Wnt signaling in the niche enforces hematopoietic stem cell quiescence and is necessary to preserve self-renewal in vivo

Wingless (Wnt) is a potent morphogen demonstrated in multiple cell lineages to promote the expansion and maintenance of stem and progenitor cell populations. Wnt effects are highly context dependent, and varying effects of Wnt signaling on hematopoietic stem cells (HSCs) have been reported. We explored the impact of Wnt signaling in vivo, specifically in the context of the HSC niche by using an osteoblast-specific promoter driving expression of the paninhibitor of canonical Wnt signaling, Dickkopf1 (Dkk1). Here we report that Wnt signaling was markedly inhibited in HSCs and, unexpectedly given prior reports, reduction in HSC Wnt signaling resulted in reduced p21Cip1 expression, increased cell cycling, and a progressive decline in regenerative function after transplantation. This effect was microenvironment determined, but irreversible if the cells were transferred to a normal host. Wnt pathway activation in the niche is required to limit HSC proliferation and preserve the reconstituting function of endogenous hematopoietic stem cells.     

Differential requirements for Wnt and Notch signaling in hematopoietic versus thymic niches

All blood cells are derived from multipotent stem cells, the so-called hematopoietic stem cells (HSCs), that in adults reside in the bone marrow. Most types of blood cells also develop there, with the notable exception of T lymphocytes that develop in the thymus. For both HSCs and developing T cells, interactions with the surrounding microenvironment are critical in regulating maintenance, differentiation, apoptosis, and proliferation. Such specialized regulatory microenvironments are referred to as niches and provide both soluble factors as well as cell-cell interactions between niche component cells and blood cells. Two pathways that are critical for early T cell development in the thymic niche are Wnt and Notch signaling. These signals also play important but controversial roles in the HSC niche. Here, we review the differences and similarities between the thymic and hematopoietic niches, with particular focus on Wnt and Notch signals, as well as the latest insights into regulation of these developmentally important pathways.     

Less Is More: Unveiling the Functional Core of Hematopoietic Stem Cells through Knockout Mice

Hematopoietic stem cells (HSCs) represent one of the first recognized somatic stem cell types. As such, nearly 200 genes have been examined for roles in HSC function in knockout mice. In this review, we compile the majority of these reports to provide a broad overview of the functional modules revealed by these genetic analyses and highlight some key regulatory pathways involved, including cell cycle control, Tgf-β signaling, Pten/Akt signaling, Wnt signaling, and cytokine signaling. Finally, we propose recommendations for characterization of HSC function in knockout mice to facilitate cross-study comparisons that would generate?a more cohesive picture of HSC biology.     

Transforming growth factor-β signaling confers hepatic stellate cells progenitor features after partial hepatectomy

Liver regeneration involves not only hepatocyte replication but progenitor aggregation and scarring. Partial hepatectomy (PH), an established model for liver regeneration, reactivates transforming growth factor-β (TGF-β) signaling. Hepatic stellate cells (HSCs) are primarily responding cells for TGF-β and resident in stem cell niche. In the current study, PH mice were treated with SB-431542, an inhibitor of TGF-β Type I receptor, aiming to address the role of TGF-β signaling on the fate determination of HSCs during liver regeneration. After PH, control mice exhibited HSCs activation, progenitor cells accumulation, and a fraction of HSCs acquired the phenotype of hepatocyte or cholangiocyte. Blocking TGF-β signaling delayed proliferation, impaired progenitor response, and scarring repair. In SB-431542 group, merely no HSCs were found coexpressed progenitor makers, such as SOX9 and AFP. Inhibition of TGF-β pathway disturbed the epithelial-mesenchymal transitions and diminished the nuclear accumulation of β-catenin as well as the expression of cytochrome P450 2E1 in HSC during liver regeneration. We identify a key role of TGF-β signaling on promoting HSC transition, which subsequently becomes progenitor for generating liver epithelial cells after PH. This process might interact with an acknowledged stem cell function signaling, Wnt/β-catenin.     

Integrin αvβ3 enhances the suppressive effect of interferon‐γ on hematopoietic stem cells

Hematopoietic homeostasis depends on the maintenance of hematopoietic stem cells (HSCs), which are regulated within a specialized bone marrow (BM) niche. When HSC sense external stimuli, their adhesion status may be critical for determining HSC cell fate. The cell surface molecule, integrin αvβ3, is activated through HSC adhesion to extracellular matrix and niche cells. Integrin β3 signaling maintains HSCs within the niche. Here, we showed the synergistic negative regulation of the pro‐inflammatory cytokine interferon‐γ (IFNγ) and β3 integrin signaling in murine HSC function by a novel definitive phenotyping of HSCs. Integrin αvβ3 suppressed HSC function in the presence of IFNγ and impaired integrin β3 signaling mitigated IFNγ‐dependent negative action on HSCs. During IFNγ stimulation, integrin β3 signaling enhanced STAT1‐mediated gene expression via serine phosphorylation. These findings show that integrin β3 signaling intensifies the suppressive effect of IFNγ on HSCs, which indicates that cell adhesion via integrin αvβ3 within the BM niche acts as a context‐dependent signal modulator to regulate the HSC function under both steady‐state and inflammatory conditions.     

