The N-glycome regulates the endothelial-to-hematopoietic transition

High Mannose

Definitive hematopoietic stem and progenitor cells (HSPCs) arise from the transdifferentiation of hemogenic endothelial cells (hemECs). The mechanisms of this endothelial-to-hematopoietic transition (EHT) are poorly understood. We show that microRNA-223 (miR-223)–mediated regulation of N-glycan biosynthesis in endothelial cells (ECs) regulates EHT. miR-223 is enriched in hemECs and in oligopotent nascent HSPCs. miR-223 restricts the EHT of lymphoid-myeloid lineages by suppressing the mannosyltransferase alg2 and sialyltransferase st3gal2, two enzymes involved in protein N-glycosylation. ECs that lack miR-223 showed a decrease of high mannose versus sialylated sugars on N-glycoproteins such as the metalloprotease Adam10. EC-specific expression of an N-glycan Adam10 mutant or of the N-glycoenzymes phenocopied miR-223 mutant defects. Thus, the N-glycome is an intrinsic regulator of EHT, serving as a key determinant of the hematopoietic fate.

The N-Glycome regulates the endothelial-to-hematopoietic transition

10.1101/602912 ◽

2019 ◽

Author(s):

Dionna M. Kasper ◽

Jared Hintzen ◽

Yinyu Wu ◽

Joey J. Ghersi ◽

Hanna K. Mandl ◽

...

Keyword(s):

Endothelial Cells ◽

Blood System ◽

Protein Glycosylation ◽

Specific Expression ◽

Hematopoietic Stem ◽

Single Cell Rna Sequencing ◽

Complex Hybrid ◽

High Mannose

AbstractHematopoietic stem and progenitor cells (HSPCs) that establish and maintain the blood system in adult vertebrates arise from the transdifferentiation of hemogenic endothelial cells (hemECs) during embryogenesis. This endothelial-to-hematopoietic transition (EHT) is tightly regulated, but the mechanisms are poorly understood. Here, we show that microRNA (miR)-223-mediated regulation of N-glycan biosynthesis in endothelial cells (ECs) regulates EHT. Single cell RNA-sequencing revealed that miR-223 is enriched in hemECs and in oligopotent nascent HSPCs. miR-223 restricts the EHT of lymphoid/myeloid lineages by suppressing the expression of mannosyltransferase alg2 and sialyltransferase st3gal2, two enzymes involved in N-linked protein glycosylation. High-throughput glycomics of ECs lacking miR-223 showed a decrease of high mannose versus sialylated complex/hybrid sugars on N-glycoproteins involved in EHT such as the metalloprotease Adam10. Endothelial-specific expression of an N-glycan Adam10 mutant or of the N-glycoenzymes phenocopied the aberrant HSPC production of miR-223 mutants. Thus, the N-glycome plays a previously unappreciated role as an intrinsic regulator of EHT, with specific mannose and sialic acid modifications serving as key endothelial determinants of their hematopoietic fate.One Sentence SummaryThe N-glycan “sugar code” governs the hematopoietic fate of endothelial cells and regulates blood stem cell production in vivo.

Endothelial Cells Promote Expansion of Long-Term Engrafting Marrow Hematopoietic Stem and Progenitor Cells in Primates

Stem Cells Translational Medicine ◽

10.5966/sctm.2016-0240 ◽

2016 ◽

Vol 6 (3) ◽

pp. 864-876 ◽

Cited By ~ 18

Author(s):

Jennifer L. Gori ◽

Jason M. Butler ◽

Balvir Kunar ◽

Michael G. Poulos ◽

Michael Ginsberg ◽

...

