Defining the hematopoietic stem cell niche: the chicken and the egg conundrum

Understanding the in vivo regulation of hematopoietic stem cells (HSCs) will be critical to identifying key factors involved in the regulation of HSC self‐renewal and differentiation. The niche (microenvironment) in which HSCs reside has recently regained attention accompanied by a dramatic increase in the understanding of the cellular constituents of the bone marrow HSC niche. The use of sophisticated genetic models allowing modulation of specific lineages has demonstrated roles for mesenchymal‐derived elements such as osteoblasts and adipocytes, vasculature, nerves, and a range of hematopoietic progeny of the HSC as being participants in the regulation of the bone marrow microenvironment. Whilst providing significant insight into the cellular composition of the niche, is it possible to manipulate any given cell lineage in vivo without impacting, knowingly or unknowingly, on those that remain? J. Cell. Biochem. 112: 1486–1490, 2011. © 2011 Wiley‐Liss, Inc.     

An EGFR/Ebi/Sno pathway promotes delta expression by inactivating Su(H)/SMRTER repression during inductive notch signaling

Stem cells within the bone marrow (BM) exist in a quiescent state or are instructed to differentiate and mobilize to circulation following specific signals. Matrix metalloproteinase-9 (MMP-9), induced in BM cells, releases soluble Kit-ligand (sKitL), permitting the transfer of endothelial and hematopoietic stem cells (HSCs) from the quiescent to proliferative niche. BM ablation induces SDF-1, which upregulates MMP-9 expression, and causes shedding of sKitL and recruitment of c-Kit+ stem/progenitors. In MMP-9-/- mice, release of sKitL and HSC motility are impaired, resulting in failure of hematopoietic recovery and increased mortality, while exogenous sKitL restores hematopoiesis and survival after BM ablation. Release of sKitL by MMP-9 enables BM repopulating cells to translocate to a permissive vascular niche favoring differentiation and reconstitution of the stem/progenitor cell pool.     

Disseminated prostate cancer cells can instruct hematopoietic stem and progenitor cells to regulate bone phenotype

Prostate cancer metastases and hematopoietic stem cells (HSC) frequently home to the bone marrow, where they compete to occupy the same HSC niche. We have also shown that under conditions of hematopoietic stress, HSCs secrete the bone morphogenetic proteins (BMP)-2 and BMP-6 that drives osteoblastic differentiation from mesenchymal precursors. As it is not known, we examined whether metastatic prostate cancer cells can alter regulation of normal bone formation by HSCs and hematopoietic progenitor cells (HPC). HSC/HPCs isolated from mice bearing nonmetastatic and metastatic tumor cells were isolated and their ability to influence osteoblastic and osteoclastic differentiation was evaluated. When the animals were inoculated with the LNCaP C4-2B cell line, which produces mixed osteoblastic and osteolytic lesions in bone, HPCs, but not HSCs, were able to induced stromal cells to differentiate down an osteoblastic phenotype. Part of the mechanism responsible for this activity was the production of BMP-2. On the other hand, when the animals were implanted with PC3 cells that exhibits predominantly osteolytic lesions in bone, HSCs derived from these animals were capable of directly differentiating into tartrate-resistant acid phosphatase-positive osteoclasts through an interleukin-6-mediated pathway. These studies for the first time identify HSC/HPCs as novel targets for future therapy involved in the bone abnormalities of prostate cancer.     

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.     

Bone Marrow Osteoblastic Niche: A New Model to Study Physiological Regulation of Megakaryopoiesis

Background

The mechanism by which megakaryocytes (Mks) proliferate, differentiate, and release platelets into circulation are not well understood. Growing evidence indicates that a complex regulatory mechanism, involving cellular interactions, composition of the extracellular matrix and physical parameters such as oxygen tension, may contribute to the quiescent or permissive microenvironment related to Mk differentiation and maturation within the bone marrow.

Methodology/Principal Findings

Differentiating human mesenchymal stem cells (hMSCs) into osteoblasts (hOSTs), we established an in vitro model for the osteoblastic niche. We demonstrated for the first time that the combination of HSCs, Mks and hypoxia sustain and promote bone formation by increasing type I collagen release from hOSTs and enhancing its fibrillar organization, as revealed by second harmonic generation microscopy. Through co-culture, we demonstrated that direct cell-cell contact modulates Mk maturation and differentiation. In particular we showed that low oxygen tension and direct interaction of hematopoietic stem cells (HSCs) with hOSTs inhibits Mk maturation and proplatelet formation (PPF). This regulatory mechanism was dependent on the fibrillar structure of type I collagen released by hOSTs and on the resulting engagement of the alpha2beta1 integrin. In contrast, normoxic conditions and the direct interaction of HSCs with undifferentiated hMSCs promoted Mk maturation and PPF, through a mechanism involving the VCAM-1 pathway.

Conclusions/Significance

By combining cellular, physical and biochemical parameters, we mimicked an in vitro model of the osteoblastic niche that provides a physiological quiescent microenvironment where Mk differentiation and PPF are prevented. These findings serve as an important step in developing suitable in vitro systems to use for the study and manipulation of Mk differentiation and maturation in both normal and diseased states.     

