Genetic Analyses in Zebrafish Reveal That Angiopoietin-Like Proteins 1 and 2 Are Required for HSC Development during Embryogenesis

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 729-729 ◽  
Author(s):  
Michelle I Lin ◽  
Leonard I. Zon
Keyword(s):  
Mrna Expression ◽  
Ex Vivo ◽  
Ectopic Expression ◽  
Hemogenic Endothelium ◽  
Cell Stage ◽  
Hematopoietic Stem ◽  
Vegf Signaling ◽  
Close Proximity ◽  
Lateral Mesoderm ◽  
Stem Cell Growth Factor

Abstract Recent studies have shown that members of the angiopoietin-like protein (angptl) family can help stimulate ex vivo expansion of mouse and human hematopoietic stem cells (HSCs). The requirement of angptl for HSC development has yet to be determined. Here, the effects of morpholino (MO) knockdown of angptl1, angptl2 or the combination of both, were assessed during zebrafish development. Single MO knockdown of either angptl1 or angptl2 resulted in a decrease in c-myb positive HSCs in the aorta-gonad-mesonephros (AGM) region, the site of definitive hematopoiesis at 36 hours post-fertilization (hpf). Embryos injected with combined angptl1 and angptl2 MOs (double morphants) had an even more severe phenotype, in which both c-myb and runx1 positive HSCs were almost completely absent. In an effort to discern the onset of the phenotype, early HSC markers were evaluated. The double morphants had decreased expression of scl and lmo2 at 8–10 somite stages (ss) in the bilateral stripes of the lateral mesoderm region from which hematopoietic and vascular progenitors are thought to arise. To determine if angptl are sufficient to expand HSCs, angptl1 and angptl2 mRNA were injected into one-cell stage embryos and HSC markers were evaluated. Angptl1 and angptl2 mRNA significantly increased c-myb and runx1 positive HSCs in the AGM. Interestingly, addition of thrombopoietin, a potent stem cell growth factor, with angptl1 and angptl2 further increased HSCs in the AGM, implying a synergistic effect of these growth factors during HSC development. Angptl regulation of HSC development may occur by stimulating the production of the hemogenic endothelium. MO knockdown of either angptl1 or angptl2 alone did not alter vascular development significantly, while MO knockdown of both angptl1 and angptl2 resulted in a disruption of intersegmental blood vessel sprouting at 28 hpf. The arterial and venous specification in these double morphants was also disrupted at 28 hpf, with decreased expression of the arterial marker ephrinB2 but increased ectopic expression of the venous marker, flt4. These results strongly suggest an early downstream effect of angptl signaling on hemogenic endothelium. Furthermore, since Vegf signaling is indispensable for proper endothelial specification and definitive HSC formation, we found that in the double morphants, somite expression of vegf165 mRNA was decreased early in development. Although angptl1 mRNA expression was undetectable at this stage, angptl2 mRNA expression was localized in the yolk sac extension and posterior spinal cord, appearing in close proximity to the vegf positive somites. This suggests that in these locations, angptl may stimulate Vegf production in the embryo to participate in the induction of hemogenic endothelium and subsequently lead to HSC formation. Taken together, our data demonstrates that the angptl are required for HSC development by coordinating the production of a functional hemogenic endothelium that can lead to the formation of HSCs.

Genetic Interaction Between Angiopoietin-Like Proteins 1 and 2 and Notch Signaling During HSC Development

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 573-573
Author(s):  
Michelle I Lin ◽  
Charles W Carspecken ◽  
Audrey Uong ◽  
Emily Price ◽  
Leonard I. Zon
Keyword(s):  
Notch Signaling ◽  
Board Of Directors ◽  
Genetic Interaction ◽  
Ectopic Expression ◽  
Receptor Interaction ◽  
Hemogenic Endothelium ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Equity Ownership ◽  
Definitive Hematopoiesis

