scholarly journals Modulation of Mesodermal Patterning Combined with High VEGFA Concentrations Promote Robust Arterial Hemogenic Endothelium Differentiation from Human iPSCs

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5093-5093
Author(s):  
Juan Pablo Ruiz ◽  
Andre Larochelle
Keyword(s):  
Stem Cell ◽  
Embryoid Body ◽  
Notch Pathway ◽  
Hemogenic Endothelium ◽  
Hematopoietic Stem ◽  
Cell Technologies ◽  
Differentiation Protocol ◽  
Human Ipscs ◽  
Arterial Endothelium ◽  
Nsg Mouse

Abstract One of the most promising objectives of clinical hematology is to derive transplantable autologous hematopoietic stem/progenitor cells (HSPCs) from human iPSCs ex vivo, but efficient and clinically relevant methodologies remain unavailable. Observations in the developing embryo indicate that definitive HSPCs arise in the dorsal aorta from hemogenic endothelium (HE) in close association with an arterial vascular endothelial niche. The Notch pathway plays a key role in arterial and HSC differentiation; in the embryo, only HE populations found in arterial regions with active Notch signaling through Delta-like ligand 4 (Dll4) and Jagged 1 (Jag1) lead to HSPCs with repopulating potential. Recent studies have also shown that modulation of mesodermal patterning through repression and activation of Activin/Nodal and Wnt/β-catenin pathways, respectively, promotes arterial programs and definitive hematopoiesis. To facilitate the development of functional HSPCs from human iPSCs, we previously developed a simple, monolayer-based, chemically-defined, and scalable differentiation protocol requiring no replating or embryoid body (EB) formation (commercially available as STEMdiffTM Hematopoietic Kit, Stem Cell Technologies). During the first 3 days, mesodermal specification is induced using morphogens (bFGF, BMP4, VEGF 10ng/mL) and, for the subsequent 18 days, cells are further differentiated into HSPCs with the addition of hematopoietic cytokines (SCF, Flt3L and TPO). As previously presented by our group, this differentiation system recapitulates the successive waves of hematopoiesis during development and leads to robust production of immunophenotypic HSC-like cells (CD34+CD38-CD90+CD45RA-CD49f+). However, these cells do not result in efficient, long-term engraftment in immunodeficient (NSG) mouse models. Characterization of the supportive monolayer from which HSPCs arise during vitro differentiation revealed limited percentages of arterial HE (CD43-CD45-CD34hiCD144+CD73-Dll4+) and arterial endothelium (CD43-CD45-CD34hiCD144+CD73midCD184+), and overabundance of stromal cells (CD43-CD45-CD34-CD144-) which upregulate MSC markers CD105, CD73, and CD90 at later stages in culture. This provides a possible explanation for the lack of engraftment potential of iPSC-derived HSPCs in this system (Panel A). To restrict stromal development and further promote differentiation and maintenance of a supportive arterial endothelial niche, we modified the standard differentiation protocol by addition of CHIR99021 (CHIR) and SB431542 (SB) during the mesodermal stage of differentiation (days 2-3) to activate Wnt/β-catenin and block of Activin/Nodal signaling, respectively. Given that VEGF acts upstream of the Notch pathway during arterial endothelial differentiation, we also increased the concentration of VEGFA 20-fold throughout differentiation (200ng/mL). Our results showed that mesodermal patterning alone (CHIR/SB) activated critical HoxA cluster genes in both early endothelial and late HSPC populations but was insufficient to repress stromal production and maintain an endothelial niche beyond early culture days. However, increased VEGF concentrations, alone or in combination with CHIR/SB, markedly reduced stromal differentiation (Panel B) and enhanced arterial endothelium formation (Panel C) compared to the standard system (control). Importantly, combination treatments also led to significantly higher percentages of arterial HE at days 5 and 7 (panel D). Current assessment of these treatments on the hematopoietic potential of the system is ongoing, and include NSG mouse transplantations. Overall, our data indicate that commercially available technologies can be further modified and improved to move closer to chemically-defined and scalable HSPC differentiation protocols. Figure. Figure. Disclosures Larochelle: Stem Cell Technologies: Patents & Royalties: StemDiff Hematopoietic Differentiation Kit.

