Identification of in vitro HSC fate regulators by differential lipid raft clustering

Most hematopoietic stem cells (HSC) in the bone marrow reside in a quiescent state and occasionally enter the cell cycle upon cytokine-induced activation. Although the mechanisms regulating HSC quiescence and activation remain poorly defined, recent studies have revealed a role of lipid raft clustering (LRC) in HSC activation. Here, we tested the hypothesis that changes in lipid raft distribution could serve as an indicator of the quiescent and activated state of HSCs in response to putative niche signals. A semi-automated image analysis tool was developed to map the presence or absence of lipid raft clusters in live HSCs cultured for just one hour in serum-free medium supplemented with stem cell factor (SCF). By screening the ability of 19 protein candidates to alter lipid raft dynamics, we identified six factors that induced either a marked decrease (Wnt5a, Wnt3a and Osteopontin) or increase (IL3, IL6 and VEGF) in LRC. Cell cycle kinetics of single HSCs exposed to these factors revealed a correlation of LRC dynamics and proliferation kinetics: factors that decreased LRC slowed down cell cycle kinetics, while factors that increased LRC led to faster and more synchronous cycling. The possibility of identifying, by LRC analysis at very early time points, whether a stem cell is activated and possibly committed upon exposure to a signaling cue of interest could open up new avenues for large-scale screening efforts.     

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.     

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.     

Tie2/angiopoietin-1 signaling regulates hematopoietic stem cell quiescence in the bone marrow niche

The quiescent state is thought to be an indispensable property for the maintenance of hematopoietic stem cells (HSCs). Interaction of HSCs with their particular microenvironments, known as the stem cell niches, is critical for adult hematopoiesis in the bone marrow (BM). Here, we demonstrate that HSCs expressing the receptor tyrosine kinase Tie2 are quiescent and antiapoptotic, and comprise a side-population (SP) of HSCs, which adhere to osteoblasts (OBs) in the BM niche. The interaction of Tie2 with its ligand Angiopoietin-1 (Ang-1) induced cobblestone formation of HSCs in vitro and maintained in vivo long-term repopulating activity of HSCs. Furthermore, Ang-1 enhanced the ability of HSCs to become quiescent and induced adhesion to bone, resulting in protection of the HSC compartment from myelosuppressive stress. These data suggest that the Tie2/Ang-1 signaling pathway plays a critical role in the maintenance of HSCs in a quiescent state in the BM niche.     

Hematopoietic stem cell niche: An interplay among a repertoire of multiple functional niches

Background

Hematopoietic stem cell (HSC) niche of the BM provides a specialized microenvironment for the regulation of HSCs. The strict control of HSCs by the niche coordinates the balance between the proliferation and the differentiation of HSCs for the homeostasis of the blood system in steady states and during stress hematopoiesis. The osteoblastic and vascular niches are the classically identified constituents of the BM niche.

Scope of review

Recent research broadens our understanding of the BM niche as an assembly of multiple niche cells within the BM. We provide an overview of the HSC niche aiming to delineate the defined and possible niche cell interactions which collectively modulate the HSC integrity.

Major conclusions

Multiple cells in the BM, including osteoblasts, vascular endothelia, perivascular mesenchymal cells and HSC progeny cells, function conjunctively as niche cells to regulate HSCs.

General significance

The study of HSC niche cells and their functions provides insights into stem cell biology and also may be extrapolated into the study of cancer stem cells. This article is part of a Special Issue entitled Biochemistry of Stem Cells.     

The endoplasmic reticulum chaperone protein GRP94 is required for maintaining hematopoietic stem cell interactions with the adult bone marrow niche

