All lineages of blood cells are generated from hematopoietic stem cells (HSCs) in the bone marrow. It has been assumed that the special microenvironments known as niches in the marrow play an essential role in maintaining hematopoietic stem and progenitor cells (HSPCs) to provide appropriate numbers of mature blood cells throughout life [1–6]. HSPC niches are microenvironments that are required for the maintenance of HSPCs, which are in contact with these microenvironments, and the identity of the HSPC niche has been a subject of long-standing debate. Bone marrow cells are morphologically and functionally heterogeneous, and efforts over the past decade have focused on determining the nature and functions of nonhematopoietic cells, which are involved in HSC maintenance. We will review the recent studies on candidates for cells, which constitute a niche for HSPCs in bone marrow, and the critical transcriptional regulators of formation and maintenance of HSPC niches.
N-CADHERIN+CD45- OSTEOBLASTIC CELLS/OSTEOBLASTS
Calvi et al. reported that PTH/PTHrP receptor-expressing osteoblasts were involved in regulation of HSCs. On the contrary, Zhang et al. have defined BrdU-label-retaining cells after 10 days BrdU administration and 70 days chase as HSCs and revealed that these cells are in contact with a population of osteoblasts lining the bone surface, termed spindle-shaped N-cadherin+CD45- osteoblastic cells, which express a high level of N-cadherin (endosteal niches). However, it has been shown that most of these BrdU-label-retaining cells are not CD150+CD48-CD41-Lin-Sca-1+c-kit+ phenotypic HSCs and most phenotypic HSCs are not included in these BrdU-label-retaining cells  and that only rare phenotypic HSCs are associated with the bone surface . Furthermore, cytokines, including CXC chemokine ligand (CXCL) 12 and stem cell factor (SCF), which are essential for HSPC maintenance [4,11,12,13▪,14▪], and N-cadherin [15,16] produced by osteoblasts are dispensable for HSC maintenance. These results argue against the functional relevance of osteoblasts in HSC maintenance and suggest that osteoblasts play a role for a smaller number of HSCs than had been previously thought.
Kiel et al. have identified highly purified HSCs as CD150+CD48-CD41-Lin- cells and shown that about 60% of these phenotypic HSCs are associated with sinusoidal endothelium. Additionally, it has been shown that CXCL12 and SCF produced by Tie2+ endothelial cells are essential for HSC maintenance [12,13▪,14▪]. These studies indicate that endothelial cells create microenvironments meeting the criteria for an HSC niche.
CXCL12-ABUNDANT RETICULAR CELLS/STEM CELL FACTOR AND LEPR-EXPRESSING CELLS
CXCL12-abundant reticular (CAR) cells are identified as a population of reticular cells, expressing a high amount of CXCL12 using mice, in which the GFP gene was knocked into the CXCL12 locus (CXCL12-GFP mice) [11,18]. CAR cells in adult bone marrow express platelet-derived growth factor receptor β (PDGFRβ) and adipogenic and osteogenic transcription factors, including peroxisome proliferator-activated receptor γ (PPARγ), Runx2 and Osterix (Osx), but not Sca-1, and have the potential to differentiate into adipocytes and osteoblasts in vitro. Recent fate-mapping revealed that CAR cells/SCF and Lepr-expressing cells gave rise to most bone and adipocytes in adult bone marrow [19▪]. It has been shown that SCF-expressing cells specifically express leptin receptor (Lepr)  and that SCF and Lepr-expressing cells are largely equivalent to CAR cells [13▪,19▪]. Most of the HSCs are in contact with processes of CAR cells and short-term ablation of CAR cells in vivo using a diphtheria toxin receptor-mediated cell knockout technique led to a reduction in the numbers of cycling cells in the long-term repopulating HSC (LT-HSC) population and lymphoid and erythroid progenitors and impaired adipogenic and osteogenic differentiation potential of bone marrow cells . In addition, when SCF was conditionally deleted from CAR cells, the numbers of LT-HSCs were severely reduced in bone marrow . Together, CAR cells are adipo-osteogenic progenitors, which create microenvironments meeting the criteria for an HSPC niche (Fig. 1).
