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Hematopoietic Microenvironment
Evolution of the Niche Concept
In 1868, Ernest Neumann first suggested that blood cells are being replenished throughout postnatal life, and this proposal led to the attempts to localize the place of hematopoiesis. His hypothesis that blood cell production takes place in the bone marrow (BM) was experimentally validated by selective lead shielding of limbs in irradiated animals almost a century later. Notably, these and other studies showed that differentiation pathways of immature blood cells are determined by their location and are different between the spleen and the BM. Based on this difference between BM and spleen, Schofield first proposed that there is a specialized place or niche where stem cells reside and are governed. He succinctly posed in 1978 that “stem cell is seen in association with other cells which determine its behavior.”
Trentin further clarified how different sites affected hematopoietic stem/progenitor cell (HSPC) differentiation. Although both spleen and marrow support multiple cell lineages (erythropoietic and granulocytopoietic, for example), the ratios of differentiating cells were distinct—spleen favored erythropoiesis, but BM predominantly supported granulopoiesis. This controlling influence of the surrounding cells was further illustrated by implanting BM stroma into the spleen and showing that hematopoietic cells abruptly changed from erythropoiesis to granulopoiesis at the spleen–BM demarcation. These observations suggest that immature differentiating progenitors require interactions with specific other cell types in a defined micro environment.
This chapter reviews the current knowledge of the hematopoietic microenvironment during development and in postnatal life in normal hematopoiesis and in myelodysplasia and leukemia. The opportunities for therapeutic manipulation of the niche in the treatment of these disorders are also discussed. For the related topics on stem cell mobilization, hematopoietic cytokines and the role of microenvironment in lymphoid malignancies, plasma cell disorders, and myeloproliferative conditions, readers are referred to other chapters of this book.
Hematopoietic Microenvironment During Development
In mammals, hematopoiesis during development takes place in distinct extra-embryonic and embryonic sites. Sequentially, it moves from the yolk sac to the aorta-gonad-mesonephros (AGM) region, fetal liver, placenta, and BM (for details, see Chapter 8 ).
The first definitive adult HSPCs emerge from the floor of the dorsal aorta, more precisely from the AGM region in the midgestation mouse embryo, and the HSPC clusters appear in close association with the aortic endothelium. Phenotypically defined HSPCs (Sca1+ c-kit + CD41+) arise directly from ventral aortic endothelial cells largely triggered by shear and pulsatile pressure forces. Although direct cellular interactions during HSPCs emergence in the embryo remain to be dissected, bone morphogenetic protein 4 (BMP4), fibroblast growth factor (FGF), transforming growth factor (TGF), vascular endothelial growth factor (VEGF)-Flk1, YAP, and piezo signaling pathways are involved.
The placenta has also been identified as a hematopoietic organ during development. Placenta is known to produce hormones that influence vascularization and therefore may affect blood cell production because hematopoiesis and vasculogenesis are tightly coupled. The hematopoiesis-promoting factors may be either produced by the placental trophoblast cells or enter via maternal circulation. Hematopoietic progenitors appear in the placenta at E9, but their number declines by E13. The cells and local factors providing placental hematopoietic support are currently unknown, but mesenchymal/stromal cells have been suggested as candidates. The placental microenvironment is thought to be geared toward supporting the expansion or maturation of HSPCs without their concomitant differentiation.
In the fetal liver, the HSPCs are first detected on day 9 of mouse embryonic development and approximately week 7 in the human. The liver remains the predominant site of blood production until the BM is invested with cells at approximately 11 weeks post conception and becomes the major blood production organ at week 20. Stromal cell lines obtained from the fetal liver are able to support primitive hematopoietic cells in ex vivo cultures. Some of these cells (termed myelosupportive stroma ) are able to differentiate in vitro into mesenchymal components (osteoblasts, chondrocytes, and adipocytes). The fetal liver niche has been characterized as Nestin expressing periportal cells and hepatic stellate cells expressing stem cell factor (SCF).
