Synergistic prostaglandin E synthesis by myeloid and endothelial cells promotes fetal hematopoietic stem cell expansion in vertebrates

During development, hematopoietic stem cells (HSCs) are produced from the hemogenic endothelium and will expand in a transient hematopoietic niche. Prostaglandin E₂ (PGE2) is essential during vertebrate development and HSC specification, but its precise source in the embryo remains elusive. Here, we show that in the zebrafish embryo, PGE2 synthesis genes are expressed by distinct stromal cell populations, myeloid (neutrophils, macrophages), and endothelial cells of the caudal hematopoietic tissue. Ablation of myeloid cells, which produce the PGE2 precursor prostaglandin H₂ (PGH2), results in loss of HSCs in the caudal hematopoietic tissue, which could be rescued by exogeneous PGE2 or PGH2 supplementation. Endothelial cells contribute by expressing the PGH2 import transporter slco2b1 and ptges3, the enzyme converting PGH2 into PGE2. Of note, differential niche cell expression of PGE2 biosynthesis enzymes is also observed in the mouse fetal liver. Taken altogether, our data suggest that the triad composed of neutrophils, macrophages, and endothelial cells sequentially and synergistically contributes to blood stem cell expansion during vertebrate development.     

G protein-coupled receptor 183 facilitates endothelial-to-hematopoietic transition via Notch1 inhibition

In vertebrates, embryonic hematopoietic stem and progenitor cells (HSPCs) are derived from a subset of endothelial cells, the hemogenic endothelium (HE), through the endothelial-to-hematopoietic transition (EHT). Notch signaling is essential for HSPC development during embryogenesis across vertebrates. However, whether and how it regulates EHT remains unclear. Here, we show that G protein-coupled receptor 183 (Gpr183) signaling serves as an indispensable switch for HSPC emergence by repressing Notch signaling before the onset of EHT. Inhibition of Gpr183 significantly upregulates Notch signaling and abolishes HSPC emergence. Upon activation by its ligand 7α-25-OHC, Gpr183 recruits β-arrestin1 and the E3 ligase Nedd4 to degrade Notch1 in specified HE cells and then facilitates the subsequent EHT. Importantly, 7α-25-OHC stimulation promotes HSPC emergence in vivo and in vitro, providing an attractive strategy for enhancing the in vitro generation of functional HSPCs.     

Pyruvate metabolism guides definitive lineage specification during hematopoietic emergence

During embryonic development, hematopoiesis occurs through primitive and definitive waves, giving rise to distinct blood lineages. Hematopoietic stem cells (HSCs) emerge from hemogenic endothelial (HE) cells, through endothelial‐to‐hematopoietic transition (EHT). In the adult, HSC quiescence, maintenance, and differentiation are closely linked to changes in metabolism. However, metabolic processes underlying the emergence of HSCs from HE cells remain unclear. Here, we show that the emergence of blood is regulated by multiple metabolic pathways that induce or modulate the differentiation toward specific hematopoietic lineages during human EHT. In both in vitro and in vivo settings, steering pyruvate use toward glycolysis or OXPHOS differentially skews the hematopoietic output of HE cells toward either an erythroid fate with primitive phenotype, or a definitive lymphoid fate, respectively. We demonstrate that glycolysis‐mediated differentiation of HE toward primitive erythroid hematopoiesis is dependent on the epigenetic regulator LSD1. In contrast, OXPHOS‐mediated differentiation of HE toward definitive hematopoiesis is dependent on cholesterol metabolism. Our findings reveal that during EHT, metabolism is a major regulator of primitive versus definitive hematopoietic differentiation.     

TGFβ inhibition enhances the generation of hematopoietic progenitors from human ES cell-derived hemogenic endothelial cells using a stepwise strategy

Embryonic hematopoiesis is a complex process. Elucidating the mechanism regulating hematopoietic differentiation from pluripotent stem cells would allow us to establish a strategy to efficiently generate hematopoietic cells. However, the mechanism governing the generation of hematopoietic progenitors from human embryonic stem cells (hESCs) remains unknown. Here, on the basis of the emergence of CD43(+) hematopoietic cells from hemogenic endothelial (HE) cells, we demonstrated that VEGF was essential and sufficient, and that bFGF was synergistic with VEGF to specify the HE cells and the subsequent transition into CD43(+) hematopoietic cells. Significantly, we identified TGFβ as a novel signal to regulate hematopoietic development, as the TGFβ inhibitor SB 431542 significantly promoted the transition from HE cells into CD43(+) hematopoietic progenitor cells (HPCs) during hESC differentiation. By defining these critical signaling factors during hematopoietic differentiation, we can efficiently generate HPCs from hESCs. Our strategy could offer an in vitro model to study early human hematopoietic development.     

RUNX1B Expression Is Highly Heterogeneous and Distinguishes Megakaryocytic and Erythroid Lineage Fate in Adult Mouse Hematopoiesis

