Complexity of bone marrow hematopoietic stem cell niche
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Hematopoietic stem cells (HSCs) that produce a variety of hematopoietic lineage cells throughout the life reside in specialized microenvironment called “niche” in the bone marrow (BM) where they are tightly regulated. With the recent advances in experimental technologies enabling the selective deletion of molecules, various types of cells in the BM have been proposed to contribute to HSC niche activity. Among these are stromal cells closely associated with the vasculature. In this review, we provide an overview of recent advances in HSC niche research, and focus on the studies describing the functional roles of perivascular cells for HSC maintenance and mobilization. Not only for physiologic state, we also discuss the recent evidences suggesting the importance of microenvironment for emergence of malignant hematopoietic diseases.
KeywordsHematopoietic stem cell Niche Perivascular niche Niche factor
Cellular components of the HSC niche
After birth, the hematopoietic system develops along with bone formation in mammals. Initial in vitro studies have revealed that osteoblasts can promote the expansion of hematopoietic progenitor cells in culture [2, 3]. Studies in which fluorescently labeled lineage marker-negative progenitor cells colonized in the vicinity of endosteal region after intravenous injection, further suggested that osteolineage cells had an ability to support hematopoietic stem/progenitor cell (HSPC) function . In 2003, two groups reported independently that the activation of osteolineage cells by pharmacologic or genetic manipulation increased HSC number in the bone marrow, which suggested an important role for osteolineage cells in HSC maintenance [5, 6]. An adhesion molecule, N-cadherin, has been proposed to promote HSC quiescence via homophilic or heterophilic interactions between HSCs and osteoblasts . However, conditional deletion of N-cadherin from osteolineage cells or from the hematopoietic compartment has not revealed defects in HSC maintenance [7, 8, 9]. N-cadherin appears dispensable for steady-state maintenance but may contribute under other conditions since the overexpression of N-cadherin in HSCs induced their quiescence . Osteopontin, mainly produced by mature osteoblast, negatively regulates the HSC pool . Recent genetic studies have questioned the contribution of osteoblast to HSC maintenance. Deletion of major niche factors, C-X-C motif ligand 12 (CXCL12) or stem cell factor (SCF), from mature osteoblasts or osteoblastic progenitor cells using Col.2.3-Cre, osteocalcin-Cre, or osterix (Osx)-Cre mice led to no reduction of HSCs in BM, which indicated that the osteolineage cells do not contribute to the maintenance of HSCs at least by producing these niche factors [12, 13, 14]. Three-dimensional bone marrow imaging studies analyzing the special relationship between phenotypic HSCs and bone marrow structures showed that HSCs do not associate with osteoblasts [15, 16].
Perivascular area as a niche complex
Since the BM extensively produces hematopoietic cells, it is highly vascularized to supply oxygen and nutrients. The nutrient arteries go into BM-penetrating compact bone through bone canal, and then give off branches into small arterioles. Arterioles transit to venous sinusoids in close proximity to endosteum, the interface between bone marrow and bone surface. Venous sinusoids compose a complex network in the bone marrow cavity and mature blood cells differentiated from progenitor cells leave the BM to systemic circulation through the sinusoids. Although initial studies in which fluorescently labeled hematopoietic progenitor cells were transplanted into irradiated mice suggested that HSPCs were colonized in the proximity to endosteal area , subsequent studies suggested that endogenous HSCs defined by phenotypic surface makers located close to BM sinusoids . The finding that perivascular cells expressed high levels of major niche factors and were significantly associated with HSCs has attracted extensive attention as a key component for HSC niche [18, 19].
Peri-sinusoidal stromal cell
Cells differentiated from mesenchymal origin that are broadly distributed through BM, mainly associated with sinusoids, and have been suggested as an important player in HSC niche. These cells include CXCL12-abundant reticular (CAR) cells , the cells marked by the green fluorescent protein (GFP) under the regulatory elements of the nestin promoter (Nes-GFP+) , leptin receptor (LepR)-expressing cells , and the stromal cells targeted by Cre promoted by transcription factor Osx  or paired related homeobox-1 (Prx-1) [12, 14]. These stromal cells have been shown to have high (~90%) redundancy.
