Junke Zheng

The Distinct Metabolic Profile of Hematopoietic Stem Cells Reflects Their Location in a Hypoxic Niche

Bone marrow transplantation is the primary therapy for numerous hematopoietic disorders. The efficiency of bone marrow transplantation depends on the function of long-term hematopoietic stem cells (LT-HSCs), which is markedly influenced by their hypoxic niche. Survival in this low-oxygen microenvironment requires significant metabolic adaptation. Here, we show that LT-HSCs utilize glycolysis instead of mitochondrial oxidative phosphorylation to meet their energy demands. We used flow cytometry to identify a unique low mitochondrial activity/glycolysis-dependent subpopulation that houses the majority of hematopoietic progenitors and LT-HSCs. Finally, we demonstrate that Meis1 and Hif-1alpha are markedly enriched in LT-HSCs and that Meis1 regulates HSC metabolism through transcriptional activation of Hif-1alpha. These findings reveal an important transcriptional network that regulates HSC metabolism.

Inhibitory receptors bind ANGPTLs and support blood stem cells and leukaemia development

How environmental cues regulate adult stem cell and cancer cell activity through surface receptors is poorly understood. Angiopoietin-like proteins (ANGPTLs), a family of seven secreted glycoproteins, are known to support the activity of haematopoietic stem cells (HSCs) in vitro and in vivo. ANGPTLs also have important roles in lipid metabolism, angiogenesis and inflammation, but were considered ‘orphan ligands’ because no receptors were identified. Here we show that the immune-inhibitory receptor human leukocyte immunoglobulin-like receptor B2 (LILRB2) and its mouse orthologue paired immunoglobulin-like receptor (PIRB) are receptors for several ANGPTLs. LILRB2 and PIRB are expressed on human and mouse HSCs, respectively, and the binding of ANGPTLs to these receptors supported ex vivo expansion of HSCs. In mouse transplantation acute myeloid leukaemia models, a deficiency in intracellular signalling of PIRB resulted in increased differentiation of leukaemia cells, revealing that PIRB supports leukaemia development. Our study indicates an unexpected functional significance of classical immune-inhibitory receptors in maintenance of stemness of normal adult stem cells and in support of cancer development.

Meis1 regulates the metabolic phenotype and oxidant defense of hematopoietic stem cells

The role of Meis1 in leukemia is well established, but its role in hematopoietic stem cells (HSCs) remains poorly understood. Previously, we showed that HSCs use glycolytic metabolism to meet their energy demands. However, the mechanism of regulation of HSC metabolism, and the importance of maintaining this distinct metabolic phenotype on HSC function has not been determined. More importantly, the primary function of Meis1 in HSCs remains unknown. Here, we examined the effect of loss of Meis1 on HSC function and metabolism. Inducible Meis1 deletion in adult mouse HSCs resulted in loss of HSC quiescence, and failure of bone marrow repopulation after transplantation. While we previously showed that Meis1 regulates Hif-1α transcription in vitro, we demonstrate here that loss of Meis1 results in down-regulation of both Hif-1α and Hif-2α in HSCs. This resulted in a shift to mitochondrial metabolism, increased reactive oxygen species production, and apoptosis of HSCs. Finally, we demonstrate that the effect of Meis1 knockout on HSCs is entirely mediated through reactive oxygen species where treatment of the Meis1 knockout mice with the scavenger N-acetylcystein restored HSC quiescence and rescued HSC function. These results uncover an important transcriptional network that regulates metabolism, oxidant defense, and maintenance of HSCs.

Congenital bone marrow failure in DNA-PKcs mutant mice associated with deficiencies in DNA repair

Phosphorylation of DNA-PKcs is essential for activation of DNA damage repair and for the maintenance of tissue stem cell populations.

Angiopoietin-like protein 3 supports the activity of hematopoietic stem cells in the bone marrow niche

The physiologic roles of angiopoietin-like proteins (Angptls) in the hematopoietic system remain unknown. Here we show that hematopoietic stem cells (HSCs) in Angptl3-null mice are decreased in number and quiescence. HSCs transplanted into Angptl3-null recipient mice exhibited impaired repopulation. Bone marrow sinusoidal endothelial cells express high levels of Angptl3 and are adjacent to HSCs. Importantly, bone marrow stromal cells or endothelium deficient in Angptl3 have a significantly decreased ability to support the expansion of repopulating HSCs. Angptl3 represses the expression of the transcription factor Ikaros, whose unregulated overexpression diminishes the repopulation activity of HSCs. Angptl3, as an extrinsic factor, thus supports the stemness of HSCs in the bone marrow niche.

Insulin‐Like Growth Factor‐Binding Protein 2 Secreted by a Tumorigenic Cell Line Supports Ex Vivo Expansion of Mouse Hematopoietic Stem Cells

Successful hematopoietic stem cell (HSC) transplantation is often limited by the numbers of HSCs, and robust methods to expand HSCs ex vivo are needed. We previously showed that angiopoietin‐like proteins (Angptls), a group of growth factors isolated from a fetal liver HSC‐supportive cell population, improved ex vivo expansion of HSCs. Here, we demonstrate that insulin‐like growth factor‐binding protein 2 (IGFBP2), secreted by a tumorigenic cell line, also enhanced ex vivo expansion of mouse HSCs. On the basis of these findings, we established a completely defined, serum‐free culture system for mouse HSCs, containing SCF, thrombopoietin, fibroblast growth factor 1, Angptl3, and IGFBP2. As measured by competitive repopulation analyses, there was a 48‐fold increase in numbers of long‐term repopulating mouse HSCs after 21 days of culture. This is the first demonstration that IGFBP2 stimulates expansion or proliferation of murine stem cells. Our finding also suggests that certain cancer cells synthesize proteins that can stimulate HSC expansion.