Young donor hematopoietic stem cells revitalize aged or damaged bone marrow niche by transdifferentiating into functional niche cells

The bone marrow niche maintains hematopoietic stem cell (HSC) homeostasis and declines in function in the physiologically aging population and in patients with hematological malignancies. A fundamental question is now whether and how HSCs are able to renew or repair their niche. Here, we show that disabling HSCs based on disrupting autophagy accelerated niche aging in mice, whereas transplantation of young, but not aged or impaired, donor HSCs normalized niche cell populations and restored niche factors in host mice carrying an artificially harassed niche and in physiologically aged host mice, as well as in leukemia patients. Mechanistically, HSCs, identified using a donor lineage fluorescence-tracing system, transdifferentiate in an autophagy-dependent manner into functional niche cells in the host that include mesenchymal stromal cells and endothelial cells, previously regarded as “nonhematopoietic” sources. Our findings thus identify young donor HSCs as a primary parental source of the niche, thereby suggesting a clinical solution to revitalizing aged or damaged bone marrow hematopoietic niche.     

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 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.     

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.     

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.     

Latexin regulates the abundance of multiple cellular proteins in hematopoietic stem cells

Latexin is the only known carboxypeptidase A inhibitor in mammals and shares structural similarity with cystatin C, suggesting that latexin regulates the abundance of as yet unidentified target proteins. A forward genetic approach revealed that latexin is involved in homeostasis of hematopoietic stem cells (HSCs) in mice; however, little is known about the mechanisms by which latexin negatively affects the numbers of HSCs. In this study, we found that latexin is preferentially expressed in hematopoietic stem/progenitor cells, and is co-localized with the molecules responsible for the interaction of HSCs with a bone marrow niche, such as N-cadherin, Tie2, and Roundabout 4. Latexin-knockout young female mice showed an increase in the numbers of KSL (c-Kit(+)/Sca-1(+)/linegae marker-negative) cells, which may be attributable to enhanced self-renewal because latexin-deficient KSL cells formed more colonies than their wild-type counterparts in methylcellulose culture. Proteomic analysis of Sca-1(+) bone marrow cells demonstrated that latexin ablation reduced the abundance of multiple cellular proteins, including N-cadherin, Tie2, and Roundabout 4. Finally, we found that latexin expression was lost or greatly reduced in approximately 50% of human leukemia/lymphoma cell lines. These results imply that latexin inhibits the self-renewal of HSCs by facilitating the lodgment of HSCs within a bone marrow niche to maintain HSC homeostasis.     

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.     

Blood cells need glia, too: a new role for the nervous system in the bone marrow niche

Regulation of hematopoietic stem cell (HSC) dormancy by specific cell types in the hematopoietic niche remains poorly understood. Yamazaki et al. (2011) now report that nerve-associated nonmyelinating Schwann cells activate TGF-β to maintain dormant HSCs, suggesting that glia are novel players in the bone marrow niche.     

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.     

Role of N-cadherin in the regulation of hematopoietic stem cells in the bone marrow niche

Cell-cell and cell-extracellular matrix interactions between hematopoietic stem cells (HSCs) and their niches are critical for the maintenance of stem cell properties. Here, it is demonstrated that a cell adhesion molecule, N-cadherin, is expressed in hematopoietic stem/progenitor cells (HSPCs) and plays a critical role in the regulation of HSPC engraftment. Furthermore, overexpression of N-cadherin in HSCs promoted quiescence and preserved HSC activity during serial bone marrow (BM) transplantation (BMT). Inhibition of N-cadherin by the transduction of N-cadherin short hairpin (sh) RNA (shN-cad) reduced the lodgment of donor HSCs to the endosteal surface, resulting in a significant reduction in long-term engraftment. shN-cad-transduced cells were maintained in the spleen for six months after BMT, indicating that N-cadherin expression in HSCs is specifically required in the BM. These findings suggest that N-cadherin-mediated cell adhesion is functionally essential for the regulation of HSPC activities in the BM niche.     

Bone marrow niche-inspired,multiphase expansion of megakaryocytic progenitors with high polyploidization potential

Background aimsMegakaryopoiesis encompasses hematopoietic stem and progenitor cell (HSPC) commitment to the megakaryocytic cell (Mk) lineage, expansion of Mk progenitors and mature Mks, polyploidization and platelet release. pH and pO₂ increase from the endosteum to sinuses, and different cytokines are important for various stages of differentiation. We hypothesized that mimicking the changing conditions during Mk differentiation in the bone marrow would facilitate expansion of progenitors that could generate many high-ploidy Mks.MethodsCD34⁺ HSPCs were cultured at pH 7.2 and 5% O₂ with stem cell factor (SCF), thrombopoietin (Tpo) and all combinations of Interleukin (IL)-3, IL-6, IL-11 and Flt-3 ligand to promote Mk progenitor expansion. Cells cultured with selected cytokines were shifted to pH 7.4 and 20% O₂ to generate mature Mks, and treated with nicotinamide (NIC) to enhance polyploidization.ResultsUsing Tpo + SCF + IL-3 + IL-11, we obtained 3.5 CD34⁺ CD41⁺ Mk progenitors per input HSPC, while increasing purity from 1% to 17%. Cytokine cocktails with IL-3 yielded more progenitors and mature Mks, although the purities were lower. Mk production was much greater at higher pH and pO₂. Although fewer progenitors were present, shifting to 20% O₂/pH 7.4 at day 5 (versus days 7 or 9) yielded the greatest mature Mk production, 14 per input HSPC. NIC more than doubled the percentage of high-ploidy Mks to 40%.ConclusionsWe obtained extensive Mk progenitor expansion, while ensuring that the progenitors could produce high-ploidy Mks. We anticipate that subsequent optimization of cytokines for mature Mk production and delayed NIC addition will greatly increase high-ploidy Mk production.     

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.