CAR cells expressing the high amount of CXCL12 and SCF mainly distributed around sinusoids and form a homogenous tangled network in the bone marrow . The depletion of CAR cells using CXCL12-diphteria toxin receptor (DTR) mice resulted in HSC reduction in the bone marrow . As the depletion of CAR cells not only depletes CXCL12 but also SCF produced by CAR cells, it may not mean that CXCL12 produced by CAR cells is required for HSC maintenance in the bone marrow. The stromal cells expressing LepR, the receptor for leptin, a hormone secreted by adipocytes, express high levels of Cxcl12 and Kitl genes . Conditional deletion of Cxcl12 from LepR-Cre-marked cells mobilized HSCs from BM to peripheral blood and spleen, but had no effect on HSC number in the BM. Given the fact that LepR+ stromal cells largely overlap with CAR cells, CXCL12 from CAR cells or LepR+ stromal cells around sinusoids regulates mobilizing pool of HSCs but is not required for maintenance . By contrast, Scf deletion from LepR-Cre-marked cells depleted HSCs in the BM, showing that LepR-Cre-marked stromal cells are essential sources of SCF indispensable for HSC maintenance . Bone marrow stromal cells marked by Osx-Cre are also associated with sinusoid and exclusively include CAR cells. As is the case with Cxcl12 deletion in LepR-Cre-marked cells, Cxcl12 deletion from Osx-Cre-marked cells showed HSPC mobilization, but no HSC reduction in the BM . Nes-GFP+ stromal cells have been identified as a significant niche player in the BM . These cells express high levels of niche factor genes, Cxcl12 and Scf. We have recently reported that neural/glial antigen 2 (NG2)-Cre-marked stromal cells exclusively overlap with Nes-GFP+ stromal cells in adult BM. Deletion of CXCL12 or SCF from NG2-Cre-marked cells results in a marked reduction of HSCs in the BM and also in the mobilization of HSC to extramedullary organs. These results suggest that stromal cells within the Nes-GFP+ population are a critical source of niche factors essential for HSC maintenance .
Peri-arteriolar stromal cell
Imaging analysis of whole-mount bone marrow revealed that Nes-GFP+ stromal cells can be divided into two distinct types according to the expression levels of GFP: Nes-GFPdim that closely associate with sinusoids and highly overlap with LepR+ and CAR cells and Nes-GFPbright stromal cells that locate around arterioles and express NG2. Detailed analysis of three-dimensional relationships between phenotypic CD150+ CD48− CD41− Lineage− HSCs and bone marrow structures uncovered that a significant subset (~35%) of HSCs exists significantly closer to arterioles . The role of peri-arteriolar stromal cells in HSC quiescence was suggested by a significant alteration of distance between HSC and arterioles following recovery after myelosuppression, polyinosinic:polycytidylic acid (pIpC), or in Pml knockout mice, all of which lead to HSC proliferation and distribution away from arterioles .
Vascular endothelial cell
Endothelial cells (ECs) lining the bone marrow blood vessels have been reported as one of the niche components. ECs isolated from bone marrow are known to promote the proliferation and differentiation of human CD34+ progenitor cells in vitro . Blocking angiogenic activity of ECs by neutralizing vascular endothelial-cadherin (VE-cadherin) and vascular endothelial growth factor receptor-2 (VEGFR2) impaired supportive function of ECs to long-term HSCs . Despite the relatively small amount of niche factor expression, conditional deletion of niche factors, CXL12 or SCF, from endothelial cells led to the decrease of HSC number in bone marrow, suggesting that ECs play indispensable roles for HSC maintenance through producing these niche factors [13, 14]. ECs have also been shown to regulate HSC quiescence through surface molecule E-selectin expression. Deletion or blockade of E-selectin promoted HSC quiescence and increased survival through undefined mechanism . While the heterogeneity of EC populations remains largely unresolved, ECs with a high expression of CD31 (CD31hi) and endomucin (Emcnhi), referred to as type H endothelium, which are found in end-terminal arterioles connecting to sinusoids, expressed Kitl encoding SCF at higher levels than sinusoidal type L ECs . The specific contribution of endothelial cell subset will require further analyses with selective genetic deletion of SCF. A differential role of ECs is reflected by the difference of vascular permeability observed between sinusoids and arterioles which has repercussions on HSC egress and homing. Arterial vessels are less permeable and maintain HSCs in a low reactive oxygen species (ROS), keeping HSCs in a quiescent state. On the contrary, more leaky sinusoids expose HSCs to blood plasma and promote high level of ROS in HSCs, augmenting the ability of differentiation and migration .
Sympathetic nervous system
Bone marrow is highly innervated by various types of nerves, and it has been well known that hematopoiesis is controlled by the neural regulation . Sympathetic catecholamine signals suppress niche functions of Nes-GFP+ stromal cells through β3 adrenaline receptor, and regulate circadian release of HSCs from the BM . Sympathetic signals are also involved in the process of HSC mobilization from the niche cells induced by granulocyte colony-stimulating factor (G-CSF) [28, 30, 31]. Non-myelinating Schwann cells, wrapping the sympathetic nerves and traveling along arteries, have been reported to maintain HSC quiescence through activating transforming growth factor-β (TGF-β) .
