Endothelial protein C receptor (EPCR) expression marks human fetal liver hematopoietic stem cells.
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ABSTRACT:
SUBMITTER: Subramaniam A
PROVIDER: S-EPMC6355479 | biostudies-literature | 2019 Feb
REPOSITORIES: biostudies-literature
Publications
Endothelial protein C receptor (EPCR) expression marks human fetal liver hematopoietic stem cells.
Subramaniam Agatheeswaran A Talkhoncheh Mehrnaz Safaee MS Magnusson Mattias M Larsson Jonas J
Haematologica 20180719 2
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Project description:Fetal liver hematopoietic stem and progenitor cells (HSPCs) have been considered appropriate for the management of aplastic anemia owing to their proliferative potential. Bone marrow recovery was possible in some cases; the engraftment potential of these cells, however was unsatisfactory, possibly due to the availability of a smaller number of these cells from a single fetus. The present study explores how we can expand fetal liver hematopoietic stem cells under in vitro conditions. We isolated mononuclear cells from fetal liver and hematopoietic stem cells were identified and analyzed by cell surface marker CD34. CD34+ fetal liver HSPCs cells were separated by magnetic cell sorting positive selection method. HSPCs (CD34+) were cultured by using 5 cytokines, stem cell factor (SCF), granulocyte macrophages-colony stimulating factor (GM-CSF), interleukin-6 (IL-6), Fms-related tyrosine kinase 3 (FLT-3) and erythropoietin (EPO), in 4 different combinations along with supplements, in serum-free culture media for 21 days. Cell viability continued to be greater than 90% throughout 21 days of culture. The cells expanded best in a combination of media, supplements and 5 cytokines, namely SCF, FLT-3, IL6, EPO and GM-CSF to yield a large number of total (CD34+ & CD34-) cells. Even though the total number of nucleated cells increased in culture significantly, levels of CD34 antigen expression declined steadily over this period.
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Project description:Despite the well-established cell-intrinsic role of epigenetic factors in normal and malignant hematopoiesis, their cell-extrinsic role remains largely unexplored. Herein we investigated the hematopoietic impact of inactivating Ezh2, a key component of polycomb repressive complex 2 (PRC2), in the fetal liver (FL) vascular niche. Hematopoietic specific (Vav-iCre) Ezh2 inactivation enhanced FL hematopoietic stem cell (HSC) expansion with normal FL erythropoiesis. In contrast, endothelium (Tie2-Cre) targeted Ezh2 inactivation resulted in embryonic lethality with severe anemia at embryonic day 13.5 despite normal emergence of functional HSCs. Ezh2-deficient FL endothelium overexpressed Mmp9, which cell-extrinsically depleted the membrane-bound form of Kit ligand (mKitL), an essential hematopoietic cytokine, in FL. Furthermore, Mmp9 inhibition in vitro restored mKitL expression along with the erythropoiesis supporting capacity of FL endothelial cells. These data establish that Ezh2 is intrinsically dispensable for FL HSCs and provides proof of principle that modulation of epigenetic regulators in niche components can exert a marked cell-extrinsic impact.
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Project description:Evidence is mounting that the nature of the lipid bound to the endothelial cell protein C receptor (EPCR) has an impact on its biological roles, as observed in anticoagulation and more recently, in autoimmune disease. Phosphatidylethanolamine and phosphatidylcholine species dominate the EPCR lipid cargo, yet, the extent of diversity in the EPCR-associated lipid repertoire is still unknown and remains to be uncovered. We undertook mass spectrometry analyses to decipher the EPCR lipidome, and identified species not yet described as EPCR ligands, such as phosphatidylinositols and phosphatidylserines. Remarkably, we found further, more structurally divergent lipids classes, represented by ceramides and sphingomyelins, both in less abundant quantities. In support of our mass spectrometry results and previous studies, high-resolution crystal structures of EPCR in three different space groups point to a prevalent diacyl phospholipid moiety in EPCR's pocket but a mobile and ambiguous lipid polar head group. In sum, these studies indicate that EPCR can associate with varied lipid classes, which might impact its properties in anticoagulation and the onset of autoimmune disease.