Abstract Abstract 573 Members of the angiopoietin-like protein (angptl) family have recently been shown to stimulate ex vivo expansion of mouse and human hematopoietic stem cells (HSCs). The requirement of angptls for HSCs during development and their mechanism of action have yet to be determined. To first recapitulate the effects of exogenous angptl on HSC expansion, we made a stable transgenic zebrafish line, Tg(hsp70:angptl2), that overexpresses angptl2 upon heatshock induction. Heatshocked Tg embryos showed a significant increase in cmyb- and runx1-positive HSCs in the aorta-gonad-mesonephros (AGM) region, the site of definitive hematopoiesis at 36 hours post-fertilization (hpf), suggesting that angptl2 can sufficiently expand definitive HSCs during development. We then assessed the effects of angptl1 and/or angptl2 morpholino (MO) knockdown. Single MO knockdown resulted in a decrease in cmyb- and runx1-positive HSCs in the AGM whereas embryos injected with combined angptl1 and 2 MOs (double morphants) exhibited more severe phenotype in which HSCs were completely absent. Angptl regulation of HSC development may occur by stimulating the production or specification of a patent hemogenic endothelium, as the double morphants displayed a disruption of vascular specification at 28 hpf, with decreased expression of the arterial marker ephrinB2 but increased ectopic expression of the venous marker, flt4 in the dorsal aorta. These double morphants also exhibited disrupted intersegmental blood vessel sprouting. Because proper patterning of the developing blood vessels is a prerequisite for subsequent HSC formation, these results strongly suggest an early downstream effect of angptl signaling on hemogenic endothelium specification. To dissect the mechanism of angptl signaling, we asked whether there is a genetic interaction between notch and angptl signaling since the observed phenotype in the angptl double morphants resembled that in the notch mutant mindbomb, mib. We first monitored notch signaling using a transgenic notch reporter zebrafish line that expresses EGFP when the active component of notch signaling, notch intracellular domain (NICD), is expressed. MO knockdown of angptls 1 and 2 resulted in an absence of notch signaling, particularly in the vasculature. Interestingly, overexpressing angptl2 by crossing Tg(hsp70:angptl2) into the mib mutant showed a significant rescue of the HSC phenotype, implying that angptl2 acts downstream of mib signaling. When MOs against angptls 1 and 2 were then injected into the NICD overexpressing Tg embryos, we found that NICD restored HSC formation in the angptl double morphants. Together, these data corroborate the hypothesis that angptls can regulate notch signaling by acting downstream of notch ligand-receptor interaction and upstream of NICD during definitive hematopoiesis. To further probe at the downstream signaling of angptls since their receptor is currently unknown, we found that both angptls can stimulate activation of akt in cultured endothelial cells. To investigate whether akt is involved in angptl signaling during development, we injected the constitutively active myr-akt into angptl double morphants and observed rescue of both HSC and vascular defects. This strongly indicates that akt is the key signaling component downstream of angptl signaling during these biological processes. Injection of myr-akt can also rescue HSC defects in mib, suggesting that notch signaling also requires akt activation for definitive HSC formation. Taken together, our data demonstrate that the angptls are required for HSC development through notch and akt signaling, by coordinating the production of a functional hemogenic endothelium. Disclosures: Zon: FATE, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Stemgent: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

In vivo proliferation advantage of genetically corrected hematopoietic stem cells in a mouse model of Fanconi anemia FA-D1

Blood ◽  
2008 ◽  
Vol 112 (13) ◽  
pp. 4853-4861 ◽  
Author(s):  
Paula Río ◽  
Néstor W. Meza ◽  
África González-Murillo ◽  
Susana Navarro ◽  
Lara Álvarez ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mouse Model ◽  
Hematopoietic Stem Cells ◽  
Fanconi Anemia ◽  
Ex Vivo ◽  
Bone Marrow Failure ◽  
Ectopic Expression ◽  
Multiple Time ◽  
Hematopoietic Stem

AbstractFanconi anemia (FA) is an inherited recessive DNA repair disorder mainly characterized by bone marrow failure and cancer predisposition. Studies in mosaic FA patients have shown that reversion of one inherited germ-line mutation resulting in a functional allele in one or a few hematopoietic stem cells (HSCs) can lead to the proliferation advantage of corrected cells, thus over time normalizing the hematologic status of the patient. In contrast to these observations, it is still unclear whether ex vivo genetic correction of FA HSCs also provides a similar proliferation advantage to FA HSCs. Using an FA mouse model with a marked hematopoietic phenotype, the FA-D1 (Brca2Δ27/Δ27) mice, we demonstrate that the lentivirus-mediated gene therapy of FA HSCs results in the progressive expansion of genetically corrected clones in mild-conditioned FA-D1 recipients. Consistent with these data, hematopoietic progenitors from FA recipients progressively became mitomycin C resistant and their chromosomal instability was reverted. No evidence of myelodysplasia, leukemias, or abnormal clonal repopulation was observed at multiple time points in primary or secondary recipients. Our results demonstrate that ectopic expression of BRCA2 confers a beneficial in vivo proliferation advantage to FA-D1 HSCs that enables the full hematopoietic repopulation of FA recipients with genetically corrected cells.