Estrogens Act As a Dorsal-Ventral Limiting Factor To Pattern The Hematopoietic Stem Cell Niche

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 465-465
Author(s):  
Kelli J. Carroll ◽  
Michael C Dovey ◽  
Maija K Garnaas ◽  
Claire C Cutting ◽  
Gregory M Frechette ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Conflicts Of Interest ◽  
Notch Pathway ◽  
Estrogen Signaling ◽  
Hemogenic Endothelium ◽  
Hematopoietic Stem ◽  
Endogenous Estrogen ◽  
The Impact ◽  
Hematopoietic Niche

While the genetic control of hematopoietic stem cell (HSC) function is increasingly understood, less is known about factors that pattern the embryonic hematopoietic niche and specify the location of HSC emergence. 17beta-estradiol (E2) and related estrogenic compounds were identified in a zebrafish chemical genetic screen as modifiers of the number of runx1+ HSCs in the Aorta-Gonad-Mesonephros region. Exposure to exogenous E2 during hematopoietic specification significantly decreased production of runx1+ HSCs by in situ hybridization (ISH) and qPCR (p<0.01); this finding was confirmed by CD41 FACS (p<0.001). Morpholino knockdown of the estrogen receptors indicated E2-mediated loss of HSCs was due to Esr2a signaling. Zebrafish that express GFP under the control of the estrogen response element (ERE:GFP) showed endogenous estrogen activity and a 1.5 fold enhancement of GFP expression by FACS upon E2 treatment (p<0.01) confirming that zebrafish embryos contain endogenous estrogen and the transcriptional machinery to respond to exogenous E2; estradiol immunoassay revealed that endogenous estrogen is preferentially located in the yolk of developing embryos. E2 treatment from 12-24 hours post fertilization (hpf), the time in which hemogenic endothelium is specified, disrupted vessel formation as shown by flk1 (kdrl) and altered the assignment of hemogenic arterial identity as assessed by the arterial markers ephrinB2a and tbx20. As arterial specification is mediated by a cascade of sonic hedgehog, VEGF, and Notch, we examined if these pathways were altered. Consistent with a defect in the specification of hemogenic endothelium, E2 decreased expression of VEGFAa and arterial Notch pathway components deltaC and notch3 by ISH and qPCR (p<0.05). Induction of VEGFAa or Notch activity via heat-shock inducible lines rescued E2-mediated hematopoietic defects, suggesting that alterations in these networks underlie observed hematopoietic phenotypes. Further, repression of endogenous estrogen by the aromatase inhibitor anastrozole or the estrogen receptor antagonist fulvestrant increased levels of runx1, the arterial markers ephrinB2a and tbx20, and VEGFAa expression. Expression of venous flt4 was correspondingly decreased, demonstrating that endogenous estrogen signaling regulates arterial/venous identity. These results suggest that maternally deposited estrogens act as a novel morphogen organizer in patterning the HSC niche during early development. It further indicates that endogenous estrogen signaling acts to limit the ventral boundary of hemogenic endothelium by antagonizing somitic VEGFAa, thereby implicating estrogen as a critical regulator of the spatially restricted induction of HSCs. To determine if E2 could impact HSCs independently of its role in specification of hemogenic endothelium, we exposed embryos to E2 from 27-34 hpf, after arterial establishment and initiation of blood flow. Here, E2 enhanced HSCs by ISH and qPCR (runx1; p<0.05). qPCR levels of the cell cycle markers cyclinD1 (p<0.01) and c-myc (p<0.001) and AGM phospho-histone H3 immunoreactivity (p<0.01) increased, suggesting E2 enhances cell cycling. To determine if the ability of E2 to enhance proliferation was restricted to HSCs or was apparent on any blood progenitor population, we assessed the impact of E2 on primitive hematopoiesis. E2 treatment from 12-36 hpf enhanced expression of gata1 by ISH, FACS, and qPCR (p<0.05) while repressing the expression of globin (p<0.05), suggesting that while E2 enhances the self-renewal of progenitors, it impairs their differentiation into mature lineages. To analyze conservation of effect, we examined hematopoietic development in murine embryos with 5-alpha reductase deficiency. These embryos fail to metabolize testosterone into dihydroxytestosterone and as a result have more testosterone available for conversion by aromatase into estradiol, leading to increased estrogen levels. Homozygote pups show fewer phenotypic HSCs by FACS; heterozygotes display increased erythrocyte progenitors but no increase in mature erythrocytes, suggesting that the impact of estrogen on HSC induction and erythropoiesis is conserved in a mammalian system and is sensitive to dose. Together, these data indicate a novel role for estrogen in the regionalization of the hematopoietic niche and identify estrogen as an enhancer of HSC proliferation and maturation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Making HSCs in vitro: don’t forget the hemogenic endothelium