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Project description:Notch signaling is essential for the emergence of definitive hematopoietic stem cells (HSCs) in the embryo and their development in the fetal liver niche. However, how Notch signaling is activated and which fetal liver cell type provides the ligand for receptor activation in HSCs is unknown. Here we provide evidence that endothelial Jagged1 (Jag1) has a critical early role in fetal liver vascular development but is not required for hematopoietic function during fetal HSC expansion. We demonstrate that Jag1 is expressed in many hematopoietic cells in the fetal liver, including HSCs, and that its expression is lost in adult bone marrow HSCs. Deletion of hematopoietic Jag1 does not affect fetal liver development; however, Jag1-deficient fetal liver HSCs exhibit a significant transplantation defect. Bulk and single-cell transcriptomic analysis of HSCs during peak expansion in the fetal liver indicates that loss of hematopoietic Jag1 leads to the downregulation of critical hematopoietic factors such as GATA2, Mllt3, and HoxA7, but does not perturb Notch receptor expression. Ex vivo activation of Notch signaling in Jag1-deficient fetal HSCs partially rescues the functional defect in a transplant setting. These findings indicate a new fetal-specific niche that is based on juxtracrine hematopoietic Notch signaling and reveal Jag1 as a fetal-specific niche factor essential for HSC function.
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Project description:The hematopoietic stem cell (HSC) is a unique cell type found in bone marrow, which has the capacity for both self-renewal and differentiation into all blood lineages. The identification of genes expressed specifically in HSCs may help identify gene products vital to the control of self-renewal and/or differentiation, as well as antigens capable of forming the basis for improved methods of stem cell isolation. In previous studies, we identified a number of genes that appeared to be differentially expressed in murine bone marrow-derived HSCs, using microarray technology. We report here that one of those genes, encoding the murine endothelial protein C receptor (EPCR), is expressed at high levels within the bone marrow in HSCs. Bone marrow cells isolated on the basis of EPCR expression alone are highly enriched for hematopoietic reconstitution activity, showing levels of engraftment in vivo comparable to that of stem cells purified using the most effective conventional methods. Moreover, evaluation of cell populations first enriched for stem cell activity by conventional methods and subsequently fractionated on the basis of EPCR expression indicates that stem cell activity is always associated with EPCR-expressing cells. Based on our findings, we believe EPCR represents the first known marker that 'explicitly' identifies hematopoietic stem cells within murine bone marrow.
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Project description:Tissue stem cells temporally change intrinsic mechanisms to meet physiological demands. However, little is known whether and how stem cells rely on distinct extrinsic maintenance mechanisms over time. Here, we found that hematopoietic stem cells (HSCs) temporally transition to depend on thrombopoietin (TPO), a key extrinsic factor, from E16.5 onward in the developing liver. Deletion of Tpo reduced mTOR activity, induced differentiation gene expression, and preferentially depleted metabolically active HSCs. Ectopic activation of the JAK2 or MAPK pathway did not rescue HSCs in Tpo-/- mice. Enforced activation of the mTOR pathway by conditionally deleting Tsc1 significantly rescued HSCs and their gene expression in Tpo-/- mice. Lin28b intrinsically promoted mTOR activation in HSCs, and its expression diminished over time. Conditional deletion of Lin28b further reduced mTOR activity and strongly exacerbated HSC depletion in Tpo-/- mice. Therefore, HSCs temporally transition from intrinsic LIN28B-dependent to extrinsic TPO-dependent maintenance in the developing liver.
