impaired, Notch1 signaling, required for both arterial specification and HSC development, was robust in cells of the endothelial clusters. Furthermore, positive staining for Runx1 and c-myb indicated that cells in the clusters had activated the hematopoietic program. Interestingly, electron microscopy demonstrated that cells in the clusters were tethered to each other via adherens junctions, a characteristic of endothelial cells. In addition, they also maintained high levels of Flk1, expressed VEGF and were actively proliferating, consistent with exposure to extended hypoxia. These data suggest that although cells in the clusters have initiated hematopoietic commitment, they are unable to down-regulate their endothelial identity and complete hematopoietic emergence, resulting in the formation of clusters of Hemogenic intermediates. These results imply that cues imparted via circulation are required to complete the commitment to a hematopoietic fate from Hemogenic Endothelium. Data from co-culture experiments suggest that prolonged Notch1 signaling impairs hematopoietic emergence from Hemogenic endothelial cells, and may account for the HSC emergence defect in the absence of blood flow. Overall, these data suggest that blood flow and circulating primitive red blood cells are critical components of the dynamic microenvironment necessary to both relieve the hypoxia required for the specification and proliferation of Hemogenic Endothelium and provide important mechanical and/or molecular signals required by HSCs to fully commit to the hematopoietic fate and complete emergence. Disclosures: No relevant conflicts of interest to declare.
Igor I Slukvin - One of the best experts on this subject based on the ideXlab platform.
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SOX17 Is Essential for Integration of Arterial and HOXA Programs in Hemogenic Endothelium
Blood, 2019Co-Authors: Ho Sun Jung, James A Thomson, Mi Ae Park, Gene Uenishi, Matthew Raymond, Igor I SlukvinAbstract:Recent advances in understanding the major bottlenecks in derivation of engraftable HSCs and lymphoid cells from pluripotent stem cells (PSC), have identified deficiencies in NOTCH and HOXA signaling as contributing factors to the observed functional deficits of PSC-derived hematopoietic progenitors. However, little is known about the mechanisms that are essential for establishing these pathways during PSC differentiation. Here, we revealed the critical role of SOX17 in linking HOXA and NOTCH-mediated arterial programs in Hemogenic Endothelium (HE) and specification of definitive lympho-myeloid hematopoiesis. Using SOX17-knockout (SOX17-/-) and SOX17 DOX-inducible (iSOX17) hESCs, we found that SOX17-deficiency substantially reduces formation of CD144+CD43-CD73-DLL4+CXCR4+/- arterial HE and definitive lympho-myeloid hematopoiesis, while SOX17 upregulation at mesodermal stage of development causes the opposite effect. Molecular profiling of HE generated from iSOX17 hESCs in DOX+ and DOX- conditions using RNAseq, SOX17 ChIPseq and ATACseq, revealed that SOX17 overexpression upregulates 522 genes enriched in NOTCH, TGFb, HEDGEHOG and WNT signaling, including DLL1, DLL4, NOTCH4, LFNG, WNT5a, WNT5b, GLI3, and genes associated with HSC development, CXCR4,KITLG and ALDH1A2. In addition, we noted significant upregulation of HOXA7,HOXA9, HOXA10, HOXB8, HOXC4 and CDX2 homeobox genes in SOX17-induced cultures, with no expression of HOXA genes observed in HE from SOX17-/- cells. ChIPSeq analysis revealed DOX+ specific SOX17 binding at transcriptional start sites (TSS) of 316 significantly upregulated genes, including ALDH1A2, CDX2, DLL1, DLL4, HEY1, HOXA7, HOXB8, HOXC4 and KITLG, suggesting that upregulation of these genes could be explained by their direct activation by SOX17. Since ALDH1A2 and CDX2 are known to play a role in the activation of HOXA genes, we investigated whether SOX17's effect on HOXA expression could also be mediated by ALDH1A2 and CDX2. We found that adding ALDH1 inhibitor to DOX+ cultures had no effect on arterial HE development and HOXA expression. In contrast, transfection of iSOX17 hPSCs cultures with CDX2 shRNA significantly decreased arterial HE formation and downregulated HOXA7, HOXA9, and HOXA10 expression. Overall, our studies indicate that SOX17 plays a critical role in the activation and integration of arterial and HOXA programs in HE, which is mediated by CDX2. These findings will be important for designing a strategy for direct HSC fate programming from hPSCs. Disclosures Uenishi: Casebia Therapeutics: Employment. Slukvin:Cynata Therapeutics: Consultancy, Other: Founder and Stockholder.