Hematopoietic stem cell (HSC) homeostasis in the adult bone marrow (BM) is regulated by both intrinsic gene expression products and interactions with extrinsic factors in the HSC niche. GRP94, an endoplasmic reticulum chaperone, has been reported to be essential for the expression of specific integrins and to selectively regulate early T and B lymphopoiesis. In GRP94 deficient BM chimeras, multipotent hematopoietic progenitors persisted and even increased, however, the mechanism is not well understood. Here we employed a conditional knockout (KO) strategy to acutely eliminate GRP94 in the hematopoietic system. We observed an increase in HSCs and granulocyte-monocyte progenitors in the Grp94 KO BM, correlating with an increased number of colony forming units. Cell cycle analysis revealed that a loss of quiescence and an increase in proliferation led to an increase in Grp94 KO HSCs. This expansion of the HSC pool can be attributed to the impaired interaction of HSCs with the niche, evidenced by enhanced HSC mobilization and severely compromised homing and lodging ability of primitive hematopoietic cells. Transplanting wild-type (WT) hematopoietic cells into a GRP94 null microenvironment yielded a normal hematology profile and comparable numbers of HSCs as compared to WT control, suggesting that GRP94 in HSCs, but not niche cells, is required for maintaining HSC homeostasis. Investigating this, we further determined that there was a near complete loss of integrin α4 expression on the cell surface of Grp94 KO HSCs, which showed impaired binding with fibronectin, an extracellular matrix molecule known to play a role in mediating HSC-niche interactions. Furthermore, the Grp94 KO mice displayed altered myeloid and lymphoid differentiation. Collectively, our studies establish GRP94 as a novel cell intrinsic factor required to maintain the interaction of HSCs with their niche, and thus regulate their physiology.     

Regulation of hematopoietic stem cells in the niche

Hematopoiesis provides a suitable model for understanding adult stem cells and their niche. Hematopoietic stem cells(HSCs) continuously produce blood cells through orchestrated proliferation, self-renewal, and differentiation in the bone marrow(BM). Within the BM exists a highly organized microenvironment termed "niche" where stem cells reside and are maintained. HSC niche is the first evidence that a microenvironment contributes to protecting stem cell integrity and functionality in mammals. Although multiple models exist, recent progress has principally elucidated the cellular complexity of the HSC niche that maintains and regulates HSCs in BM. Here we introduce the development and summarize the achievements of HSC niche studies.     

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.     

Thrombopoietin/MPL signaling regulates hematopoietic stem cell quiescence and interaction with the osteoblastic niche

Maintenance of hematopoietic stem cells (HSCs) depends on interaction with their niche. Here we show that the long-term (LT)-HSCs expressing the thrombopoietin (THPO) receptor, MPL, are a quiescent population in adult bone marrow (BM) and are closely associated with THPO-producing osteoblastic cells. THPO/MPL signaling upregulated beta1-integrin and cyclin-dependent kinase inhibitors in HSCs. Furthermore, inhibition and stimulation of THPO/MPL pathway by treatments with anti-MPL neutralizing antibody, AMM2, and with THPO showed reciprocal regulation of quiescence of LT-HSC. AMM2 treatment reduced the number of quiescent LT-HSCs and allowed exogenous HSC engraftment without irradiation. By contrast, exogenous THPO transiently increased quiescent HSC population and subsequently induced HSC proliferation in vivo. Altogether, these observations suggest that THPO/MPL signaling plays a critical role of LT-HSC regulation in the osteoblastic niche.     

Regulation of Cell Cycle in Hematopoietic Stem Cells by the Niche

The quiescent state is thought to be an indispensable property for themaintenance of hematopoietic stem cells (HSCs). Interaction of HSCs with theirparticular microenvironments, known as the stem cell niches, is critical for cell cycleregulation of HSCs. Monitoring of the quiescence of HSCs using by a new stem cellmarker, Side Population (SP), revealed that the cell cycle status of HSCs is dynamicallycontrolled by the microenvironments. We have recently revealed a molecularmechanism in which cell cycle of HSCs is regulated by the niche. HSCs expressing thereceptor tyrosine kinase Tie2 are adhere to osteoblasts (OBs) in the BM niche. Theinteraction of Tie2 and its ligand Angiopoietin-1 (Ang-1) leads to tight adhesion ofHSCs to stromal cells, resulting in maintainance of long-term repopulating activity ofHSCs. Thus, Tie2/Ang-1 signaling pathway plays a critical role in the maintenance ofHSCs in a quiescent state in the BM niche. The understanding of cell cycle control instem cells leads to development of new strategy for progress in regenerative medicine.     