Hematopoietic stem cells: generation and self-renewal

Adult stem cells hold great promise for future therapeutic applications. Hematopoietic stem cells (HSCs) are among the best-characterized adult stem cells. As such, these cells provide a conceptual framework for the study of adult stem cells from other organs. Here, we review the current knowledge of HSC generation during embryonic development and HSC maintenance in the bone marrow (BM) during adult life. Recent scientific progress has demonstrated that the development of HSCs involves many anatomical sites in the embryo, but the relative contribution of each of these sites to the adult HSC pool remains controversial. Specialized anatomical sites in the BM have been identified as stem cell niches, and these play essential roles in regulating the self-renewal and differentiation of HSCs through recently identified signaling pathways. Extracellular signaling from stem cell niches must integrate with the intracellular molecular machinery and/or genetic programs to regulate HSC fate choice. The exact cellular and/or molecular mechanisms defining stem cell niche and 'stemness' of HSC is largely unknown although substantial progress has been made recently. Hence, many questions remain to be answered even in this relatively well-defined model of stem cell biology.     

Membrane-bound SCF and VCAM-1 synergistically regulate the morphology of hematopoietic stem cells

Membrane-bound factors expressed by niche stromal cells constitute a unique class of localized cues and regulate the long-term functions of adult stem cells, yet little is known about the underlying mechanisms. Here, we used a supported lipid bilayer (SLB) to recapitulate the membrane-bound interactions between hematopoietic stem cells (HSCs) and niche stromal cells. HSCs cluster membrane-bound stem cell factor (mSCF) at the HSC-SLB interface. They further form a polarized morphology with aggregated mSCF under a large protrusion through a synergy with VCAM-1 on the bilayer, which drastically enhances HSC adhesion. These features are unique to mSCF and HSCs among the factors and hematopoietic populations we examined. The mSCF–VCAM-1 synergy and the polarized HSC morphology require PI3K signaling and cytoskeletal reorganization. The synergy also enhances nuclear retention of FOXO3a, a crucial factor for HSC maintenance, and minimizes its loss induced by soluble SCF. Our work thus reveals a unique role and signaling mechanism of membrane-bound factors in regulating stem cell morphology and function.     

Canonical wnt signaling regulates hematopoiesis in a dosage-dependent fashion

Canonical Wnt signaling has been implicated in the regulation of hematopoiesis. By employing a Wnt-reporter mouse, we observed that Wnt signaling is differentially activated during hematopoiesis, suggesting an important regulatory role for specific Wnt signaling levels. To investigate whether canonical Wnt signaling regulates hematopoiesis in?a dosage-dependent fashion, we analyzed the effect of different mutations in the Adenomatous polyposis coli gene (Apc), a negative modulator of the canonical Wnt pathway. By combining different targeted hypomorphic alleles and a conditional deletion allele of Apc, a gradient of five different Wnt signaling levels was obtained in?vivo. We here show that different, lineage-specific Wnt dosages regulate hematopoietic stem cells (HSCs), myeloid precursors, and T lymphoid precursors during hematopoiesis. Differential, lineage-specific optimal Wnt dosages provide a unifying concept that explains the differences reported among inducible gain-of-function approaches, leading to either HSC expansion or depletion of the HSC pool.     

The role of Wnt signaling in hematopoietic stem cell development

Hematopoietic stem cells (HSCs) can self-renew and differentiate into all cell types of the blood. This is therapeutically important as HSC transplants can provide a curative effect for blood cancers and disorders. The process by which HSCs develop has been the subject of extensive research in a variety of model organisms; however, efforts to produce bonafide HSCs from pluripotent precursors capable of long-term multilineage reconstitution have fallen short. Studies in zebrafish, chicken, and mice have been instrumental in guiding efforts to derive HSCs from human pluripotent stem cells and have identified a complex set of molecular signals and cellular interactions mediated by such developmental regulators as fibroblast growth factor, Notch, transforming growth factor beta (TGFβ), and Wnt, which collectively promote the stepwise developmental progression toward mature HSCs. Tight temporal and spatial control of these signals is critical to generate the appropriate numbers of HSCs needed for the life of the organism. The role of the Wnt family of signaling proteins in hematopoietic development has been the subject of many studies owing in part to the complex nature of its signaling mechanisms. By integrating cell fate specification with cell polarity establishment, Wnt is uniquely capable of controlling complex biological processes, including at multiple stages of embryonic HSC development, from HSC specification to emergence from the hemogenic epithelium to subsequent expansion. This review highlights key signaling events where specific Wnt signals instruct and guide hematopoietic development in both zebrafish and mice and extend these findings to current efforts of generating HSCs in vitro.     

