Perisinusoidal Niche Research Articles

Endosteal stem cells at the bone-blood interface: A double-edged sword for rapid bone formation: Bone marrow endosteal stem cells provide a robust source of bone-making osteoblasts both in normal and abnormal bone formation.

Endosteal stem cells are a subclass of bone marrow skeletal stem cell populations that are particularly important for rapid bone formation occurring in growth and regeneration. These stem cells are strategically located near the bone surface in a specialized microenvironment of the endosteal niche. These stem cells are abundant in young stages but eventually depleted and replaced by other stem cell types residing in a non-endosteal perisinusoidal niche. Single-cell molecular profiling and in vivo cell lineage analyses play key roles in discovering endosteal stem cells. Importantly, endosteal stem cells can transform into bone tumor-making cells when deleterious mutations occur in tumor suppressor genes. The emerging hypothesis is that osteoblast-chondrocyte transitional identities confer a special subset of endosteal stromal cells with stem cell-like properties, which may make them susceptible for tumorigenic transformation. Endosteal stem cells are likely to represent an important therapeutic target of bone diseases caused by aberrant bone formation.

Haematopoietic stem cells in perisinusoidal niches are protected from ageing

With ageing, intrinsic haematopoietic stem cell (HSC) activity decreases, resulting in impaired tissue homeostasis, reduced engraftment following transplantation and increased susceptibility to diseases. However, whether ageing also affects the HSC niche, and thereby impairs its capacity to support HSC function, is still widely debated. Here, by using in-vivo long-term label-retention assays we demonstrate that aged label-retaining HSCs, which are, in old mice, the most quiescent HSC subpopulation with the highest regenerative capacity and cellular polarity, reside predominantly in perisinusoidal niches. Furthermore, we demonstrate that sinusoidal niches are uniquely preserved in shape, morphology and number on ageing. Finally, we show that myeloablative chemotherapy can selectively disrupt aged sinusoidal niches in the long term, which is linked to the lack of recovery of endothelial Jag2 at sinusoids. Overall, our data characterize the functional alterations of the aged HSC niche and unveil that perisinusoidal niches are uniquely preserved and thereby protect HSCs from ageing.

1005 - THE IDENTIFICATION OF A PERI-ARTERIOLAR NICHE FOR LYMPHOID PROGENITORS AND OSTEOGENIC PROGENITORS IN THE BONE MARROW

Leptin Receptor-expressing (LepR+) stromal cells in adult bone marrow are highly enriched for skeletal stem cells and are the main source of osteoblasts and adipocytes in addition to SCF and Cxcl12 for HSC maintenance (Nature 481:457; Nature 495:231; Cell Stem Cell 15:154). We discovered that a subset of these cells produce a previously uncharacterized bone-forming growth factor, Clec11a/Osteolectin, which promotes the osteogenic differentiation of LepR+ cells and is required for the maintenance of adult skeletal bone mass (eLife 5:e18782). Osteolectin promotes osteogenic differentiation by binding to a11b1 integrin and promoting Wnt pathway activation (eLife 8:e42274). To test if Osteolectin expression could help resolve some of the heterogeneity among LepR+ cells in vivo, we generated a knock-in tdTomato reporter. We found that Osteolectin was exclusively expressed by peri-arteriolar LepR+ cells. The Osteolectin+ cells expressed osteogenic genes while the Osteolectin− cells expressed adipogenic genes. Fate mapping showed that the Osteolectin+ cells gave rise to osteoblasts but not adipocytes in vivo. Conditional deletion of Scf in Osteolectin+ cells did not affect the frequency or function of hematopoietic stem cells (HSCs) or most restricted hematopoietic progenitors in the bone marrow, consistent with our observation that most HSCs and restricted progenitors are associated with sinusoidal blood vessels (Nature 526:126; Cell Stem Cell 24:477); however, common lymphoid progenitors were depleted. This suggests the existence of a peri-arteriolar niche for a subset of lymphoid progenitors in the bone marrow. HSCs are maintained primarily in peri-sinusoidal niches while lymphoid progenitors are maintained in peri-arteriolar and endosteal niches.