EMP Emergence from Hemogenic Endothelium in the Mammalian Yolk Sac Is Independent of Flow and Arterial Identity, but Is Regulated By Canonical Wnt Signaling

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 768-768
Author(s):  
Jenna M. Frame ◽  
Kathleen E McGrath ◽  
Katherine H. Fegan ◽  
James Palis
Keyword(s):  
Endothelial Cells ◽  
Blood Flow ◽  
Wnt Signaling ◽  
Ex Vivo ◽  
Yolk Sac ◽  
Beta Catenin ◽  
Hemogenic Endothelium ◽  
Canonical Wnt Signaling ◽  
Hematopoietic Stem ◽  
Canonical Wnt

Abstract Hematopoietic stem cells (HSCs) emerge from arterial vessels of the mouse embryo through a Runx1-dependent process of endothelial-to-hematopoietic transition beginning at embryonic day 10.5 (E10.5). This arterial endothelial-to-hematopoietic transition is known to require embryonic circulation as well as beta-catenin signaling within the endothelial precursor, known as hemogenic endothelium. However, embryonic survival is dependent on the earlier emergence of a robust wave of yolk sac-derived definitive erythro-myeloid progenitors (EMPs), which have unilineage as well as multilineage potential, including high-proliferative potential colony forming cell (HPP-CFC) potential (Palis et al., PNAS, 2001). Like HSCs, EMP specification is dependent on Runx1, suggesting that they also emerge from a hemogenic endothelial precursor. However, the spatial localization of EMPs in the yolk sac and the mechanisms governing their emergence are not well understood. To visualize emerging EMPs in the yolk sac, we performed whole-mount immunohistochemistry for Kit, which we have demonstrated to contain nearly all EMP potential at E9.5. Kit+ cells coexpress Runx1 and CD31, and a subset have a polygonal/endothelial morphology, appear integrated into the vascular network, and are associated with rounded Kit+ cells in clusters, features consistent with an endothelial-to-hematopoietic transition. However, unlike HSCs, which emerge from major embryonic arteries, clusters of EMPs are located in larger and smaller caliber vessels in branches of both the arterial and venous vasculature, which is spatially organized within the yolk sac. To determine if EMP emergence from the vasculature is dependent on embryonic blood flow, which is required for HSC emergence, we analyzed the yolk sacs of Ncx1-null embryos, which fail to initiate heart contractions and subsequently lack embryonic circulation. Despite the lack of vascular remodeling in these circulation-deficient yolk sacs, Ncx1-null EMPs displayed normal cluster morphology, including both polygonal and rounded kit+ cells, indicating the endothelial-to-hematopoietic transition can occur without the mechanical influence of blood flow. To address whether EMP formation is responsive to other developmental signals, we utilized a yolk sac explant culture to evaluate the propensity of hemogenic endothelial cells to commit to hematopoiesis ex vivo. Culture of intact E8.5 yolk sacs for 48 hours with the canonical Wnt ligand Wnt3a resulted in an increase in both day 6-7 colony forming cells and day 13-14 HPP-CFC when compared with control yolk sacs. Preliminary treatment with Dkk1 alone did not adversely affect colony-forming activity when compared with untreated yolk sacs, and potentiation of endogenous canonical Wnt signaling with HLY78 did not augment colony production, suggesting that low levels of endogenous Wnt ligands are produced ex vivo. Despite the positive effect of Wnt3a on whole yolk sacs, treatment of isolated E9.5 Kit+CD41+CD16/32+ EMPs with Wnt3a did not increase colony formation, suggesting that Wnt signaling augments progenitor production at, or prior to, the hemogenic endothelial stage. Preliminary results utilizing imaging flow cytometry demonstrated increased beta-catenin intensity within the nuclear region in E9.5 Kit+VE-Cadherin/AA4.1+ endothelium following Wnt3a treatment, suggesting that hemogenic endothelial cells in the yolk sac are Wnt responsive. Consistent with this finding, in vitro Wnt3a treatment on primary E8.5-9.5 VE-Cadherin/AA4.1+CD16/32- endothelial cells resulted in upregulation of the beta-catenin target gene Axin2. To address whether Wnt signaling is endogenously active in vivo, we analyzed E8.5-E9 yolk sacs of BAT-gal reporter mice (Maretto et al., PNAS, 2003), and visualized a subset of cells with endothelial morphology expressing LacZ. Taken together, these data support the concept that EMPs, like HSCs, emerge from hemogenic endothelium. Surprisingly, this earlier endothelial-to-hematopoietic transition in the yolk sac is not dependent on blood flow or an arterial identity. However, similar to HSC emergence, EMP emergence from hemogenic endothelium is positively regulated by canonical Wnt signaling. These data highlight the presence of spatially, temporally, and functionally heterogeneous populations of hemogenic endothelium in the mammalian conceptus. Disclosures No relevant conflicts of interest to declare.