Nonmyelinating Schwann cells maintain hematopoietic stem cell hibernation in the bone marrow niche

Hematopoietic stem cells (HSCs) reside and self-renew in the bone marrow (BM) niche. Overall, the signaling that regulates stem cell dormancy in the HSC niche remains controversial. Here, we demonstrate that TGF-β type II receptor-deficient HSCs show low-level Smad activation and impaired long-term repopulating activity, underlining the critical role of TGF-β/Smad signaling in HSC maintenance. TGF-β is produced as a latent form by a variety of cells, so we searched for those that express activator molecules for latent TGF-β. Nonmyelinating Schwann cells in BM proved responsible for activation. These glial cells ensheathed autonomic nerves, expressed HSC niche factor genes, and were in contact with a substantial proportion of HSCs. Autonomic nerve denervation reduced the number of these active TGF-β-producing cells and led to rapid loss of HSCs from BM. We propose that glial cells are components of a BM niche and maintain HSC hibernation by regulating activation of latent TGF-β.     

Hematopoietic stem cell development and regulatory signaling in zebrafish

Background

Hematopoietic stem cells (HSCs) are a population of multipotent cells that can self-renew and differentiate into all blood lineages. HSC development must be tightly controlled from cell fate determination to self-maintenance during adulthood. This involves a panel of important developmental signaling pathways and other factors which act synergistically within the HSC population and/or in the HSC niche. Genetically conserved processes of HSC development plus many other developmental advantages make the zebrafish an ideal model organism to elucidate the regulatory mechanisms underlying HSC programming.

Scope of review

This review summarizes recent progress on zebrafish HSCs with particular focus on how developmental signaling controls hemogenic endothelium-derived HSC development. We also describe the interaction of different signaling pathways during these processes.

Major conclusions

The hematopoietic stem cell system is a paradigm for stem cell studies. Use of the zebrafish model to study signaling regulation of HSCs in vivo has resulted in a great deal of information concerning HSC biology in vertebrates.

General significance

These new findings facilitate a better understanding of molecular mechanisms of HSC programming, and will provide possible new strategies for the treatment of HSC-related hematological diseases, such as leukemia. This article is part of a Special Issue entitled Biochemistry of Stem Cells.     

Wnt signaling in the niche

There is much interest in understanding the signals in the bone marrow niche that keep hematopoietic stem cells (HSCs) in a quiescent state. In the current issue of Cell Stem Cell, Fleming et al. (2008) report that blocking Wnt signaling in the niche increases the number of proliferating HSCs and reduces their ability to reconstitute the hematopoietic system of irradiated recipient mice. These findings show that Wnt/beta-catenin activity is crucial for the maintenance of HSC quiescence in the bone marrow niche.     

Osteopontin attenuates aging-associated phenotypes of hematopoietic stem cells

Upon aging, hematopoietic stem cells (HSCs) undergo changes in function and structure, including skewing to myeloid lineages, lower reconstitution potential and loss of protein polarity. While stem cell intrinsic mechanisms are known to contribute to HSC aging, little is known on whether age-related changes in the bone marrow niche regulate HSC aging. Upon aging, the expression of osteopontin (OPN) in the murine bone marrow stroma is reduced. Exposure of young HSCs to an OPN knockout niche results in a decrease in engraftment, an increase in long-term HSC frequency and loss of stem cell polarity. Exposure of aged HSCs to thrombin-cleaved OPN attenuates aging of old HSCs, resulting in increased engraftment, decreased HSC frequency, increased stem cell polarity and a restored balance of lymphoid and myeloid cells in peripheral blood. Thus, our data suggest a critical role for reduced stroma-derived OPN for HSC aging and identify thrombin-cleaved OPN as a novel niche informed therapeutic approach for ameliorating HSC phenotypes associated with aging.     