Robo4 cooperates with CXCR4 to specify hematopoietic stem cell localization to bone marrow niches

Specific bone marrow (BM) niches are critical for hematopoietic stem cell (HSC) function during both normal hematopoiesis and in stem cell transplantation therapy. We demonstrate that the guidance molecule Robo4 functions to specifically anchor HSCs to BM niches. Robo4-deficient HSCs displayed poor localization to BM niches and drastically reduced long-term reconstitution capability while retaining multilineage potential. Cxcr4, a critical regulator of HSC location, is upregulated in Robo4(-/-) HSCs to compensate for Robo4 loss. Robo4 deletion led to altered HSC mobilization efficiency, revealing that inhibition of both Cxcr4- and Robo4-mediated niche interactions are necessary for efficient HSC mobilization. Surprisingly, we found that WT HSCs express very low levels of Cxcr4 and respond poorly to Cxcr4 manipulation relative to other hematopoietic cells. We conclude that Robo4 cooperates with Cxcr4 to endow HSCs with competitive access to limited stem cell niches, and we propose Robo4 as a therapeutic target in HSC transplantation therapy.     

Temporal modulation of calcium sensing in hematopoietic stem cells is crucial for proper stem cell expansion and engraftment

Hematopoietic stem cells (HSCs) are known to reside in a bone marrow (BM) niche, which is associated with relatively higher calcium content. HSCs sense and respond to calcium changes. However, how calcium-sensing components modulate HSC function and expansion is largely unknown. We investigated temporal modulation of calcium sensing and Ca²⁺ homeostasis during ex vivo HSC culture and in vivo. Murine BM-HSCs, human BM, and umbilical cord blood (UCB) mononuclear cells (MNCs) were treated with store-operated calcium entry (SOCE) inhibitors SKF 96365 hydrochloride (abbreviated as SKF) and 2-aminoethoxydiphenyl borate (2-APB). Besides, K⁺ channel inhibitor TEA chloride (abbreviated as TEA) was used to compare the relationship between calcium-activated potassium channel activities. Seven days of SKF treatment induced mouse and human ex vivo BM-HSC expansion as well as UCB-derived primitive HSC expansion. SKF treatment induced the surface expression of CaSR, CXCR4, and adhesion molecules on human hematopoietic stem and progenitor cells. HSCs expanded with SKF successfully differentiated into blood lineages in recipient animals and demonstrated a higher repopulation capability. Furthermore, modulation of SOCE in the BM-induced HSC content and differentially altered niche-related gene expression profile in vivo. Intriguingly, treatments with SOCE inhibitors SKF and 2-APB boosted the mouse BM mesenchymal stem cell (MSC) and human adipose-derived MSCs proliferation, whereas they did not affect the endothelial cell proliferation. These findings suggest that temporal modulation of calcium sensing is crucial in expansion and maintenance of murine HSCs, human HSCs, and mouse BM-MSCs function.     

Regulation of hematopoietic and leukemic stem cells by the immune system

Hematopoietic stem cells (HSCs) are rare, multipotent cells that generate via progenitor and precursor cells of all blood lineages. Similar to normal hematopoiesis, leukemia is also hierarchically organized and a subpopulation of leukemic cells, the leukemic stem cells (LSCs), is responsible for disease initiation and maintenance and gives rise to more differentiated malignant cells. Although genetically abnormal, LSCs share many characteristics with normal HSCs, including quiescence, multipotency and self-renewal. Normal HSCs reside in a specialized microenvironment in the bone marrow (BM), the so-called HSC niche that crucially regulates HSC survival and function. Many cell types including osteoblastic, perivascular, endothelial and mesenchymal cells contribute to the HSC niche. In addition, the BM functions as primary and secondary lymphoid organ and hosts various mature immune cell types, including T and B cells, dendritic cells and macrophages that contribute to the HSC niche. Signals derived from the HSC niche are necessary to regulate demand-adapted responses of HSCs and progenitor cells after BM stress or during infection. LSCs occupy similar niches and depend on signals from the BM microenvironment. However, in addition to the cell types that constitute the HSC niche during homeostasis, in leukemia the BM is infiltrated by activated leukemia-specific immune cells. Leukemic cells express different antigens that are able to activate CD4⁺ and CD8⁺ T cells. It is well documented that activated T cells can contribute to the control of leukemic cells and it was hoped that these cells may be able to target and eliminate the therapy-resistant LSCs. However, the actual interaction of leukemia-specific T cells with LSCs remains ill-defined. Paradoxically, many immune mechanisms that evolved to activate emergency hematopoiesis during infection may actually contribute to the expansion and differentiation of LSCs, promoting leukemia progression. In this review, we summarize mechanisms by which the immune system regulates HSCs and LSCs.     