Blood ◽  
2018 ◽  
Vol 132 (13) ◽  
pp. 1372-1378 ◽  
Author(s):  
Bradley W. Blaser ◽  
Leonard I. Zon
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Autologous Transplantation ◽  
Cell Types ◽  
Therapeutic Modality ◽  
Hemogenic Endothelium ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell Ontogeny

Generating a hematopoietic stem cell (HSC) in vitro from nonhematopoietic tissue has been a goal of experimental hematologists for decades. Until recently, no in vitro–derived cell has closely demonstrated the full lineage potential and self-renewal capacity of a true HSC. Studies revealing stem cell ontogeny from embryonic mesoderm to hemogenic endothelium to HSC provided the key to inducing HSC-like cells in vitro from a variety of cell types. Here we review the path to this discovery and discuss the future of autologous transplantation with in vitro–derived HSCs as a therapeutic modality.

Role of Notch Signaling in Hematopoietic Stem Cell Function

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. SCI-15-SCI-15
Author(s):  
Lluis Espinosa ◽  
Anna Bigas
Keyword(s):  
Stem Cell ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Notch Pathway ◽  
Cell Types ◽  
Loss Of Function ◽  
Hematopoietic Stem ◽  
On Chip ◽  
Stem Cell Populations

Abstract Abstract SCI-15 The Notch pathway controls the generation of different cell types in most tissues including blood, and dysregulation of this pathway is strongly associated with oncogenic processes. In many systems, Notch is also required for the maintenance of the stem cell populations. However, in the adult hematopoietic system this link between Notch and stemness has not been established. Instead, work of several groups, including ours, has clearly demonstrated that Notch has a prominent role in the generation of hematopoietic stem cells (HSC) during embryonic development. Although the first wave of blood cells appears in the mouse embryo around day 7.5 of development and is independent of Notch function, embryonic HSC are formed around day 10 of development from endothelial-like progenitors that reside in the embryonic aorta surrounded by the gonad and mesonephros, also called AGM region. By analyzing different Notch pathway mutant mouse embryos, we have demonstrated the involvement of the Jagged1-Notch1-GATA2 axis in this event. However, the formal demonstration that Notch regulates the GATA2 gene during HSC generation is still lacking. We have now found that GATA2 is a direct Notch target in vivo during embryonic HSC generation. However, whereas Notch positively activates GATA2 transcription in the HSC precursors, it simultaneously activates hes1 transcription, which acts a repressor of the same GATA2 gene. This finding directly implicates hes1 in the regulation of HSC development although further studies using loss-of-function mutant embryos are still needed. Altogether, our results indicate that both Notch and hes1 are required to finely regulate the levels, distribution, and likely the timing of GATA2 expression through an incoherent feed-forward loop. In parallel, we have identified other downstream targets of Notch in the AGM region by ChIP-on-chip and expression microarray analysis that we are currently characterizing. Disclosures: No relevant conflicts of interest to declare.

Genetic Basis For Hematopoietic Stem Cell Generation In The Mammalian Embryo

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3667-3667
Author(s):  
Xin Gao ◽  
Kirby D Johnson ◽  
Yuan-I Chang ◽  
Meghan E Boyer ◽  
Colin N Dewey ◽  
...  
Keyword(s):  
Stem Cell ◽  
Knockout Mice ◽  
Molecular Mechanisms ◽  
Single Cells ◽  
Genetic Network ◽  
Dorsal Aorta ◽  
Hemogenic Endothelium ◽  
Mammalian Embryo ◽  
Hematopoietic Stem ◽  
Hematopoietic System