Nestin-GFP+NG2+ cells are found in contact with arteries at much lower frequencies than CAR cells in the bone marrow from transgenic mice in which GFP is expressed under the control of the neural-specific regulatory elements of nestin gene (Fig. 1) [20,21▪]. Nestin-GFP+NG2+ cells contain cells that form mesenchymal spheres and colony-forming units-fibroblasts [21▪]. Kunisaki et al.[21▪] reported that the mean distance to arteries (52 mm) was highly statistically different from that of randomly placed CD150+CD48-Lin- HSCs (78 mm) and that HSC numbers were reduced in the marrow after in-vivo depletion of NG2-expressing cells using a diphtheria toxin receptor-mediated cell knockout technique. On the basis of these results, Kunisaki et al. concluded that nestin-expressing cells were involved in HSC maintenance in the bone marrow; however, Zhou et al.[19▪] revealed that NG2 was also expressed in nonmyelinating schwann cells lining arteries and osteoblasts, raising the possibility that some periarteriolar Nestin-GFP+NG2+ cells overlap schwann cells, which have been reported to be involved in HSC maintenance .
It has been reported that PDGFRα+Sca-1+CD31-CD45-Ter119- (PαS) cells are located along arteries in bone marrow . PαS cells are distinct from CAR cells, which express PDGFRα but not Sca-1 . When CXCL12 was conditionally deleted from PαS cells, all CAR cells and osteoblasts, the numbers of LT-HSCs were severely reduced in bone marrow [14▪]. In contrast, when CXCL12 was conditionally deleted from the majority of CAR cells and osteoblasts, a modest decrease in LT-HSC number was observed. On the basis of these findings, Greenbaum et al.[14▪] reported that CXCL12 produced by PαS cells was involved in HSC maintenance. However, these findings do not rule out the possibility that CXCL12 production from a small subset of CAR cells contributes to HSC maintenance. Studies using mice, in which CXCL12 was conditionally deleted from PαS cells, will provide direct evidence for a role of PαS cells in HSC maintenance.
THE MOLECULAR BASIS OF HEMATOPOIETIC STEM AND PROGENITOR CELL NICHE DEVELOPMENT AND IDENTIFICATION OF CXCL12-ABUNDANT RETICULAR CELL PROGENITORS
Despite years of investigation on candidate niches for HSPCs, critical and specific transcriptional regulators of the development and maintenance of cells creating HSPC niches had remained unclear although critical intracellular regulators of the hematopoietic microenvironments have been shown (Table 1[24–28,29▪▪,30▪▪,31–33]).
To address this issue, progenitors of CAR cells, which create microenvironments meeting the criteria for HSPC niche, were analyzed [29▪▪]. During development, Osx-GFP+PDGFRβhi putative CAR cell progenitors were observed from E16.5 transgenic mice, in which expression of GFP reporter gene was placed under transcriptional regulation of the Osx promoter (Osx-GFP mice). In addition, CXCL12-GFP+ cells from newborn CXCL12-GFP mice expressed PDGFRβ, leptin receptor (Lepr), CXCL12 and SCF, which were preferentially expressed in CAR cells, but distinctly lower than adult CAR cells and thus were termed as primordial CAR cells [29▪▪].
OSX, EBF2 AND BMI1 ARE CRITICAL BUT NOT SPECIFIC TRANSCRIPTIONAL REGULATORS OF HEMATOPOIETIC STEM AND PROGENITOR CELL NICHE DEVELOPMENT
Osx contains a DNA-binding domain consisting of three zinc fingers that share sequence identity with motifs in Sp1, Sp3 and Sp4, and is expressed in perichondrial cells, CAR cell progenitors, primordial CAR cells, CAR cells and osteoblasts. Coşkun et al.[30▪▪] reported that Osx was essential for colonization of bone marrow by HSCs as well as bone formation during development, suggesting that primordial CAR cells require Osx for creating niches for HSC homing during ontogeny .