Keyword(s):

Endothelial Cells ◽

Progenitor Cells ◽

Hematopoietic Stem ◽

Investigating The Role Of ARAP3 In The Regulation Of Hematopoietic Stem and Progenitor Cells

Blood ◽

10.1182/blood.v122.21.2409.2409 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2409-2409

Author(s):

Yiwen Song ◽

Sonja Vermeren ◽

Wei Tong

Keyword(s):

Endothelial Cells ◽

Plasma Membrane ◽

Progenitor Cells ◽

Hematopoietic Cells ◽

Conditional Knockout ◽

Ph Domain ◽

Hematopoietic Stem ◽

Normal Peripheral Blood ◽

Abstract ARAP3 is a member of the dual Arf-and-Rho GTPase-activating proteins (GAP) family, functioning specifically to inactivate its substrates Arf6 and RhoA GTPases. ARAP3 is translocated to the plasma membrane after PIP3 binding to the first two of its five PH domains, facilitating its GAP activity in a PI3K-mediated manner. Rho family GTPases are found to play critical roles in many aspects of hematopoietic stem and progenitor cells (HSPCs), such as engraftment and migration, while a role for Arf family GTPases in hematopoiesis is less defined. Previous studies found that either exogenous ARAP3 expression in epithelial cells or RNAi-mediated ARAP3 depletion in endothelial cells disrupts F-actin or lamellipodia formation, respectively, resulting in a cell rounding phenotype and failure to spread. This implies that ARAP3 control of Arf6 and RhoA is tightly regulated, and maintaining precise regulation of ARAP3 levels is crucial to actin organization in the cell. Although ARAP3 was first identified in porcine leukocytes, its function in the hematopoietic system is incompletely understood. Germline deletion of Arap3 results in embryonic lethality due to angiogenic defects. Since endothelial cells are important for the emergence of HSCs during embryonic development, early lethality precludes further studying the role of ARAP3 in definitive hematopoiesis. Therefore, we generated several transgenic mouse models to manipulate ARAP3 in the hematopoietic compartment: (1) Arap3fl/fl;Vav-Cretg conditional knockout mice (CKO) deletes ARAP3 specifically in hematopoietic cells, (2) Arap3fl/fl;VE-Cadherin -Cretg CKO mice selectively deletes ARAP3 in embryonic endothelial cells and thereby hematopoietic cells, and (3) Arap3R302,3A/R302,3A germline knock-in mice (KI/KI) mutates the first PH domain to ablate PI3K-mediated ARAP3 activity in all tissues. We found an almost 100% and 90% excision efficiency in the Vav-Cretg- and VEC-Cretg- mediated deletion of ARAP3 in the bone marrow (BM), respectively. However, the CKO mice appear normal in steady-state hematopoiesis, showing normal peripheral blood (PB) counts and normal distributions of all lineages in the BM. Interestingly, we observed an expansion of the Lin-Scal+cKit+ (LSK) stem and progenitor compartment in the CKO mice. This is due to an increase in the multi-potent progenitor (MPP) fraction, but not the long-term or short-term HSC (LT- or ST-HSC) fractions. Although loss of ARAP3 does not alter the frequency of phenotypically-characterized HSCs, we performed competitive BM transplantation (BMT) studies to investigate the functional impact of ARAP3 deficiency. 500 LSK cells from Arap3 CKO (Arap3fl/fl;Vav-Cretg and Arap3fl/fl;VEC-Cretg) or Arap3fl/fl control littermate donors were transplanted with competitor BM cells into irradiated recipients. We observed similar donor-derived reconstitution and lineage repopulation in the mice transplanted with Arap3fl/fl and Arap3 CKO HSCs. Moreover, Arap3 CKO HSCs show normal reconstitution in secondary transplants. Arap3 KI/KI mice are also grossly normal and exhibit an expanded MPP compartment. Importantly, Arap3KI/KI LSKs show impaired reconstitution compared to controls in the competitive BMT assays. Upon secondary and tertiary transplantation, reconstitution in both PB and BM diminished in the Arap3KI/KI groups, in contrast to sustained reconstitution in the control group. Additionally, we observed a marked skewing towards the myeloid lineage in Arap3KI/KI transplanted secondary and tertiary recipients. These data suggest a defect in HSC function in Arap3KI/KI mice. Myeloid-skewed reconstitution also points to the possibility of selection for “myeloid-primed” HSCs and against “balanced” HSCs, as HSCs exhaust during aging or upon serial transplantation. Taken together, our data suggest that ARAP3 plays a non-cell-autonomous role in HSCs by regulating HSC niche cells. Alternatively, the ARAP3 PH domain mutant that is incapable of locating to the plasma membrane in response to PI3K may exert a novel dominant negative function in HSCs. We are investigating mechanistically how ARAP3 controls HSC engraftment and self-renewal to elucidate the potential cell-autonomous and non-cell-autonomous roles of ARAP3 in HSCs. In summary, our studies identify a previously unappreciated role of ARAP3 as a regulator of hematopoiesis and hematopoietic stem and progenitor cell function. Disclosures: No relevant conflicts of interest to declare.