3180 – INVESTIGATING IRRADIATION DAMAGE TO HAEMATOPOIETIC NICHES IN THE SPLEEN

Haematopoietic stem cell transplantation (HSCT) is used to treat patients with blood disorders such as leukaemia. Intravenous HSCT allows cells to home to the bone marrow niche and initiate long-term haematopoietic reconstitution. However, HSC can also home to extramedullary organs such as the spleen. Under these circumstances, the spleen possesses the capacity to support extramedullary haematopoiesis (EMH) and immune cell production. This secondary function of the spleen could be targeted to enhance early haematopoietic reconstitution after HSCT. To ensure the success of HSCT, patients must first be conditioned to clear niche space for transplanted HSC to engraft. Radiotherapy used to condition HSCT recipients not only causes myeloablation but also results in non-specific damage to multiple tissues including bone marrow and stem cell niches. It is known that sinusoidal endothelial cells in the bone marrow are both important HSC niche regulators and are damaged following irradiation. In the murine spleen, Scf-expressing endothelial cells and Tcf21+ cells have been shown to form perisinusoidal niches in the red pulp for HSCs to reside in. This project investigates whether damage caused by lethal irradiation extends to the spleen, by examining stromal and endothelial cells using markers such as CD105, MAdCAM-1, MECA-32, VCAM-1, PDGFRβ and CD31 in order to resolve multiple vascular and mesenchymal structures including splenic HSC niches. If the stromal niches regulating extramedullary haematopoiesis during the early period of haematopoietic recovery can be better understood, it may lead to therapies which accelerate immune reconstitution and ultimately lessen rates of transplant-associated mortality.

Nidogen-1 Contributes to the Interaction Network Involved in Pro-B Cell Retention in the Peri-sinusoidal Hematopoietic Stem Cell Niche

In the bone marrow, CXCL12 and IL-7 are essential for B cell differentiation, whereas hematopoietic stem cell (HSC) maintenance requires SCF and CXCL12. Peri-sinusoidal stromal (PSS) cells are themain source of IL-7, but their characterization as a pro-B cell niche remains limited. Here, we characterize pro-B cell supporting stromal cells and decipher the interaction network allowing pro-B cell retention. Preferential contacts are found between pro-B cells and PSS cells, which homogeneously express HSC and B cell niche genes. Furthermore, pro-B cells are frequently located in the vicinity of HSCs in the same niche. Using an interactome bioinformatics pipeline, we identify Nidogen-1 as essential for pro-B cell retention in the peri-sinusoidal niche as confirmed in Nidogen-1-/- mice. Finally, human pro-B cells and hematopoietic progenitors are observed close to similar IL-7+ stromal cells. Thus, a multispecific niche exists in mouse and human supporting both early progenitors and committed hematopoietic lineages.