Megakaryocytes are essential for HSC quiescence through the production of thrombopoietin

Tissue homeostasis demands regulatory feedback, suggesting that hematopoietic stem cell (HSC) activity is controlled in part by HSC progeny. Yet, cell extrinsic HSC regulation has been well characterized only in niche cells of non-hematopoietic origin. Here we identify feedback regulation of HSCs by megakaryocytes (Mks), which are mature hematopoietic cells, through production of thrombopoietin (Thpo), a cytokine pertinent for HSC maintenance. Induced ablation of Mk cell population in mice perturbed quiescent HSCs in bone marrow (BM). The ablation of Mks resulted in decreased intra-BM Thpo concentration presumably due to Thpo production by Mks. Thpo administration Mk ablated mice restored HSC functions. Overall, our study establishes Mk as an essential cellular component of the HSC niche and delineates cytokine-oriented regulation of HSCs by their own progeny.     

Niche-to-niche migration of bone-marrow-derived cells

During ontogenesis, haematopoietic stem cells (HSCs) relocate between extra-embryonic and embryonic compartments. Similarly, site-specific homing of HSCs is ongoing during adulthood. With the expanding knowledge of HSC physiology, a new paradigm emerges in which HSCs and haematopoietic progenitor cells (HPCs) migrate to defined microenvironments within the bone marrow (BM) and to 'activated' or 'inducible' niches elsewhere. Here, we summarize current understanding of HSC niche characteristics, and the physiological and pathological mechanisms that guide HSC homing both within the BM and to distant niches in the periphery, promoting new vessel growth in tumours and ischaemia. Recent observations suggest that features of the HSC niche might also be recapitulated in pre-metastatic sites. Clusters of BM-derived HPCs promote invasion of disseminating cancer cells. Clear clinical benefits can be foreseen by modulating HSCs and their microenvironments, in promoting tissue regeneration, and inhibiting tumourigenesis and cancer metastasis.     

Fetal liver: an ideal niche for hematopoietic stem cell expansion

Fetal liver (FL) is an intricate and highly vascularized hematopoietic organ, which can support the extensive expansion of hematopoietic stem cells (HSCs) without loss of stemness, as well as of the downstream lineages of HSCs. This powerful function of FL largely benefits from the niche (or microenvironment), which provides a residence for HSC expansion. Numerous studies have demonstrated that the FL niche consists of heterogeneous cell populations that associate with HSCs spatially and regulate HSCs functionally. At the molecular level, a complex of cell extrinsic and intrinsic signaling network within the FL niche cells maintains HSC expansion. Here, we summarize recent studies on the analysis of the FL HSCs and their niche, and specifically on the molecular regulatory network for HSC expansion. Based on these studies, we hypothesize a strategy to obtain a large number of functional HSCs via 3D reconstruction of FL organoid ex vivo for clinical treatment in the future.     

Regulation of hematopoiesis and the hematopoietic stem cell niche by Wnt signaling pathways

Hematopoietic stem cells （HSCs） are a rare population of cells that are responsible for life-long generation of blood cells of all lineages. In order to maintain their numbers, HSCs must establish a balance between the opposing cell fates of self-renewal （in which the ability to function as HSCs is retained） and initiation of hematopoietic differentiation. Multiple signaling pathways have been implicated in the regulation of HSC cell fate. One such set of pathways are those activated by the Wnt family of ligands. Wnt signaling pathways play a crucial role during embryogenesis and deregulation of these pathways has been implicated in the formation of solid tumors. Wnt signaling also plays a role in the regulation of stem cells from multiple tissues, such as embryonic, epidermal, and intestinal stem cells. However, the function of Wnt signaling in HSC biology is still controversial. In this review, we will discuss the basic characteristics of the adult HSC and its regulatory microenvironment, the ＂niche＂, focusing on the regulation of the HSC and its niche by the Wnt signaling pathways.