Blood Flow Is Required for the Release of Hematopoietic Stem- and Progenitor Cells From Hemogenic Endothelium in the Placenta.

Blood ◽

10.1182/blood.v114.22.698.698 ◽

2009 ◽

Vol 114 (22) ◽

pp. 698-698

Author(s):

Katrin E Rhodes ◽

Ben Van Handel ◽

Michele Wang ◽

Yanling Wang ◽

Akanksha Chhabra ◽

...

Keyword(s):

Endothelial Cells ◽

Blood Flow ◽

Progenitor Cells ◽

Conflicts Of Interest ◽

Yolk Sac ◽

Positive Staining ◽

Hemogenic Endothelium ◽

Hematopoietic Stem ◽

Notch1 Signaling ◽

Abstract 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 CD41medckit+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.

Latent Cardiogenic Potential in Endocardium and Hemogenic Endothelium Revealed in the Absence of Scl/tal1

Blood ◽

10.1182/blood.v118.21.2362.2362 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2362-2362

Author(s):

Amelie Montel-Hagen ◽

Ben Van Handel ◽

Roberto Ferrari ◽

Rajkumar Sasidharan ◽

Tonis Org ◽

...

Keyword(s):

Endothelial Cells ◽

Progenitor Cells ◽

Conflicts Of Interest ◽

Lineage Tracing ◽

Bhlh Transcription Factor ◽

Wild Type ◽

Transcriptional Program ◽

Hemogenic Endothelium ◽

Hematopoietic Stem ◽

Abstract 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.

Liver Sinusoidal Endothelial Cells Promote the Expansion of Human Cord Blood Hematopoietic Stem and Progenitor Cells

International Journal of Molecular Sciences ◽

10.3390/ijms20081985 ◽

2019 ◽

Vol 20 (8) ◽

pp. 1985 ◽

Cited By ~ 3

Author(s):

Huilin Li ◽

Haiyun Pei ◽

Xiaoyan Xie ◽

Sihan Wang ◽

Yali Jia ◽

...

Keyword(s):

Endothelial Cells ◽

Cord Blood ◽

Progenitor Cells ◽

Ex Vivo ◽

Liver Sinusoidal Endothelial Cells ◽

Sinusoidal Endothelial Cells ◽

Differentiation Potential ◽

Hematopoietic Stem ◽

Cord blood (CB) is an attractive source of hematopoietic stem cells (HSCs) for hematopoietic cell transplantation. However, its application remains limited due to the low number of HSCs/progenitors in a single CB unit and its notoriously difficulty in expanding ex vivo. Here, we demonstrated that the human fetal liver sinusoidal endothelial cells engineered to constitutively express the adenoviral E4orf1 gene (hFLSECs-E4orf1) is capable of efficient expansion ex vivo for human CB hematopoietic stem and progenitor cells (HSPCs). Coculture of CD34+ hCB cells with hFLSECs-E4orf1 resulted in generation of substantially more total nucleated cells, CD34+CD38− and CD34+ CD38−CD90+ HSPCs in comparison with that of cytokines alone after 14 days. The multilineage differentiation potential of the expanded hematopoietic cells in coculture condition, as assessed by in vitro colony formation, was also significantly heightened. The CD34+ hCB cells amplified on hFLSECs-E4orf1 were capable of engraftment in vivo. Furthermore, hFLSECs-E4orf1 highly expressed hematopoiesis related growth factor and Notch receptors. Accordingly, the CD34+ hCB cells amplified on hFLSECs-E4orf1 exhibited Notch signaling activation. Taken together, our findings indicated that FLSECs may potentially be the crucial component of the microenvironment to support recapitulation of embryonic HSC amplification in vitro and allow identification of new growth factors responsible for collective regulation of hematopoiesis.