One of the mature cell types, the macrophage, has been identified as important niche-modulating cells in the BM. Deletion of macrophages using macrophage-Fas-induced apoptosis transgenic mice or clodronate-loaded liposomes leads to HSPC mobilization into the blood accompanied with the reduction of niche factor-encoding genes . Independent studies have further established the roles of macrophage in HSC regulation that CD169+ macrophages promote the secretion of CXCL12 from Nes-GFP+ stromal cells, which led to the retention of HSCs in the niche . Selective overexpression of Csf3r in monocytes/macrophage in mice lacking Csf3r could rescue G-CSF-induced HSPC mobilization, suggesting an important role for Csf3r signaling in monocyte/macrophages . Thus, these results indicate that bone marrow macrophages participate in HSC regulation through bone marrow microenvironment.
Platelet-producing megakaryocytes (MKs) have been suggested to comprise one of the components of HSC niche. Three-dimensional whole-mount imaging analysis showed that a subset of HSCs specifically locates adjacent to MKs . Selective depletion of MKs led to a loss of quiescence of HSCs, and the injection of chemokine C-X-C motif ligand 4 (CXCL4) that is produced by MKs increased quiescence resulting in HSC reduction. Both Cxcl4 knockout mice and Cxcl4-Cre driving inducible DTR (iDTR) transgenic mice showed increased proliferation of HSCs . In another study conducted by Zhao et al., the ablation of MKs using Pf4-Cre driving iDTR mice led to an increased HSC number and proliferation accompanied with a reduction of TGF-β1 protein and nuclear-localized phosphorylated SMAD2/3 in HSCs . Nakamura-Ishizu et al. identified that MKs regulate HSC quiescence through producing thrombopoietin (TPO), crucial cytokine for HSC quiescence, mediated by the membrane protein C-type lectin-like receptor-2 (CLEC-2) signaling [38, 39].
In humans, active hematopoietic tissues in BM are progressively replaced by adipocytes with aging. Adipocytes have been considered as “space filler” for a long time; however, Naveiras et al. found that the number of HSCs is negatively correlated with the abundance of adipose tissue in the marrow and bone marrow recovery was accelerated after irradiation in lipoatrophic A-ZIP/F1 fatless mice or mice treated with peroxisome proliferator-activated receptor-γ (PPAR-γ) inhibitor bisphenol A diglycidyl ether (BADGE), which inhibits adipogenesis . A subsequent study showed that the inhibition of adipogenesis by BADGE treatment enhanced hematopoietic recovery after chemotherapy, suggesting the roles of adipocytes in hematopoietic cell regeneration . On the contrary, the enhanced adipogenesis by triglitazone treatment in vivo had no effect on HSC frequency in BM . Further studies will be needed to investigate the roles of adipocytes in HSC niche in steady state.
Differential cytokine contributions of peri-arteriolar and peri-sinusoidal stromal cells
A subset of endogenous HSCs locates significantly closer to arterioles and arteriole-associated NG2-expressing pericytes maintain HSC quiescence . The fact that significant alteration in HSC association with arterioles after 5-FU treatment, after the administration of pIpC, or in animals genetically deficient of Pml, all of which induce HSC proliferation, suggested the role of peri-arteriolar stromal cells in the regulation of HSC quiescence . However, other studies have argued that HSCs are randomly distributed in the BM and peri-arteriolar stromal cells do not contribute to HSC niche function . Another study using HoxB5-marked HSCs has also suggested a uniform distribution among bones but the relationship with vessels was not reported , and another recent report has argued for distinct vascular niche contributions . Thus, the location of endogenous HSCs and the functional differences between peri-arteriolar niche cells and peri-sinusoidal niche cells remained unclear [45, 46]. Functional deletion study of the niche factors in perivascular niche cells is necessary to define the mechanism by which each perivascular stromal cell type regulates HSC function.
We recently reported that cytokines from different perivascular niche cells differentially regulate HSCs in BM . Firstly, to evaluate the contributions of NG2+ peri-arteriolar cells, we analyzed constitutive NG2-Cre; Nes-GFP+; tdTomato mice and found that NG2-Cre exclusively targets the entire Nes-GFP+ stromal cells (97%) including both Nes-GFPdim cells that are broadly distributed around sinusoids and Nes-GFPbright cells that are tightly associated with arterioles. Consistent with the high overlap with Nes-GFP+ cells, a high proportion (89%) of NG2-Cre-marked cells expressed LepR and peri-arteriolar NG2-expressing pericytes were also labeled by NG2-Cre, suggesting that NG2-Cre targets both Nes-GFPdim LepR+ peri-sinusoidal stromal cells and NG2-expressing peri-arteriolar stromal cells.