Abstract The generation of hematopoietic stem cells (HSCs) via endothelial-to-hematopoietic transition within the aorta-gonad-mesonephros (AGM) region of the mammalian embryo is crucial for development of the adult hematopoietic system. Many questions remain unanswered regarding the molecular program in hemogenic endothelium that promotes the budding of hematopoietic cell clusters containing HSCs. Previously, we described a deletion of a Gata2 cis-element (+9.5) that depletes fetal liver HSCs, is lethal at E13-14 of embryogenesis, and is mutated in an immunodeficiency that progresses to myelodysplasia (MDS)/leukemia. In contrast to Gata2 knockout mice, which die around E10.5 because of anemia, the prolonged embryonic development of +9.5 site knockout mice provides a unique model system to investigate the potential roles for GATA-2 in HSC production, migration and function, and more specifically, the requirement for the +9.5 element to regulate Gata2 expression during these processes. Using an ex vivo system involving culturing intact AGM, or AGM dissociated into single cells and then reaggregated into an organoid, we demonstrated that the +9.5 deletion reduced Gata2 expression in uncultured AGM (1.4 fold, p<0.05), cultured intact AGM (4 fold, p<0.001) and cultured AGM reaggregates (3.4 fold, p<0.001). The importance of the +9.5 element for Gata2 expression in the AGM suggested that it might control the function of hemogenic endothelium and/or the HSC progeny. The homozygous +9.5 mutation resulted in a complete loss of progenitors and long-term repopulating HSCs in the AGM, as determined by quantitative colony assays and competitive transplantation assays, respectively. To determine whether the ablation of HSC repopulating activity in the +9.5-/- mutant AGM reflects a +9.5 element requirement for HSC genesis from hemogenic endothelium, we used a whole-mount three-dimensional embryo immunostaining assay to visualize HSC genesis in +9.5+/+ and +9.5-/- AGMs. Imaging of E10.5 embryos revealed CD31+c-Kit+ hematopoietic clusters in +9.5+/+ dorsal aorta, while clusters were absent from the +9.5-/- embryos. The absence of hematopoietic clusters in the +9.5-/- dorsal aorta, and the ablation of HSC repopulating activity, demonstrated that the +9.5 element is required for hemogenic endothelium to generate HSCs in the AGM. In principle, the +9.5-dependent genetic network should reveal clues regarding the molecular mechanisms underlying the defective HSC generation in +9.5-/- AGMs. We conducted RNA-seq to define +9.5+/+ and +9.5-/- AGM explant transcriptomes, and this genomic analysis indicated that the +9.5 element instigates a stem cell-regulatory genetic network consisting of genes encoding established regulators of hemogenic endothelium and HSCs, and genes not implicated previously in hematopoiesis. We investigated whether the +9.5 element contributes to the transcriptome of AGM endothelium. Quantitative RT-PCR analysis revealed a similar impact of +9.5 deletion on representative genes in the fraction enriched in endothelial cells (CD31+c-Kit-) from the AGM. These studies establish a new model whereby a composite cis-regulatory element induces Gata2 expression and instigates a complex genetic network in the AGM, which controls the transition of hemogenic endothelium to HSCs in the AGM. Studies are ongoing to establish the genetic network in hemogenic endothelium that mediates the development of the adult hematopoietic system and the applicability of the respective mechanisms to distinct biological and pathological contexts. Disclosures: No relevant conflicts of interest to declare.

Notch Signaling Is Necessary and Sufficient for Runx1-Dependent Stem Cell Induction in the Embryonic Aorta-Gonad-Mesonephros (AGM) Region.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4161-4161
Author(s):  
Caroline Erter Burns ◽  
Leonard I. Zon
Keyword(s):  
Stem Cell ◽  
Notch Signaling ◽  
Cell Fate ◽  
Notch Pathway ◽  
Notch Receptor ◽  
Dorsal Aorta ◽  
Loss Of Function ◽  
Cardinal Vein ◽  
Hematopoietic Stem