The Regulation of Hematopoietic Stem Cells and Erythropoiesis

Hematopoietic stresses mobilize hematopoietic stem cells (HSCs) from the bone marrow to the spleen and induce extramedullary hematopoiesis (EMH), including erythropoiesis. We assessed the sources of the key niche factors, SCF and CXCL12, in the mouse spleen after EMH induction by cyclophosphamide plus granulocyte colony-stimulating factor, blood loss, or pregnancy. In each case, Scf was expressed by endothelial cells and Tcf21+ stromal cells, primarily around sinusoids in the red pulp, while Cxcl12 was expressed by a subset of Tcf21+ stromal cells (Nature 527:466, 2015). EMH induction markedly expanded the Scf-expressing endothelial cells and stromal cells by inducing their proliferation. Most splenic HSCs were adjacent to Tcf21+ stromal cells in red pulp. Conditional deletion of Scf from spleen endothelial cells or Scf or Cxcl12 from Tcf21+ stromal cells severely reduced spleen EMH and reduced blood cell counts without affecting bone marrow hematopoiesis. Endothelial cells and Tcf21+ stromal cells thus create a perisinusoidal EMH niche in the spleen, which is necessary for the physiological response to diverse hematopoietic stresses.During pregnancy, the blood volume increases relatively quickly, requiring increased erythropoiesis to maintain red blood cell counts. We discovered that HSCs in female mice divide significantly more frequently than in males as a result of estrogen/estrogen receptor signaling in HSCs (Nature 505:555). Estrogen levels increase during pregnancy, increasing HSC division, HSC frequency, cellularity, and erythropoiesis in the spleen. Conditional deletion of Estrogen Receptor α from HSCs reduced HSC division in female, but not male, mice and attenuated the increases in HSC division, HSC frequency, and erythropoiesis during pregnancy. Treatment of mice with estradiol induces HSC proliferation in the bone marrow but not mobilization. Treatment with the alternative ERα ligand, 27-hydroxycholesterol (27HC), induces ERα-dependent HSC mobilization and EMH but not HSC division in the bone marrow (JCI 127:3392). During pregnancy, 27HC levels increased in hematopoietic stem/progenitor cells as a result of Cyp27a1, a cholesterol hydroxylase. Cyp27a1-deficient mice have significantly reduced 27HC levels, HSC mobilization, and EMH during pregnancy. Two different ERα ligands, estradiol and 27HC, thus work together to promote EMH during pregnancy.We have also examined the bone marrow to better understand the niche for erythropoiesis. HSCs in the bone marrow are maintained in a perivascular niche created by Leptin Receptor+ (LepR) stromal cells and endothelial cells. An important question is why LepR+ cells are one hundred times more frequent than HSCs. We discovered that SCF from LepR+ cells is also necessary for the maintenance of many c-kit+ restricted hematopoietic progenitors. Conditional deletion of Scf from LepR+ cells depleted CMPs, CLPs, GMPs, MEPs, PreMegEs, and CFU-Es, depleting myeloid and erythroid cells from the blood. This was not a consequence of HSC depletion as many other restricted progenitors were unaffected. Moreover, conditional deletion of Scf from endothelial cells depleted HSCs, but not restricted progenitors. Imaging showed that early erythroid progenitors were closely associated with perisinusoidal LepR+ cells. Our data reveal cellular specialization within the niche: SCF from LepR+ cells is broadly required by HSCs and c-kit+ restricted progenitors while SCF from endothelial cells is required only by HSCs. Erythroid progenitors depend upon SCF synthesized by LepR+ cells in perisinusoidal niches. DisclosuresMorrison:OncoMed Pharmaceuticals: Equity Ownership; GI Therapeutics: Consultancy, Equity Ownership; Frequency Therapeutics: Consultancy.

2008 - Hematopoietic Stem Cells in Perisinusoidal Niches are Protected From Aging

Bone marrow (BM) hematopoietic stem cell (HSC) niches are constituted by multiple hematopoietic and non-hematopoietic cells interacting in a complex 3-dimensional (3D) architecture. With aging, intrinsic HSC activity decreases, resulting in impaired tissue homeostasis, reduced engraftment following transplantation and increased susceptibility to diseases. However, whether aging affects also the HSC niches impairing their capacity to support HSC function is still largely unknown.

Deep imaging of bone marrow shows non-dividing stem cells are mainly perisinusoidal.