Deletion of CXCL12 or SCF from NG2-Cre-marked cells led to dramatic reductions of HSCs in BM, confirming that Nes-GFP+ stromal cells are important sources of these niche factors for HSC maintenance. To delineate the function of niche factors secreted from distinct types of perivascular niche cells, we utilized NG2-CreERTM or Myh11-CreERT2, or LepR-Cre to delete CXCL12 or SCF in peri-arteriolar stromal cells or peri-sinusoidal stromal cells, respectively. While CXCL12 deletion from peri-arteriolar niche cells (NG2-CreERTM; Cxcl12 flox/− and Myh11-CreERT2; Cxcl12 flox/− mice) led to a significant reduction of HSC number and alteration of HSC location from arterioles in BM, LepR-Cre; Cxcl12 flox/− mice in which CXCL12 was deleted from peri-sinusoidal niche cells showed no significant changes in HSC number nor HSC distribution from arterioles in BM but mobilization to spleen and blood. These data thus suggest that CXCL12 from peri-arteriolar plays a predominant role in HSC maintenance. On the other hand, SCF deletion in peri-sinusoidal but not peri-arteriolar niche cells impaired HSC maintenance in BM. Of interest, despite dramatic reduction of HSCs in LepR-Cre; Scf flox/− or NG2-Cre; Scf flox/− mice, HSC location from arterioles was not altered in these Scf-depleted mice. These results are consistent with the previous report showing that peri-sinusoidal niche cells are the main source of SCF essential for HSC maintenance . It remains unclear whether CXCL12 from peri-arteriolar niche cells directly controls HSCs or not. Since the major function of CXCL12 is to mediate HSC migration and retention to the niche cells , it is possible that CXCL12 from peri-arteriolar niche cells anchor HSCs in a specific microenvironment in the vicinity of arterioles. In the context of direct interaction between HSCs and niche cells, LepR+ peri-sinusoidal or NG2+ stromal cells express high levels of VCAM-1, a key adhesion molecule for HSPC homing. VCAM-1 may regulate the mobilizable HSC populations .
Role of LSC niche in initiation and progression of hematological malignancies
In the opposite direction of the notion above that hematological malignancies induce changes of HSC niche, the finding that leukemia patients undergoing allogeneic stem cell transplantation can develop donor-derived leukemia  hints at an idea that an abnormality in HSC niche could directly initiate hematologic diseases. A proof-of-concept experiment shows that while constitutive deletion of retinoic acid receptor gamma (Rarγ) gene in mice results in the development of MPN-like disease, only transplantation of normal hematopoietic cells into Rarγ-deficient mice, and not transplantation of Rarγ-deficient hematopoietic cells into wild-type mice, was able to reconstitute this disease . Along the same lines, genetic deletion of the miRNA processing Dicer1 in osteoprogenitors  and expression of stabilized nuclear β-catenin in OBCs  were reported to drive the development of MDS- and AML-like disease, respectively. Taken together, these findings indicate that genetic lesion in bone marrow microenvironment indeed has the potential to promote hematologic diseases (Fig. 3b).
These two modes of relationships between LSCs and their niche are not mutually exclusive and they can synergize to predispose organisms to more aggressive diseases by forming a self-reinforcing loop. Together with an evidence that stromal cell populations isolated from individuals with myeloid malignancies can harbor genetic abnormalities that are different from mutations in leukemic clones , LSC niche should be added to the target of treatment for hematological malignancies. At least two strategies can be envisioned to implement such notion: (1) detachment of LSCs from their niche and (2) targeting MSC remodeling and/or inflammation in the microenvironment. The efficacy of CXCR4 inhibitor AMD3100 (plerixafor) was already tested in AML patients in a non-randomized phase I/II study, which shows that the rate of overall complete remission or complete remission with incomplete blood count recovery was 46% in patients treated with the combination of this drug and the standard chemotherapy, indicating that this approach is feasible in AML . Regarding the second strategy, there are currently no FDA-approved drugs that are able to directly target MSC remodeling. Considering that several cytokines including tumor necrosis factor α, IL-1, and IL-6 are involved in niche remodeling , it will be exciting to test the effect of blocking these cytokines with the use of antibodies that are available in the clinics.
Recent advances of experimental technologies including genetically modified mice and imaging techniques have enabled researchers to investigate the impact of specific deletion from various cell types on endogenous HSCs and their niche. Since HSCs must be conserved for the entire lifetime, the mechanisms by which niche cells regulate HSCs should be diverse and complex. While intensive studies have been conducted, the location of endogenous HSCs and the exact role of perivascular cells and other niche cells remain controversial. Further studies clarifying the detailed character of each type of niche cells will provide deeper understandings of the complexity and lead to new methods to cure hematologic disorders.
This work was supported by R01 Grants from the National Institute of Health (NIH) (DK056638, HL116340, HL097819 to P.S.F.). N.A. and S.T. are supported by JSPS Postdoctoral Fellowships for Research Abroad.
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Conflict of interest
The authors declare that they have no conflict of interest.