IGF binding protein 2 supports the survival and cycling of hematopoietic stem cells

The role of IGF binding protein 2 (IGFBP2) in cell growth is intriguing and largely undefined. Previously we identified IGFBP2 as an extrinsic factor that supports ex vivo expansion of hematopoietic stem cells (HSCs). Here we showed that IGFBP2-null mice have fewer HSCs than wild-type mice. While IGFBP2 has little cell-autonomous effect on HSC function, we found decreased in vivo repopulation of HSCs in primary and secondary transplanted IGFBP2-null recipients. Importantly, bone marrow stromal cells that are deficient for IGFBP2 have significantly decreased ability to support the expansion of repopulating HSCs. To investigate the mechanism by which IGFBP2 supports HSC activity, we demonstrated that HSCs in IGFBP2-null mice had decreased survival and cycling, down-regulated expression of antiapoptotic factor Bcl-2, and up-regulated expression of cell cycle inhibitors p21, p16, p19, p57, and PTEN. Moreover, we found that the C-terminus, but not the RGD domain, of extrinsic IGFBP2 was essential for support of HSC activity. Defective signaling of the IGF type I receptor did not rescue the decreased repopulation of HSCs in IGFBP2-null recipients, suggesting that the environmental effect of IGFBP2 on HSCs is independent of IGF-IR mediated signaling. Therefore, as an environmental factor, IGFBP2 supports the survival and cycling of HSCs.

Paired immunoglobulin-like receptor B regulates platelet activation.

Murine paired immunoglobulin-like receptors B (PIRB), as the ortholog of human leukocyte immunoglobulin-like receptor B2 (LILRB2), is involved in a variety of biological functions. Here, we found that PIRB and LILRB2 were expressed in mouse and human platelets, respectively. PIRB intracellular domain deletion (PIRB-TM) mice had thrombocythemia and significantly higher proportions of megakaryocytes in bone marrow. Agonist-induced aggregation and spreading on immobilized fibrinogen were facilitated in PIRB-TM platelets. The rate of clot retraction in platelet-rich plasma containing PIRB-TM platelets was also increased. Characterization of signaling confirmed that PIRB associated with phosphatases Shp1/2 in platelets. The phosphorylation of Shp1/2 was significantly downregulated in PIRB-TM platelets stimulated with collagen-related peptide (CRP) or on spreading. The results further revealed that the phosphorylation levels of the linker for activation of T cells, SH2 domain-containing leukocyte protein of 76kDa, and phospholipase C were enhanced in PIRB-TM platelets stimulated with CRP. The phosphorylation levels of FAK Y397 and integrin β3 Y759 were also enhanced in PIRB-TM platelet spread on fibrinogen. The PIRB/LILRB2 ligand angiopoietin-like-protein 2 (ANGPTL2) was expressed and stored in platelet α-granules. ANGPTL2 inhibited agonist-induced platelet aggregation and spreading on fibrinogen. The data presented here reveal that PIRB and its ligand ANGPTL2 possess an antithrombotic function by suppressing collagen receptor glycoprotein VI and integrin αIIbβ3-mediated signaling.

Sclerotherapy of voluminous venous malformation in head and neck with absolute ethanol under digital subtraction angiography guidance

Objectives Venous malformation (VM) is the most common symptomatic low-flow vascular malformation, which predominantly occurs in the head and neck region. The aim of this paper was to evaluate the results of endovascular sclerotherapy of voluminous VM, when the lesion is either ≥15 cm in maximum diameter or the lesion invades more than one anatomical space, in the head and neck region using absolute ethanol under digital subtraction angiography (DSA) guidance. Methods A total of 23 patients with head and neck VMs between October 2005 and December 2008 were retrospectively reviewed. All patients received direct puncture ethanol sclerotherapy under DSA guidance. Follow-up assessments were performed at 3–25 months after therapies were completed, and complications were reported in some cases. Results All patients were satisfied with the results of therapy. Seventeen patients (73.9%) achieved excellent responses and six patients (26.1%) achieved good responses in magnetic resonance imaging assessments. Minor complications developed during the procedures, all of which were successfully managed with full recovery during follow-ups. Serious complications such as acute pulmonary hypertension, cardiovascular collapse and pulmonary embolism were not encountered. Conclusion It is concluded that sclerotherapy with absolute ethanol under DSA guidance is an important alternative therapy for voluminous and extensive VM, as the procedure is reasonably safe and offers good therapeutic results.

Profilin 1 is essential for retention and metabolism of mouse hematopoietic stem cells in bone marrow

How stem cells interact with the microenvironment to regulate their cell fates and metabolism is largely unknown. Here we demonstrated that the deletion of the cytoskeleton-modulating protein profilin 1 (pfn1) in hematopoietic stem cell (HSCs) led to bone marrow failure, loss of quiescence, and mobilization and apoptosis of HSCs in vivo. A switch from glycolysis to mitochondrial respiration with increased reactive oxygen species (ROS) level was also observed in HSCs on pfn1 deletion. Importantly, treatment of pfn1-deficient mice with the antioxidant N-acetyl-l-cysteine reversed the ROS level and loss of quiescence of HSCs, suggesting that the metabolism is mechanistically linked to the cell cycle quiescence of stem cells. The actin-binding and proline-binding activities of pfn1 are required for its function in HSCs. Our study provided evidence that pfn1 at least partially acts through the axis of pfn1/Gα13/EGR1 to regulate stem cell retention and metabolism in the bone marrow.