Hematopoietic stem cells (HSCs) reside in a perivascular niche but the location remains controversial1. HSCs are rare and few can be found in thin tissue sections2,3 or upon live imaging4, making it difficult to comprehensively localize dividing and non-dividing HSCs. We discovered that α-catulinGFP/+ was expressed by only 0.02% of bone marrow hematopoietic cells, including virtually all HSCs. One in 3.5 α-catulin-GFP+c-kit+ cells gave long-term multilineage reconstitution of irradiated mice, indicating that α-catulin-GFP+c-kit+ cells contain HSCs with a purity comparable to the best markers available. We were able to optically clear the bone marrow to perform deep confocal imaging, making it possible to image thousands of α-catulin-GFP+c-kit+ cells and to digitally reconstruct large segments of bone marrow. The distribution of α-catulin-GFP+c-kit+ cells indicated that HSCs were more common in central marrow than near bone surfaces and in the diaphysis relative to the metaphysis. Nearly all HSCs contacted Leptin Receptor+ and Cxcl12high niche cells. Approximately 85% of HSCs were within 10μm of a sinusoidal blood vessel. Most HSCs were distant from arterioles, transition zone vessels, and bone surfaces. This was true of Ki-67+ dividing HSCs and Ki-67− non-dividing HSCs. Dividing and non-dividing HSCs thus reside mainly in perisinusoidal niches with Leptin Receptor+Cxcl12high cells throughout the bone marrow.

Arteriolar niches maintain haematopoietic stem cell quiescence

Cell cycle quiescence is a critical feature contributing to haematopoietic stem cell (HSC) maintenance. Although various candidate stromal cells have been identified as potential HSC niches, the spatial localization of quiescent HSCs in the bone marrow (BM) remains unclear. Here, using a novel approach that combines whole-mount confocal immunofluorescence imaging techniques and computational modelling to analyse significant tridimensional associations among vascular structures, stromal cells and HSCs, we show that quiescent HSCs associate specifically with small arterioles that are preferentially found in endosteal BM. These arterioles are ensheathed exclusively by rare NG2+ pericytes, distinct from sinusoid-associated LepR+ cells. Pharmacological or genetic activation of HSC cell cycle alters the distribution of HSCs from NG2+ peri-arteriolar niches to LepR+ peri-sinusoidal niches. Conditional depletion of NG2+ cells induces HSC cycling and reduces functional long-term repopulating HSCs in BM. These results thus indicate that arteriolar niches are indispensable to maintain HSC quiescence.

N-cadherin+ HSCs in fetal liver exhibit higher long-term bone marrow reconstitution activity than N-cadherin− HSCs

Adult hematopoietic stem cells (HSCs) are maintained in a microenvironment known as the stem cell niche. The regulation of HSCs in fetal liver (FL) and their niche, however, remains to be elucidated. In this study, we investigated the role of N-cadherin (N-cad) in the maintenance of HSCs during FL hematopoiesis. By using anti-N-cad antibodies (Abs) produced by our laboratory, we detected high N-cad expression in embryonic day 12.5 (E12.5) mouse FL HSCs, but not in E15.5 and E18.5 FL. Immunofluorescence staining revealed that N-cad+c-Kit+ and N-cad+ endothelial protein C receptor (EPCR)+ HSCs co-localized with Lyve-1+ sinusoidal endothelial cells (ECs) in E12.5 FL and that some of these cells also expressed N-cad. However, N-cad+ HSCs were also observed to detach from the perisinusoidal niche at E15.5 and E18.5, concomitant with a down-regulation of N-cad and an up-regulation of E-cadherin (E-cad) in hepatic cells. Moreover, EPCR+ long-term (LT)-HSCs were enriched in the N-cad+Lin−Sca-1+c-Kit+ (LSK) fraction in E12.5 FL, but not in E15.5 or E18.5 FL. In a long-term reconstitution (LTR) activity assay, higher engraftment associated with N-cad+ LSK cells versus N-cad− LSK cells in E12.5 FL when transplanted into lethally irradiated recipient mice. However, the higher engraftment of N-cad+ LSK cells decreased subsequently in E15.5 and E18.5 FL. It is possible that N-cad expression conferred higher LTR activity to HSCs by facilitating interactions with the perisinusoidal niche, especially at E12.5. The down-regulation of N-cad during FL hematopoiesis may help us better understand the regulation and mobility of HSCs before migration into BM.