Despite the differences in the hematopoietic microenvironment between the sites of fetal and adult hematopoiesis, the key components of the molecular milieu are likely to be shared, as evidenced by successful (although limited) engraftment of HSPCs across developmental barriers. For example, AGM- or fetal liver–derived HSPCs are able to engraft in the adult BM. Notably, they have a competitive advantage over their BM-derived counterparts, with the long-term repopulating ability exceeding that of the BM by fivefold. Vice versa, BM HSPCs engraft in the fetal liver when transplanted in utero, although at low efficiency (less than 5% for the whole BM and 0.43% for highly enriched HSPCs), which may be partly attributable to the absence of pre-transplant conditioning.
Multilineage hematopoiesis during development occurs largely by the virtue of sequential HSPC migration from the AGM region to the fetal liver and the BM, as opposed to de novo HSPC generation. Failure of migration to the “next niche,” as exemplified by the targeted disruption of the guanine-nucleotide–binding protein stimulatory α-subunit (GS-α), calcium-sensing receptor, or CXCL12/CXCR4 axis (discussed in detail in Chapter 11 on HSPC migration) leads to severe impairment in hematopoiesis. This suggests even in the absence of cell-intrinsic HSPC defects, proper progression of blood cell production throughout developmental critically depends on the ability of the HSPC to sequentially move to the appropriate microenvironmental compartments.
Adult Bone Marrow Microenvironment
Hematopoietic Stem Cell Niches
Location of the Hematopoietic Stem/Progenitor Cell Niche
In mammals, BM is a major site of hematopoiesis throughout life. The niche preserves and dynamically regulates the HSPC pool by providing signals required for maintenance, quiescence, and retention of HSPCs in the BM. However, the location of the HSPC niche within the marrow has been a subject of controversy. The endosteal surface has long been considered the zone in which HSPCs are preferentially located. In the setting of irradiation conditioning, this has been directly demonstrated by intravital imaging studies that allow dynamic assessment of the interaction between transplanted HSPC and the niche. Currently, in vivo imaging is limited to calvarial BM, an area in the mouse skull where the bone is very thin, thus permitting penetration of the laser beam into the BM cavity. Using this technique and simultaneous multicolor fluorescent labeling of osteolineage cells (OLCs), HSPCs, and the vasculature, studies showed that in irradiated recipients, transplanted HSPCs home closest to the endosteal surface and individual OLCs, as compared with more differentiated progenitors, and that they are “anchored” to their niches at least through 72 hours. Preferential localization of primitive hematopoietic cells to the endosteal surface under the homeostatic conditions has been also demonstrated, although this analysis was performed using immunostaining of histological BM sections of either femoral bones or the sternum.
However, other studies performed under steady-state (not transplant) conditions indicate that most HSPCs are located in the central marrow in a perivascular position. Vascular integrity is largely lost with transplant conditioning, so it is not surprising that there might be distinctive sites of hematopoiesis under homeostasis. The positioning of cells near vasculature is to some extent inevitable, as the marrow is richly endowed with sinusoidal vessels. Indeed, approximately 40% of the BM is vascular. Nonetheless, selective deletion of factors in primitive mesenchymal cells and endothelial cells provide evidence for them providing a regulatory and therefore, niche role. Positioning of HSPC in BM by detailed 3-D analysis has indicated that the cells distribute equivalently to random dots when quiescent. Only upon activation do they have distinctive localization near CXCL12 cells. Thus, the niche may be more macroarchitectural, with multiple cells contributing to the functional regulation of HSPC.
Functional roles for specific cells have been defined by the deletion of a key niche factor, such as SCF (kit-ligand), from either endothelial or perivascular cells. This was found to lead to decreases in HSPC providing an experimental basis for the notion of a perivascular niche. While the debate about the location of the HSPC niche—and consequently, cell types that serve as niche participants—continues, it is important to bear in mind that HSPCs themselves are molecularly and functionally heterogeneous and that several distinct niches may co-exist to support this heterogeneity, particularly under different conditions such as the stress of transplantation.