Endodermal Cells Differentiate Into Endothelial Cells to Function as a Vascular Niche for Hematopoietic Stem and Progenitor Cells

SSRN Electronic Journal ◽

10.2139/ssrn.3970227 ◽

2021 ◽

Author(s):

Hiroyuki Ishikawa ◽

Hiroyuki Nakajima ◽

Takuya yamamoto ◽

Ayano Chiba ◽

Hajime Fukui ◽

...

Keyword(s):

Endothelial Cells ◽

Progenitor Cells ◽

Hematopoietic Stem ◽

Vascular Niche ◽

Endodermal Cells

Identification of Pleiotrophin as a Novel Growth Factor for Hematopoietic Stem and Progenitor Cells.

Blood ◽

10.1182/blood.v110.11.1404.1404 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1404-1404

Author(s):

Heather Himburg ◽

Garrett Muramoto ◽

Sarah Meadows ◽

Alice Salter ◽

Ningyi Zi ◽

...

Keyword(s):

Endothelial Cells ◽

Growth Factor ◽

Progenitor Cells ◽

Dependent Manner ◽

Cell Content ◽

Hematopoietic Stem ◽

Vascular Niche ◽

Human Hscs ◽

Dose Dependent

Abstract Hematopoietic stem and progenitor cells have been demonstrated to reside in association with bone marrow sinusoidal endothelial cells in vivo but the mechanisms through which the BM vascular niche regulates hematopoiesis remains incompletely defined. In an attempt to identify candidate soluble growth factors for hematopoietic stem cells (HSCs) which are produced by endothelial cells (Ecs), we have performed comparative genome-wide expression analysis of a primary human brain-derived Ecs which we have previously demonstrated to support a 10-fold expansion of human SCID-repopulating cells (SRCs) in non-contact cultures. This analysis revealed that pleiotrophin (PTN), a heparin binding growth factor, was 32-fold overexpressed in HUBECs that supported human HSC expansion as compared to non-supportive EC lines. Pleiotrophin has anti-apoptotic, mitogenic and transforming activities, suggesting a role in tumorigenesis. No role for pleiotrophin in hematopoiesis has been described. In order to determine if PTN contributes to the demonstrated HSC-supportive activity of HUBECs, the activity of PTN was blocked by adminstration of a neutralizing anti-PTN mAb to the coculture model. Three doses of PTN neutralizing antibody (5, 25, and 50 ug/ml) were added to non-contact cocultures of HUBECs with human cord blood CD34+CD38-lin- HSCs. The anti-PTN treatment caused a decrease in the numbers of phenotypic HSCs in culture in a dose dependent manner. Relative to control HUBEC cultures, the % and expansion of CD34(+) progenitors decreased signficantly at the two higher doses of anti-PTN (p<0.05, respectively). Additionally, CD34(+)CD38(−) lin(−) progenitors were decreased by 33% at the highest dose of anti-PTN compared to control HUBEC cultures (p<0.05). Transplantation studies of anti-PTN treated versus control HUBEC-cultured progeny into NOD/SCID mice are ongoing to confirm the central role of PTN in the expansion of human HSCs in culture. In a correlative study, the addition of recombinant PTN to cultures of human HSCs with thrombopoietin, SCF and flt-3 ligand was shown to significantly increase CD34+ cell content and CD34+CD38- cell content compared to cytokines alone, again in a dose-dependent manner. Taken together these results suggest that PTN, a growth factor with no previously ascribed hematopoietic activity, is a soluble growth factor for human hematopoietic progenitor cells. Since blockade of PTN activity significantly inhibits the soluble activity of HUBECs to support human HSC proliferation and expansion, PTN is also a candidate growth factor for further study in the context of understanding the function of the BM vascular niche.