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Project description:Although adult mouse hematopoietic stem cells (HSCs) have been purified to near homogeneity, it remains impossible to achieve this with fetal HSCs. Adult HSC purity recently has been enhanced using the SLAM family receptors CD150, CD244, and CD48. These markers are expressed at different stages of the hematopoiesis hierarchy, making it possible to highly purify adult HSCs as CD150(+)CD48(-)CD244(-) cells. We found that SLAM family receptors exhibited a similar expression pattern in fetal liver. Fetal liver HSCs were CD150(+)CD48(-)CD244(-), and the vast majority of colony-forming progenitors were CD48(+)CD244(-)CD150(-) or CD48(+)CD244(+)CD150(-), just as in adult bone marrow. SLAM family markers enhanced the purification of fetal liver HSCs. Whereas 1 (11%) of every 8.9 Thy(low)Sca-1(+)lineage(-)Mac-1(+) fetal liver cells gave long-term multilineage reconstitution in irradiated mice, 1 (18%) of every 5.7 CD150(+)CD48(-)CD41(-) cells and 1 (37%) of every 2.7 CD150(+)CD48(-)Sca-1(+)lineage(-)Mac-1(+) fetal liver cells gave long-term multilineage reconstitution. These data emphasize the robustness with which SLAM family markers distinguish progenitors at different stages of the hematopoiesis hierarchy and enhance the purification of definitive HSCs from diverse contexts. Nonetheless, CD150, CD244, and CD48 are not pan-stem cell markers, as they were not detectably expressed by stem cells in the fetal or adult nervous system.
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Project description:During ontogeny, fetal liver (FL) acts as a major site for hematopoietic stem cell (HSC) maturation and expansion, whereas HSCs in the adult bone marrow (ABM) are largely quiescent. HSCs in the FL possess faster repopulation capacity as compared with ABM HSCs. However, the molecular mechanism regulating the greater self-renewal potential of FL HSCs has not yet extensively been assessed. Recently, we published RNA sequencing-based gene expression analysis on FL HSCs from 14.5-day mouse embryo (E14.5) in comparison to the ABM HSCs. We reanalyzed these data to identify key transcriptional regulators that play important roles in the expansion of HSCs during development. The comparison of FL E14.5 with ABM HSCs identified more than 1,400 differentially expressed genes. More than 200 genes were shortlisted based on the gene ontology (GO) annotation term "transcription." By morpholino-based knockdown studies in zebrafish, we assessed the function of 18 of these regulators, previously not associated with HSC proliferation. Our studies identified a previously unknown role for tdg, uhrf1, uchl5, and ncoa1 in the emergence of definitive hematopoiesis in zebrafish. In conclusion, we demonstrate that identification of genes involved in transcriptional regulation differentially expressed between expanding FL HSCs and quiescent ABM HSCs, uncovers novel regulators of HSC function.
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Project description:BackgroundEndothelial protein C receptor (EPCR) was recently identified as a key receptor for Plasmodium falciparum erythrocyte membrane protein 1 mediating sequestration of P. falciparum-infected erythrocytes in patients suffering from severe malaria. Soluble EPCR (sEPCR) inhibits binding of P. falciparum to EPCR in vitro and increased levels of sEPCR have been associated with the H3 haplotype of the EPCR encoding PROCR gene. It has been hypothesized that elevated sEPCR levels, possibly linked to the PROCR H3 genetic variant, may confer protection against severe forms of malaria. This study determined the frequencies of PROCR haplotypes H1-4 and plasma levels of sEPCR in a Tanzanian study population to investigate a possible association with severe malaria.MethodsStudy participants were children under 5 years of age admitted at the Korogwe District Hospital (N = 143), and diagnosed as having severe malaria (N = 52; including cerebral malaria N = 17), uncomplicated malaria (N = 24), or an infection other than malaria (N = 67). In addition, blood samples from 71 children living in nearby villages were included. The SNPs defining the haplotypes of PROCR gene were determined by post-PCR ligation detection reaction-fluorescent microsphere assay.ResultsIndividuals carrying at least one H3 allele had significantly higher levels of sEPCR than individuals with no H3 alleles (P < 0.001). No difference in the frequency of H3 was found between the non-malaria patients, malaria patients or the village population (P > 0.1). Plasma levels of sEPCR differed between these three groups, with higher sEPCR levels in the village population compared to the hospitalized patients (P < 0.001) and higher levels in malaria patients compared to non-malaria patients (P = 0.001). However, no differences were found in the distribution of H3 (P = 0.2) or levels of sEPCR (P = 0.8) between patients diagnosed with severe and uncomplicated malaria.ConclusionFrequencies of SNPs determining PROCR haplotypes were in concordance with other African studies. The PROCR H3 allele was associated with higher levels of sEPCR, confirming earlier findings, however, in this Tanzanian population; neither PROCR haplotype nor level of sEPCR was associated with severe malaria, however, larger studies are needed to confirm these findings.
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