Endothelial protein C receptor–expressing hematopoietic stem cells reside in the perisinusoidal niche in fetal liver

AbstractHematopoietic stem cells (HSCs) are maintained in specialized niches in adult bone marrow. However, niche and HSC maintenance mechanism in fetal liver (FL) still remains unclear. Here, we investigated the niche and the molecular mechanism of HSC maintenance in mouse FL using HSCs expressing endothelial protein C receptor (EPCR). The antiapoptotic effect of activated protein C (APC) on EPCR+ HSCs and the expression of protease-activated receptor 1 (Par-1) mRNA in these cells suggested the involvement of the cytoprotective APC/EPCR/Par-1 pathway in HSC maintenance. Immunohistochemistry revealed that EPCR+ cells were localized adjacent to, or integrated in, the Lyve-1+ sinusoidal network, where APC and extracellular matrix (ECM) are abundant, suggesting that HSCs in FL were maintained in the APC- and ECM-rich perisinusoidal niche. EPCR+ HSCs were in a relatively slow cycling state, consistent with their high expression levels of p57 and p18. Furthermore, the long-term reconstitution activity of EPCR+ HSCs decreased significantly after short culture but not when cocultured with feeder layer of FL-derived Lyve-1+ cells, which suggests that the maintenance of the self-renewal activity of FL HSCs largely depended on the interaction with the perisinusoidal niche. In conclusion, EPCR+ HSCs resided in the perisinusoidal niche in mouse FL.

The impact of cell re‐entry into the primary lymphoid organs on lymphocyte repertoire and functionality

The impact of cell re-entry into the primary lymphoid organs on lymphocyte repertoire and functionality

The bone marrow perisinusoidal niche for recirculating B cells and the positive selection of bone marrow‐derived B lymphocytes

A unique 'second' niche for follicular B cells has been described in the extravascular compartment of the bone marrow surrounding vascular sinusoids. The occupancy of this niche by B cells presumably evolved to facilitate humoral immune responses to blood-borne pathogens. B cells appear to be sustained in this niche by bone marrow dendritic cells and are lost from this compartment in certain mutant mice. We discuss here what is known regarding the mechanisms of entry and egress of B cells from the perisinusoidal niche and also consider the function of the bone marrow as a secondary lymphoid organ. Although immature B cells can mature into follicular B cells in this niche as well as in the spleen, the lineage commitment event that accompanies positive selection of B cells occurs only in the spleen.

Naive recirculating B cells mature simultaneously in the spleen and bone marrow

AbstractWe have recently demonstrated that IgDhi B cells can occupy an extravascular perisinusoidal niche in the bone marrow in addition to the well-established follicular niche in conventional secondary lymphoid organs. The spleen has long been considered to be the site at which newly formed B lymphocytes mature into IgDhi naive recirculating B cells, but the existence of mutant mice that have selectively lost mature B cells in the bone marrow prompted an examination of B-cell maturation at this latter site. Following a single pulse of BrdU in intact mice, sequential labeling of more mature B-cell populations in the bone marrow suggested ongoing maturation at this site. Further evidence for B-cell maturation in the bone marrow was obtained from analyses of transitional B cells in splenectomized lymphotoxin α-deficient mice that lack all secondary lymphoid organs. In these mice, antibody-secreting cells recognizing multivalent antigens were also observed in the bone marrow following an intravenous microbial challenge. These data suggest that newly formed B cells mature into IgDhi B cells simultaneously in the spleen and the bone marrow and establish in a stringent manner that humoral immune responses can be initiated in situ in the bone marrow.

Perisinusoidal B Cells in the Bone Marrow Participate in T-Independent Responses to Blood-Borne Microbes

Mature recirculating B cells are generally assumed to exist in follicular niches in secondary lymphoid organs, and these cells mediate T-dependent humoral immune responses. We show here that a large proportion of mature B lymphocytes occupy an anatomically and functionally distinct perisinusoidal niche in the bone marrow. Perisinusoidal B cells circulate freely, as revealed by parabiosis studies. However, unlike their counterparts in the follicular niche, these cells are capable of being activated in situ by blood-borne microbes in a T-independent manner to generate specific IgM antibodies. The bone marrow represents a unique type of secondary lymphoid organ in which mature B cells are strategically positioned in the path of circulating microbes.