Ebf2 is a member of the early B cell factor (Ebf) family of transcription factors, which binds DNA through a zinc finger domain and a helix-loop-helix-like domain. The bone marrow of mice lacking Ebf2 had increased numbers of adipocytes and severely reduced numbers of functional and phenotypic CD34-CD150+Lin-Sca1+c-kit+(LSK) LT-HSCs, Lin-Sca1loc-kitloIL-7Rα+ common lymphoid progenitors (CLPs), B220+CD43+ pro-B cells and B220+CD43- B cells in the marrow as well as thymocytes . However, the numbers of c-kit+Sca-1-Lin-CD34-FcγRII/IIIlo megakaryocyte/erythrocyte progenitors and c-kit+Sca-1-Lin-CD34+FcγRII/IIIhi granulocyte/macrophage progenitors (GMPs) and the expression of CXCL12 in osteoblastic cells were normal in the marrow  and the expression of Ebf2 was very low in CAR cell progenitors or primordial CAR cells and not specific to these cells in developing bone marrow (Y. Omatsu and T. Nagasawa, unpublished observation).
The polycomb protein Bmi1 is a component of polycomb-repressive complex 1. The expression of Bmi1 was not specific to CAR cell progenitors or primordial CAR cells in the marrow (Y. Omatsu and T. Nagasawa, unpublished observation) and observed in hematopoietic cells, including HSCs . The bone marrow of mice lacking Bmi1 in nonhematopoietic cells had increased numbers of adipocytes, decreased numbers of osteoblasts and severely reduced numbers of phenotypic HSCs, CLPs, pro-B cells and pre-B cells [25,26].
FOXC1 IS THE CRITICAL AND SPECIFIC TRANSCRIPTIONAL REGULATOR OF THE DEVELOPMENT AND MAINTENANCE OF CELLS CREATING HEMATOPOIETIC STEM AND PROGENITOR CELL NICHES
In contrast to Osx, Ebf2 and Bmi1, the transcription factor Foxc1 is expressed preferentially in CAR cell progenitors or primordial CAR cells in the developing bone marrow [29▪▪]. Foxc1 protein belongs to a family of transcription factors characterized by the presence of a forkhead box (Fox) DNA-binding domain. Quantitative real-time polymerase chain reaction with reverse transcription analysis of adult bone marrow from CXCL12-GFP mice revealed that CAR cells expressed substantially higher levels of Foxc1 than other bone marrow populations, including osteoblasts, PαS cells and endothelial cells, as well as hematopoietic cells [29▪▪]. These results indicate that Foxc1 is expressed preferentially in CAR cells in the developing and adult bone marrow.
Foxc1 null mutants die perinatally with hemorrhagic hydrocephalus and calvarial defects . When Foxc1 was conditionally deleted from PαS cells, all CAR cells and osteoblasts, or most CAR cells, the numbers of functional and phenotypic CD34-CD150+CD48-LSK LT-HSCs and hematopoietic progenitors, including CD34+CD48-LSK short-term repopulating HSCs (ST-HSCs), Flt3+ LSK multipotent progenitors (MPPs), Lin-IL-7Rα+Flt3+ common lymphoid progenitors (Flt3+CLPs), c-kit+CD19+IgM- pro-B cells, c-kit-CD19+IgM- pre-B cells and c-kit+CD71+Ter119lo proerythroblasts and GMPs, were severely reduced in the 3-week-old bone marrow [29▪▪]. Histological analysis of 3-week-old bone marrow of Foxc1 conditionally deficient mice revealed that most marrow volumes were occupied by adipocytes with few hematopoietic cells and that CAR cells were severely reduced but osteoblasts lining the bone surface, cortical thickness of the femoral diaphysis and PαS cells appeared normal [29▪▪]. Residual CAR cells in Foxc1 conditionally deficient mice had lower levels of CXCL12 and SCF expression than control animals. Consistent with these, enforced expression of Foxc1 in sorted CAR cells increased mRNA expression of CXCL12 and SCF and markedly decreased their adipogenic potential in the culture [29▪▪].