Wnt modulators, SFRP-1, and SFRP-2 are expressed in osteoblasts and differentially regulate hematopoietic stem cells

Wnt signaling has been implicated in the self-renewal of hematopoietic stem cells (HSCs). Secreted frizzled-related proteins (SFRPs) are a family of soluble proteins containing a region homologous to a receptor for Wnt, Frizzled, and are thought to act as endogenous modulators for Wnt signaling. This study examined the role of SFRPs in HSC regulation. Among the four family members, SFRP-1 and SFRP-2 are specifically induced in the bone marrow in response to myelosuppression, and immunostaining revealed that both proteins were expressed in osteoblasts. Interestingly, SFRP-1 reduced the number of multipotent progenitors in in vitro culture of CD34⁻KSL cells, while SFRP-2 did not. Furthermore, SFRP-1 compromised the long-term repopulating activity of HSCs, whereas SFRP-2 did not affect or even enhanced it in the same setting. These results indicate that although both SFRP-1 and SFRP-2 act as inhibitors for Wnt signaling in vitro, they differentially affect the homeostasis of HSCs.     

The elements of stem cell self-renewal: a genetic perspective

Every day, the body produces billions of new blood cells. Each of these is derived from a rare cell in the bone marrow called the hematopoietic stem cell (HSC). Because most mature blood cells have a limited lifespan, the ability of HSCs to self-renew and replenish the mature cell compartment is critical to sustaining life. While great progress has been made in isolating HSCs and defining their functional and phenotypic characteristics, the molecular mechanisms that regulate their self-renewal remain a mystery. Over the last few years, alterations in HSC frequency and self-renewal capacity in transgenic and knock-out mice have led to the identification of novel mediators of HSC homeostasis in vivo. These genetically modified mice have revealed that maintenance of survival, proliferation, quiescence, and normal telomere length all contribute to the self-renewal of HSCs. They also highlight the need to test in context of the normal microenvironment the role of signaling molecules such as Notch and Wnt, which have emerged recently as important regulators of HSC self-renewal. The emerging picture these data provide of the regulation of self-renewal in HSCs has provided a better understanding of the basic biology of stem cells and holds promise for designing strategies to improve bone marrow transplantation.     

Thrombopoietin and hematopoietic stem cells

Thrombopoietin (TPO) is the cytokine that is chiefly responsible for megakaryocyte production but increasingly attention has turned to its role in maintaining hematopoietic stem cells (HSCs). HSCs are required to initiate the production of all mature hematopoietic cells, but this differentiation needs to be balanced against self-renewal and quiescence to maintain the stem cell pool throughout life. TPO has been shown to support HSC quiescence during adult hematopoiesis, with the loss of TPO signaling associated with bone marrow failure and thrombocytopenia. Recent studies have shown that constitutive activation mutations in Mpl contribute to myeloproliferative disease. In this review, we will discuss TPO signaling pathways, regulation of TPO levels and the role of TPO in normal hematopoiesis and during myeloproliferative disease.     

Understanding hematopoietic stem-cell microenvironments

The hematopoietic system is the paradigm for adult mammalian stem-cell research. Recent advances have improved our understanding of the cellular and molecular components of the microenvironment - or niche - that regulates hematopoietic stem cells (HSCs). Here, we summarize the molecular and cellular properties of two types of niche, namely the osteoblastic and the vascular niche, in homeostatic regulation of HSC behavior, including its maintenance, proliferation, differentiation, mobilization and homing. We highlight the most recent findings and point to an important trend to the study of niche activity in cancers. Knowledge of the basic features of the HSC niches, including physical location, cell type and various signaling pathways, should provide insights into other stem-cell systems and benefit clinical applications.     

Identification of adiponectin as a novel hemopoietic stem cell growth factor

The hemopoietic microenvironment consists of a diverse repertoire of cells capable of providing signals that influence hemopoietic stem cell function. Although the role of osteoblasts and vascular endothelial cells has recently been characterized, the function of the most abundant cell type in the bone marrow, the adipocyte, is less defined. Given the emergence of a growing number of adipokines, it is possible that these factors may also play a role in regulating hematopoiesis. Here, we investigated the role of adiponectin, a secreted molecule derived from adipocytes, in hemopoietic stem cell (HSC) function. We show that adiponectin is expressed by components of the HSC niche and its receptors AdipoR1 and AdipoR2 are expressed by HSCs. At a functional level, adiponectin influences HSCs by increasing their proliferation, while retaining the cells in a functionally immature state as determined by in vitro and in vivo assays. We also demonstrate that adiponectin signaling is required for optimal HSC proliferation both in vitro and in long term hemopoietic reconstitution in vivo. Finally we show that adiponectin stimulation activates p38 MAPK, and that inhibition of this pathway abrogates adiponectin's proliferative effect on HSCs. These studies collectively identify adiponectin as a novel regulator of HSC function and suggest that it acts through a p38 dependent pathway.