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arterial identity of Hemogenic Endothelium a key to unlock definitive hematopoietic commitment in human pluripotent stem cell cultures
Experimental Hematology, 2019Co-Authors: Igor I Slukvin, Gene UenishiAbstract:Human pluripotent stem cells (hPSCs) have been suggested as a potential source for the de novo production of blood cells for transfusion, immunotherapies, and transplantation. However, even with advanced hematopoietic differentiation methods, the primitive and myeloid-restricted waves of hematopoiesis dominate in hPSC differentiation cultures, whereas cell surface markers to distinguish these waves of hematopoiesis from lympho-myeloid hematopoiesis remain unknown. In the embryo, hematopoietic stem cells (HSCs) arise from Hemogenic Endothelium (HE) lining arteries, but not veins. This observation led to a long-standing hypothesis that arterial specification is an essential prerequisite to initiate the HSC program. It has also been established that lymphoid potential in the yolk sac and extraembryonic vasculature is mostly confined to arteries, whereas myeloid-restricted hematopoiesis is not specific to arterial vessels. Here, we review how the link between arterialization and the subsequent definitive multilineage hematopoietic program can be exploited to identify HE enriched in lymphoid progenitors and aid in in vitro approaches to enhance the production of lymphoid cells and potentially HSCs from hPSCs. We also discuss alternative models of hematopoietic specification at arterial sites and recent advances in our understanding of hematopoietic development and the production of engraftable hematopoietic cells from hPSCs.
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gata2 is dispensable for specification of Hemogenic Endothelium but promotes endothelial to hematopoietic transition
Stem cell reports, 2018Co-Authors: Hyunjun Kang, Walattatseyon Mesquitta, Ho Sun Jung, Oleg V Moskvin, James A Thomson, Igor I SlukvinAbstract:Summary The transcriptional factor GATA2 is required for blood and hematopoietic stem cell formation during the Hemogenic Endothelium (HE) stage of development in the embryo. However, it is unclear if GATA2 controls HE lineage specification or if it solely regulates endothelial-to-hematopoietic transition (EHT). To address this problem, we innovated a unique system, which involved generating GATA2 knockout human embryonic stem cell (hESC) lines with conditional GATA2 expression (iG2 −/− hESCs). We demonstrated that GATA2 activity is not required for VE-cadherin + CD43 − CD73 + non-HE or VE-cadherin + CD43 − CD73 – HE generation and subsequent HE diversification into DLL4 + arterial and DLL4 – non-arterial lineages. However, GATA2 is primarily needed for HE to undergo EHT. Forced expression of GATA2 in non-HE failed to induce blood formation. The lack of GATA2 requirement for generation of HE and non-HE indicates the critical role of GATA2-independent pathways in specification of these two distinct endothelial lineages.
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activation of the arterial program drives development of definitive Hemogenic Endothelium with lymphoid potential
Cell Reports, 2018Co-Authors: Mi Ae Park, Ho Sun Jung, Oleg V Moskvin, James A Thomson, Akhilesh Kumar, Gene Uenishi, Igor I SlukvinAbstract:Summary Understanding the pathways guiding the development of definitive hematopoiesis with lymphoid potential is essential for advancing human pluripotent stem cell (hPSC) technologies for the treatment of blood diseases and immunotherapies. In the embryo, lymphoid progenitors and hematopoietic stem cells (HSCs) arise from Hemogenic Endothelium (HE) lining arteries but not veins. Here, we show that activation of the arterial program through ETS1 overexpression or by modulating MAPK/ERK signaling pathways at the mesodermal stage of development dramatically enhanced the formation of arterial-type HE expressing DLL4 and CXCR4. Blood cells generated from arterial HE were more than 100-fold enriched in T cell precursor frequency and possessed the capacity to produce B lymphocytes and red blood cells expressing high levels of BCL11a and β-globin. Together, these findings provide an innovative strategy to aid in the generation of definitive lymphomyeloid progenitors and lymphoid cells from hPSCs for immunotherapy through enhancing arterial programming of HE.
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direct induction of Hemogenic Endothelium and blood by overexpression of transcription factors in human pluripotent stem cells
Journal of Visualized Experiments, 2015Co-Authors: Irina Elcheva, Vera Brokvolchanskaya, Igor I SlukvinAbstract:During development, hematopoietic cells arise from a specialized subset of endothelial cells, Hemogenic Endothelium (HE). Modeling HE development in vitro is essential for mechanistic studies of the endothelial-hematopoietic transition and hematopoietic specification. Here, we describe a method for the efficient induction of HE from human pluripotent stem cells (hPSCs) by way of overexpression of different sets of transcription factors. The combination of ETV2 and GATA1 or GATA2 TFs is used to induce HE with pan-myeloid potential, while a combination of GATA2 and TAL1 transcription factors allows for the production of HE with erythroid and megakaryocytic potential. The addition of LMO2 to GATA2 and TAL1 combination substantially accelerates differentiation and increases erythroid and megakaryocytic cells production. This method provides an efficient and rapid means of HE induction from hPSCs and allows for the observation of the endothelial-hematopoietic transition in a culture dish. The protocol includes hPSCs transduction procedures and post-transduction analysis of HE and blood progenitors.