Cellular Components of the Hematopoietic Stem/Progenitor Cell Niche
Over recent years, animal studies revealed marked complexity in the cellular and molecular organization of the HSPC BM niche. Furthermore, single-cell analyses of stroma characterizing it by gene or protein expression has provided a complete inventory of the cell types present in the marrow and begun to define changes they undergo with stress. Major cellular components of the HSPC niche and the factors that they produce (summarized in Table 14.1 ) are discussed in the following sections.
Table 14.1
HSPC Niche Factors and Their Cellular Sources
Updated and modified from Kfoury Y, Mercier F, Scadden DT. Snapshot: the hematopoietic stem cell niche. Cell . 2014;3;158(1).
| Factor | Source | Effect and reference |
|---|---|---|
| Membrane-bound SCF | Lepr+ perivascular cells, Tie2 + endothelial cells | Maintenance of HSC in the bone marrow |
| CXCL12 | Lepr + perivascular cells, Tie-2 + endothelial cells | Retention of HSC in the bone marrow (Ding and Morrison, 2013) |
| Prx-1 + osteoprogenitors | Retention of HSC, myeloid and lymphoid progenitors in the bone marrow (Ding and Morrison, 2013; Greenbaum et al., 2013) | |
| Osx + osteoprogenitors and Col-2.3 + osteoblastic cells | Retention of lymphoid progenitors in the bone marrow (Ding and Morrison, 2013; Greenbaum et al., 2013) | |
| Notch signaling (Jagged1) | VE-cadherin + endothelial cells, osteoblastic cells (in PPR model) |
|
| Wnt signaling (Canonical) | Col-2.3 + osteoblastic cells (Wnt inhibitor DKK1) | Maintenance of HSC and quiescence (Fleming et al., 2008) |
| Wnt signaling (Noncanonical) | N-cadherin + osteoblastic cells | Maintenance of HSC and quiescence (Sugimura, 2012) |
| E-Selectin | CD-31 + endothelial cells | Promotion of HSC cycling (Winkler et al., 2012) |
| Pleiotrophin | VEGFR3 + /VE-cadherin + endothelial cells and CXCL12 perivascular cells | Maintenance of HSC and retention in the bone marrow (Himburg et al., 2010, 2012) |
| Thrombopoietin | Alkaline phosphatase + osteoblastic cells | Maintenance of HSC and quiescence (Qian et al., 2007; Yoshihara et al., 2007) |
| Osteopontin | Osteoblastic and other microenvironmental cells | Negative regulation of HSC numbers and maintenance of quiescence (Nilsson et al., 2005; Stier et al., 2005) |
| TGF-β | Nonmyelinating Schwann cells, megakaryocytes | HSC quiescence (Yamazaki et al., 2011) |
| Angiopoietin-1 | Osteocalcin + osteoblasts | Promotion of HSC quiescence (Arai et al., 2004) |
| Robo-4 ligand(s) (Slit2 a ) | Bone marrow stromal cells ( a Slit2 identified at the mRNA level) | Promotion of HSC homing to the bone marrow (Smith-Berdan et al., 2011, 2012) |
| Junction adhesion molecule B (JamB) | Osteoblasts, MSCs, endothelial cells | HSC adhesion and quiescence (Archangeli, 2011) |
| CXCL4 | Megakaryocytes | Promotion of HSC quiescence |
HSPC , Hematopoietic stem/progenitor cell; MSC , mesenchymal stem cell; TGF , transforming growth factor.
Osteolineage Cells
OLCs are a heterogeneous population of mesenchymal cells that line the endosteal surfaces of flat and trabeculated bones at the interface between the bone and the BM and become embedded within the bone matrix upon terminal differentiation. OLCs are thought to originate from mesenchymal stem cells (MSCs) and gradually progress from the early immature progenitors that express OLC-specific transcription factors Runx2 and osterix to mature osteoblasts expressing extracellular matrix protein osteocalcin and eventually to osteocytes.