Abstract Vertebrate hematopoiesis can be divided into two embryonic phases: a short primitive wave predominantly generating erythrocytes and a definitive (fetal/adult) wave producing long-term hematopoietic stem cells (HSCs). The definitive wave occurs in the embryonic aorta-gonad-mesonephros (AGM) region through the asymmetric induction of HSCs from the ventral, but not dorsal, aortic endothelial wall. Since Notch signaling is critical for orchestrating a variety of developmental cell fate choices from invertebrates to humans and has been implicated in affecting the differentiation of some hematopoietic lineages, we analyzed whether the Notch pathway regulates definitive HSC induction in vivo. The zebrafish mutant mindbomb harbors a mutation in an essential E3 ligase that ubiquitylates Delta, which in turn allows the Notch intercellular domain to be released and activate downstream target gene transcription. Thus, in the absence of Mindbomb function Notch signaling does not occur. We found that although mindbomb mutants show normal primitive hematopoiesis, definitive c-myb and runx1 HSC expression is lacking. Since embryos injected with synthetic morpholinos designed to inhibit proper splicing of runx1 RNA ( runx morphants) show the same hematopoietic phenotype as mindbomb mutants, we next addressed the epistatic relationship between notch and runx1 using classic gain-of-function and loss-of-function analyses. In runx1 morphants expression of a notch receptor, notch3, and a delta ligand, deltaC, in the developing dorsal aorta was normal. Moreover, injection of runx1 RNA rescued HSCs in the AGM of mindbomb mutants. Together, these results suggest that Runx1 functions downstream of Notch in promoting HSC fate. We next analyzed whether a constitutively activated form of Notch (NICD) is sufficient for HSC specification in the AGM using an inducible binary transgenic system. Zebrafish carrying the heat-shock promoter driving the activator gal4 were mated to animals carrying 6 gal4 -responsive tandem upstream activating sequences (UAS) driving NICD. At the 10 somite-stage the embryos were heat-shocked at 37°C for 1 hour to activate NICD throughout the double transgenic animals. Surprisingly, expression of both HSC markers, c-myb and runx1, were expanded from their normal restricted domain in the ventral endothelium to the entire circumference of the dorsal aorta. Most interestingly, the presence of ectopic c-myb and runx1 transcripts were observed in the developing post-cardinal vein, a vessel that normally does not produce HSCs. These data imply that activation of the Notch pathway generates increased numbers of HSCs in vivo. When runx1 RNA is injected into wild-type embryos a similar expansion of c-myb transcripts is seen throughout the entire dorsal aorta and post-cardinal vein, further indicating that Runx1 functions downstream of Notch in HSC induction. In summary, discovery of the molecular programs essential and sufficient for fetal/adult hematopoietic ontogeny will lead to a further understanding of the physiologic and pathologic processes regulating stem cell homeostasis and translate into more effective therapies for blood disorders.

scholarly journals Hematopoietic stem cells: to be or Notch to be

Blood ◽  
2012 ◽  
Vol 119 (14) ◽  
pp. 3226-3235 ◽  
Author(s):  
Anna Bigas ◽  
Lluis Espinosa
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Development ◽  
Cell Fate ◽  
Cell Biology ◽  
Adult Life ◽  
Notch Pathway ◽  
Hematopoietic Stem ◽  
Experimental Systems ◽  
Cell Diversity

Abstract Notch is a well-conserved signaling pathway and its function in cell fate determination is crucial in embryonic development and in the maintenance of tissue homeostasis during adult life. Notch activation depends on cell-cell interactions that are essential for the generation of cell diversity from initially equivalent cell populations. In the adult hematopoiesis, Notch is undoubtedly a very efficient promoter of T-cell differentiation, and this has masked for a long time the effects of Notch on other blood lineages, which are gradually being identified. However, the adult hematopoietic stem cell (HSC) remains mostly refractory to Notch intervention in experimental systems. In contrast, Notch is essential for the generation of the HSCs, which takes place during embryonic development. This review summarizes the knowledge accumulated in recent years regarding the role of the Notch pathway in the different stages of HSC ontology from embryonic life to fetal and adult bone marrow stem cells. In addition, we briefly examine other systems where Notch regulates specific stem cell capacities, in an attempt to understand how Notch functions in stem cell biology.

Zebrafish Cbfb Is Required For The Mobilization, But Not The Emergence, Of Hematopoietic Stem Cells In Embryos

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 464-464
Author(s):  
Erica Bresciani ◽  
Blake Carrington ◽  
Stephen Wincovitch ◽  
Aniket Gore ◽  
Brant M. Weinstein ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Notch Pathway ◽  
Targeted Mutagenesis ◽  
Dorsal Aorta ◽  
Human Leukemia ◽  
Hemogenic Endothelium ◽  
Hematopoietic Stem ◽  
Definitive Hematopoiesis