Strategies to Protect Hematopoietic Stem Cells from Culture-Induced Stress Conditions

2020 ◽  
Vol 15 ◽  
Author(s):  
Fatima Aerts-Kaya
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Ex Vivo ◽  
Stress Conditions ◽  
Stress Factors ◽  
Hematopoietic Stem ◽  
Induced Stress

: In contrast to their almost unlimited potential for expansion in vivo and despite years of dedicated research and optimization of expansion protocols, the expansion of Hematopoietic Stem Cells (HSCs) in vitro remains remarkably limited. Increased understanding of the mechanisms that are involved in maintenance, expansion and differentiation of HSCs will enable the development of better protocols for expansion of HSCs. This will allow procurement of HSCs with long-term engraftment potential and a better understanding of the effects of the external influences in and on the hematopoietic niche that may affect HSC function. During collection and culture of HSCs, the cells are exposed to suboptimal conditions that may induce different levels of stress and ultimately affect their self-renewal, differentiation and long-term engraftment potential. Some of these stress factors include normoxia, oxidative stress, extra-physiologic oxygen shock/stress (EPHOSS), endoplasmic reticulum (ER) stress, replicative stress, and stress related to DNA damage. Coping with these stress factors may help reduce the negative effects of cell culture on HSC potential, provide a better understanding of the true impact of certain treatments in the absence of confounding stress factors. This may facilitate the development of better ex vivo expansion protocols of HSCs with long-term engraftment potential without induction of stem cell exhaustion by cellular senescence or loss of cell viability. This review summarizes some of available strategies that may be used to protect HSCs from culture-induced stress conditions.

A Review of Evaluating Hematopoietic Stem Cells Derived from Umbilical Cord Blood's Expansion and Homing

2020 ◽  
Vol 15 (3) ◽  
pp. 250-262
Author(s):  
Maryam Islami ◽  
Fatemeh Soleimanifar
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Umbilical Cord ◽  
Stromal Cells ◽  
Ex Vivo ◽  
Hematologic Malignancies ◽  
Future Directions ◽  
Hematopoietic Stem ◽  
Efficient Procedure ◽  
Hematopoietic Recovery

Transplantation of hematopoietic stem cells (HSCs) derived from umbilical cord blood (UCB) has been taken into account as a therapeutic approach in patients with hematologic malignancies. Unfortunately, there are limitations concerning HSC transplantation (HSCT), including (a) low contents of UCB-HSCs in a single unit of UCB and (b) defects in UCB-HSC homing to their niche. Therefore, delays are observed in hematopoietic and immunologic recovery and homing. Among numerous strategies proposed, ex vivo expansion of UCB-HSCs to enhance UCB-HSC dose without any differentiation into mature cells is known as an efficient procedure that is able to alter clinical treatments through adjusting transplantation-related results and making them available. Accordingly, culture type, cytokine combinations, O2 level, co-culture with mesenchymal stromal cells (MSCs), as well as gene manipulation of UCB-HSCs can have effects on their expansion and growth. Besides, defects in homing can be resolved by exposing UCB-HSCs to compounds aimed at improving homing. Fucosylation of HSCs before expansion, CXCR4-SDF-1 axis partnership and homing gene involvement are among strategies that all depend on efficiency, reasonable costs, and confirmation of clinical trials. In general, the present study reviewed factors improving the expansion and homing of UCB-HSCs aimed at advancing hematopoietic recovery and expansion in clinical applications and future directions.

scholarly journals Non-viral ex vivo genome-editing in mouse bona fide hematopoietic stem cells with CRISPR/Cas9

2021 ◽  
Vol 20 ◽  
pp. 451-462
Author(s):  
Suvd Byambaa ◽  
Hideki Uosaki ◽  
Tsukasa Ohmori ◽  
Hiromasa Hara ◽  
Hitoshi Endo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Genome Editing ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Bona Fide

scholarly journals Human amniotic mesenchymal stromal cells support the ex vivo expansion of cord blood hematopoietic stem cells