Several lines of in vivo evidence support a functional role of the OLCs in the regulation of primitive hematopoietic cells. In the studies providing the first experimental evidence for a mammalian niche in vivo, mice with genetically modified OLCs had an increase in the number of activated OLCs and a corresponding increase in the number of HSPCs. This effect was associated with an increased trabecular bone area and an elevated number of trabecular osteoblasts that expressed the Notch ligand Jagged 1. When the OLCs were depleted, there was a reduction in the BM cellularity and migration of hematopoiesis to the extra-medullary sites. Thus, OLCs play a role in the regulation of HSPC pool size. In addition, OLCs participate in controlling HSPC quiescence through contributing to the production of CXCL12, Angiopoietin 1, thrombopoietin, pan-inhibitor of canonical Wnt signaling Dickkopf1 (Dkk1), noncanonical Wnt ligands, and ECM protein osteopontin. Finally, OLCs govern HSPC localization by controlling their egress into blood and return to the BM, a process that forms the basis for clinical peripheral blood stem cell collection for transplantation. When mature OLCs are deleted from bone, there is an increase in the number of circulating progenitors and a decrease in the mobilization of HSPC with G-CSF, indicating that OLC-derived signals retain primitive hematopoietic cells in the marrow. Following G-CSF-induced mobilization, the OLCs in the trabecular bone adapt a flattened morphology with short projections, which is associated with HSPC egress from the niche. Similar changes are seen after treatment with NSAIDs, which are known to enhance G-CSF-induced HSPC mobilization. When CXCL12, a major HSPC chemo-attractant and retention factor, is deleted from the osteoprogenitors using osterix-Cre promoter, increased HSPC mobilization is also observed. Thus, OLCs participate in the BM niche by regulating HSPC number, quiescence, and retention in the BM space.
Despite the evidence presented above, several studies have argued against the role of OLCs in the niche, citing the absence of HSPC changes either in genetic models associated with reduced OLC number or following OLC-specific deletion of HSPC regulators such as kit-ligand or CXCL12. Several experimental factors are likely to account for this discrepancy, including developmental adaptation when the genetic modification is present throughout the ontogeny or inability of genetic tools to target a specific subset within the OLC compartment that serves as a nodal point of the HSPC regulation: several studies suggest that immature OLCs are important for the niche function, while mature OLCs are dispensable. However, these latter cells appear to play a significant role in supporting lymphoid progenitors. They are critical for B-cell maturation, and osteocalcin-expressing cells regulate the production of T-competent progenitors (as shown in the following sections).
Endothelial Cells
Endothelial cells are known to secrete hematopoietic cytokines and express several adhesion molecules such as E-selectin, P-selectin, vascular cell adhesion molecule 1 (VCAM-1), and intercellular adhesion molecule 1 (ICAM-1) that have been shown to participate in cellular interactions within the HSPC niche. The existence of vascular niche for the HSPCs has been supported by in vivo imaging studies showing early homing of transplanted BM progenitors to specific subdomains of the vascular tree as well as by histologic assessment of the BM using CD150 antibody when HSPCs were found to be in close proximity to the BM sinusoids.
Endothelium-derived factors have diverse effects on HSPCs in vivo. For example, E-selectin (which is expressed exclusively in the endothelial cells) negatively regulates HSPC quiescence; consequently, HSPCs from E-selectin KO mice are more quiescent and resistant to irradiation. On the other hand, endothelial-specific deletion of SCF or CXCL12 leads to the respective reduction of the HSPC pool and loss of repopulating capacity. Similar changes are seen upon endothelial-specific deletion of Notch ligand Jagged 1, which in contrast to E-selectin, promotes HSPC quiescence. The heparin-binding growth factor, pleiotrophin, that is produced by sinusoidal endothelial cells also plays a role in the retention and self-renewal of HSPC in BM. It is not established that among endothelial subpopulations it is the arteriolar cells that secrete the predominant regulator of HSPC, SCF.
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