CBFβ and RUNX1 form a DNA-binding heterodimer that plays a crucial role during definitive hematopoiesis at the stage of hematopoietic stem cells (HSCs). Both of them are targets of recurrent chromosomal translocations in human leukemia. In mammals and zebrafish, RUNX1 is required for the emergence of definitive HSCs from the hemogenic endothelium. Mouse knockouts for either Runx1 or Cbfb show similar phenotypes with complete lack of definitive hematopoiesis. Therefore, the impairment of all definitive hematopoietic lineages in both Runx1-/- and Cbfb-/- embryos suggested that the CBF heterodimer is required for HSC formation. However the exact role of the CBF complex in the development of HSCs remains unclear. The cellular mechanisms and the genetic pathways driving the HSC generation are highly conserved across vertebrates. Thus, we used the zebrafish model to dissect the role of cbfb and the CBF complex in the emergence and the maintenance of HSCs. We generated two independent cbfb knockouts (cbfb-/-) by zinc-finger nuclease (ZFN) - mediated targeted mutagenesis. The analysis of cbfb-/- embryos revealed a previously unknown role of cbfb during definitive hematopoiesis. Similar to the published zebrafish runx1 mutant embryos (runx1W84X/W84X), cbfb-/- embryos underwent primitive hematopoiesis and developed erythromyeloid progenitors (EMPs), but they lacked definitive hematopoiesis as the expression of markers for differentiated blood lineages such as rag1, lplastin and αe1globin was completely abrogated. Moreover, circulating thrombocytes were almost undetectable in cbfb-/-/tg(cd41:GFP) embryos. Unlike the runx1 mutants in which HSCs are not formed, however, the emergence of runx1+/c-myb+ HSCs from the hemogenic endothelium along the ventral wall of the dorsal aorta was unaffected in the cbfb-/- mutants. Rather, the subsequent translocation of the HSCs from aorta-gonad-mesonephros (AGM) to the caudal hematopoietic tissue (CHT) was blocked, as evidenced by the accumulation of runx1+ HSCs in the AGM and the concomitant absence of such cells in the CHT. Live imaging analysis of cbfb-/-/tg(c-myb:eGFP) embryos confirmed that HSCs egressed from the dorsal aorta but did not enter circulation through the axial vein. Moreover, embryos treated with a specific inhibitor of RUNX1-CBFβ interaction, Ro5-3335, phenocopied the hematopoietic defects observed in the cbfb-/- mutants, confirming that the function of RUNX1 and CBFβ during HSC development could be uncoupled. The Notch-Runx1 pathway is critical for the initial specification of HSCs during definitive hematopoiesis. Therefore, in order to gain insight into the genetic mechanisms that regulate cbfb expression we investigated the Notch pathway. We found that transient Notch activation enhanced cbfb expression and expanded it ectopically. On the other hand, in the Notch signaling mutant mind bomb, cbfb expression in hematopoietic regions was abrogated. Thus, our results suggest that cbfb is also downstream of the Notch pathway during hematopoiesis. Overall our data indicate that CBFβ and functional CBFβ-RUNX1 heterodimers are not required for the emergence of HSCs, but are essential for the mobilization of HSCs during early definitive hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Gata2 cis-element is required for hematopoietic stem cell generation in the mammalian embryo

2013 ◽  
Vol 210 (13) ◽  
pp. 2833-2842 ◽  
Author(s):  
Xin Gao ◽  
Kirby D. Johnson ◽  
Yuan-I Chang ◽  
Meghan E. Boyer ◽  
Colin N. Dewey ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Fetal Liver ◽  
Hemogenic Endothelium ◽  
Mammalian Embryo ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Cis Element ◽  
Genomic Analyses

The generation of hematopoietic stem cells (HSCs) from hemogenic endothelium within the aorta, gonad, mesonephros (AGM) region of the mammalian embryo is crucial for development of the adult hematopoietic system. We described a deletion of a Gata2 cis-element (+9.5) that depletes fetal liver HSCs, is lethal at E13–14 of embryogenesis, and is mutated in an immunodeficiency that progresses to myelodysplasia/leukemia. Here, we demonstrate that the +9.5 element enhances Gata2 expression and is required to generate long-term repopulating HSCs in the AGM. Deletion of the +9.5 element abrogated the capacity of hemogenic endothelium to generate HSC-containing clusters in the aorta. Genomic analyses indicated that the +9.5 element regulated a rich ensemble of genes that control hemogenic endothelium and HSCs, as well as genes not implicated in hematopoiesis. These results reveal a mechanism that controls stem cell emergence from hemogenic endothelium to establish the adult hematopoietic system.

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