2021 ◽  
Author(s):  
Valentina Orticelli ◽  
Andrea Papait ◽  
Elsa Vertua ◽  
Patrizia Bonassi Signoroni ◽  
Pietro Romele ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cord Blood ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Stem

scholarly journals Cardioprotective Effects of PPARβ/δ Activation against Ischemia/Reperfusion Injury in Rat Heart Are Associated with ALDH2 Upregulation, Amelioration of Oxidative Stress and Preservation of Mitochondrial Energy Production

2021 ◽  
Vol 22 (12) ◽  
pp. 6399
Author(s):  
Ioanna Papatheodorou ◽  
Eleftheria Galatou ◽  
Georgios-Dimitrios Panagiotidis ◽  
Táňa Ravingerová ◽  
Antigone Lazou
Keyword(s):  
Oxidative Stress ◽  
Mrna Expression ◽  
Energy Production ◽  
Citrate Synthase ◽  
Ischemia Reperfusion Injury ◽  
Ex Vivo ◽  
Uncoupling Protein ◽  
Ischemia Reperfusion ◽  
Left Ventricular ◽  
Isocitrate Dehydrogenase 2

Accumulating evidence support the cardioprotective properties of the nuclear receptor peroxisome proliferator activated receptor β/δ (PPARβ/δ); however, the underlying mechanisms are not yet fully elucidated. The aim of the study was to further investigate the mechanisms underlying PPARβ/δ-mediated cardioprotection in the setting of myocardial ischemia/reperfusion (I/R). For this purpose, rats were treated with PPARβ/δ agonist GW0742 and/or antagonist GSK0660 in vivo and hearts were subjected to ex vivo global ischemia followed by reperfusion. PPARβ/δ activation improved left ventricular developed pressure recovery, reduced infarct size (IS) and incidence of reperfusion-induced ventricular arrhythmias while it also up-regulated superoxide dismutase 2, catalase and uncoupling protein 3 resulting in attenuation of oxidative stress as evidenced by the reduction in 4-hydroxy-2-nonenal protein adducts and protein carbonyl formation. PPARβ/δ activation also increased both mRNA expression and enzymatic activity of aldehyde dehydrogenase 2 (ALDH2); inhibition of ALDH2 abrogated the IS limiting effect of PPARβ/δ activation. Furthermore, upregulation of PGC-1α and isocitrate dehydrogenase 2 mRNA expression, increased citrate synthase activity as well as mitochondrial ATP content indicated improvement in mitochondrial content and energy production. These data provide new mechanistic insight into the cardioprotective properties of PPARβ/δ in I/R pointing to ALDH2 as a direct downstream target and suggesting that PPARβ/δ activation alleviates myocardial I/R injury through coordinated stimulation of the antioxidant defense of the heart and preservation of mitochondrial function.

scholarly journals ZMYND11-MBTD1 induces leukemogenesis through hijacking NuA4/TIP60 acetyltransferase complex and a PWWP-mediated chromatin association mechanism

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jie Li ◽  
Phillip M. Galbo ◽  
Weida Gong ◽  
Aaron J. Storey ◽  
Yi-Hsuan Tsai ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Fusion Gene ◽  
Ex Vivo ◽  
Chromosomal Translocation ◽  
Regulatory Elements ◽  
Hematopoietic Stem ◽  
Its Gene ◽  
Human Acute Myeloid Leukemia ◽  
Bromodomain Proteins

AbstractRecurring chromosomal translocation t(10;17)(p15;q21) present in a subset of human acute myeloid leukemia (AML) patients creates an aberrant fusion gene termed ZMYND11-MBTD1 (ZM); however, its function remains undetermined. Here, we show that ZM confers primary murine hematopoietic stem/progenitor cells indefinite self-renewal capability ex vivo and causes AML in vivo. Genomics profilings reveal that ZM directly binds to and maintains high expression of pro-leukemic genes including Hoxa, Meis1, Myb, Myc and Sox4. Mechanistically, ZM recruits the NuA4/Tip60 histone acetyltransferase complex to cis-regulatory elements, sustaining an active chromatin state enriched in histone acetylation and devoid of repressive histone marks. Systematic mutagenesis of ZM demonstrates essential requirements of Tip60 interaction and an H3K36me3-binding PWWP (Pro-Trp-Trp-Pro) domain for oncogenesis. Inhibitor of histone acetylation-‘reading’ bromodomain proteins, which act downstream of ZM, is efficacious in treating ZM-induced AML. Collectively, this study demonstrates AML-causing effects of ZM, examines its gene-regulatory roles, and reports an attractive mechanism-guided therapeutic strategy.

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