The Core Binding Factor (CBF) protein RUNX1 is a master regulator of definitive hematopoiesis, crucial for hematopoietic stem cell (HSC) emergence during ontogeny. RUNX1 also plays vital roles in adult mice, in regulating the correct specification of numerous blood lineages. Akin to the other mammalian Runx genes, Runx1 has two promoters P1 (distal) and P2 (proximal) which generate distinct protein isoforms. The activities and specific relevance of these two promoters in adult hematopoiesis remain to be fully elucidated. Utilizing a dual reporter mouse model we demonstrate that the distal P1 promoter is broadly active in adult hematopoietic stem and progenitor cell (HSPC) populations. By contrast the activity of the proximal P2 promoter is more restricted and its upregulation, in both the immature Lineage^- Sca1^high cKit^high (LSK) and bipotential Pre-Megakaryocytic/Erythroid Progenitor (PreMegE) populations, coincides with a loss of erythroid (Ery) specification. Accordingly the PreMegE population can be prospectively separated into “pro-erythroid” and “pro-megakaryocyte” populations based on Runx1 P2 activity. Comparative gene expression analyses between Runx1 P2⁺ and P2^- populations indicated that levels of CD34 expression could substitute for P2 activity to distinguish these two cell populations in wild type (WT) bone marrow (BM). Prospective isolation of these two populations will enable the further investigation of molecular mechanisms involved in megakaryocytic/erythroid (Mk/Ery) cell fate decisions. Having characterized the extensive activity of P1, we utilized a P1-GFP homozygous mouse model to analyze the impact of the complete absence of Runx1 P1 expression in adult mice and observed strong defects in the T cell lineage. Finally, we investigated how the leukemic fusion protein AML1-ETO9a might influence Runx1 promoter usage. Short-term AML1-ETO9a induction in BM resulted in preferential P2 upregulation, suggesting its expression may be important to establish a pre-leukemic environment.     

Dicer independent small RNAs associate with telomeric heterochromatin

Small RNAs play important roles in the establishment and maintenance of heterochromatin structures. We show the presence of telomere specific small RNAs (tel-sRNAs) in mouse embryonic stem cells that are ∼24 nucleotides in length, Dicer-independent, and 2′-O-methylated at the 3′ terminus. The tel-sRNAs are asymmetric with specificity toward telomere G-rich strand, and evolutionarily conserved from protozoan to mammalian cells. Furthermore, tel-sRNAs are up-regulated in cells that carry null mutation of H3K4 methyltransferase MLL (Mll^(−/−)) and down-regulated in cells that carry null mutations of histone H3K9 methyltransferase SUV39H (Suv39h1/h2^(−/−)), suggesting that they are subject to epigenetic regulation. These results support that tel-sRNAs are heterochromatin associated pi-like small RNAs.     

Osteoblast lineage cells expressing high levels of Runx2 enhance hematopoietic progenitor cell proliferation and function

Although osteoblasts (OB) play a key role in the hematopoietic stem cell (HSC) niche, little is known as to which specific OB lineage cells are critical for the enhancement of stem and progenitor cell function. Unlike hematopoietic cells, OB cell surface phenotypic definitions are not well developed. Therefore, to determine which OB lineage cells are most important for hematopoietic progenitor cell (HPC) function, we characterized OB differentiation by gene expression and OB function, and determined whether associations existed between OB and HPC properties. OB were harvested from murine calvariae, used immediately (fresh OB) or cultured for 1, 2, or 3 weeks prior to their co‐culture with Lin^?Sca1⁺c‐kit⁺ (LSK) cells for 1 week. OB gene expression, alkaline phosphatase activity, calcium deposition, hematopoietic cell number fold increase, CFU fold increase, and fold increase of Lin^?Sca1⁺ cells were determined. As expected, HPC properties were enhanced when LSK cells were cultured with OB compared to being cultured alone. Initial alkaline phosphatase and calcium deposition levels were significantly and inversely associated with an increase in the number of LSK progeny. Final calcium deposition levels and OB culture duration were inversely associated with all HPC parameters, while Runx2 levels were positively associated with all HPC properties. Since calcium deposition is associated with OB maturation and high levels of Runx2 are associated with less mature OB lineage cells, these results suggest that less mature OB better promote HPC proliferation and function than do more mature OB. J. Cell. Biochem. 111: 284–294, 2010. © 2010 Wiley‐Liss, Inc.     

Runx1 Phosphorylation by Src Increases Trans-activation via Augmented Stability,Reduced Histone Deacetylase (HDAC) Binding,and Increased DNA Affinity,and Activated Runx1 Favors Granulopoiesis

Src phosphorylates Runx1 on one central and four C-terminal tyrosines. We find that activated Src synergizes with Runx1 to activate a Runx1 luciferase reporter. Mutation of the four Runx1 C-terminal tyrosines to aspartate or glutamate to mimic phosphorylation increases trans-activation of the reporter in 293T cells and allows induction of Cebpa or Pu.1 mRNAs in 32Dcl3 myeloid cells, whereas mutation of these residues to phenylalanine to prevent phosphorylation obviates these effects. Three mechanisms contribute to increased Runx1 activity upon tyrosine modification as follows: increased stability, reduced histone deacetylase (HDAC) interaction, and increased DNA binding. Mutation of the five modified Runx1 tyrosines to aspartate markedly reduced co-immunoprecipitation with HDAC1 and HDAC3, markedly increased stability in cycloheximide or in the presence of co-expressed Cdh1, an E3 ubiquitin ligase coactivator, with reduced ubiquitination, and allowed DNA-binding in gel shift assay similar to wild-type Runx1. In contrast, mutation of these residues to phenylalanine modestly increased HDAC interaction, modestly reduced stability, and markedly reduced DNA binding in gel shift assays and as assessed by chromatin immunoprecipitation with the −14-kb Pu.1 or +37-kb Cebpa enhancers after stable expression in 32Dcl3 cells. Affinity for CBFβ, the Runx1 DNA-binding partner, was not affected by these tyrosine modifications, and in vitro translated CBFβ markedly increased DNA affinity of both the translated phenylalanine and aspartate Runx1 variants. Finally, further supporting a positive role for Runx1 tyrosine phosphorylation during granulopoiesis, mutation of the five Src-modified residues to aspartate but not phenylalanine allows Runx1 to increase Cebpa and granulocyte colony formation by Runx1-deleted murine marrow.