After induction of Foxc1 deletion in adult mice, the numbers of LT-HSCs, ST-HSCs, MPPs, CLPs, pro-B cells, pre-B cells and proerythroblasts were severely reduced but normal numbers of CAR cells and only small numbers of adipocytes were observed in the bone marrow [29▪▪]. In addition, residual CAR cells had lower levels of CXCL12 and SCF expression in the mutants. These results indicate that Foxc1 is essential for CXCL12 and SCF expression in CAR cells and maintenance of HSPC niches in adult bone marrow (Fig. 2) [29▪▪]. Considering that Foxn1 is essential for the development of thymic epithelium, which supports T-cell production , Foxc1 might play an important role in creating HSPC niches by other types of cells in hematopoietic organs, and Fox proteins play critical but distinct roles in the formation of different hematopoietic microenvironments.
POSSIBLE INTERACTION BETWEEN FOXC1, OSX AND EBF2
The transcription factor Ebf2 has been suggested to be essential for the development of cells creating bone marrow niches for HSCs and lymphoid progenitors . In contrast to Foxc1, Ebf2 is dispensable for maintenance of erythroid and myeloid progenitors , and Ebf2 expression was not specific to CAR cells in developing bone marrow, raising the possibility that Ebf2 expression is enhanced by Foxc1 in CAR cells (Fig. 2).
Considering that Osx is expressed in perichondrial cells, CAR cell progenitors, primordial CAR cells, CAR cells and osteoblasts and is essential for both bone and HSC niche formation during ontogeny [30▪▪,34], it may be said that during ontogeny parts of Osx-expressing perichondrial cells begin to upregulate PDGFRβ and Foxc1 expression, becoming primordial CAR cells, and other Osx-expressing perichondrial cells increase Osx expression and become osteoblasts [29▪▪] (Fig. 2).
Omatsu and colleagues have clearly demonstrated that the transcription factor Foxc1 is expressed preferentially in CAR cells and is essential for CAR cell development and maintenance of bone marrow niches for HSCs and lymphoid, myeloid and erythroid progenitor cells upregulating CXCL12 and SCF expression and that Foxc1 inhibits adipogenic processes in CAR progenitors (Fig. 2). Among various kinds of cells, which have the potential to differentiate into adipocytes and osteoblasts, including muscle PDGFRα+Sca-1+CD31-CD45- cells [18,37], only CAR cells express high levels of Foxc1 and hematopoietic cytokines and create a niche for HSPCs and thus, Foxc1 is the first transcriptional regulator that is required for development and maintenance of cells creating HSPC niches, including a specialized population of adipo-osteogenic progenitors in bone marrow. Considering that Fox proteins can act as terminal effectors of major signal transduction pathways, extracellular cues may function to stimulate or inhibit the expression and activity of Foxc1 in CAR cells. Further investigation will be important to determine the signaling pathways acting upstream of Foxc1 and other critical transcriptional regulators in cells creating HSPC niches to control hematopoiesis during homeostasis and in response to bone-marrow stressors, including inflammation. Notch signaling and/or Wnt signaling might be involved in these processes [38,39]. It may be possible that these molecules can be used therapeutically to drive the formation of hematopoietic niches from nonniche cells, such as primary dermal fibroblasts or induced pluripotent stem-derived mesenchymal cells in vitro or from impaired marrow microenvironments in vivo. Studies on critical transcriptional regulators of HSPC niches provide considerable new insights into the biology and pathology of hematopoietic microenvironments.
Financial support and sponsorship
This work was supported by Japan Science and Technology Agency (JST), Core Research for Evolutional Science and Technology (CREST).
Conflicts of interest
There are no conflicts of interest.
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