Cadherin-6 Mediates the Heterotypic Interactions between the Hemopoietic Osteoclast Cell Lineage and Stromal Cells in a Murine Model of Osteoclast Differentiation

Osteoclasts are multinucleated cells of hemopoietic origin that are responsible for bone resorption during physiological bone remodeling and in a variety of bone diseases. Osteoclast development requires direct heterotypic cell–cell interactions of the hemopoietic osteoclast precursors with the neighboring osteoblast/stromal cells. However, the molecular mechanisms underlying these heterotypic interactions are poorly understood. We isolated cadherin-6 isoform, denoted cadherin-6/2 from a cDNA library of human osteoclast-like cells. The isolated cadherin-6/2 is 3,423 bp in size consisting of an open reading frame of 2,115 bp, which encodes 705 amino acids. This isoform lacks 85 amino acids between positions 333 and 418 and contains 9 different amino acids in the extracellular domain compared with the previously described cadherin-6. The human osteoclast-like cells also expressed another isoform denoted cadherin-6/1 together with the cadherin-6. Introduction of cadherin-6/2 into L-cells that showed no cell–cell contact caused evident morphological changes accompanied with tight cell–cell association, indicating the cadherin-6/2 we isolated here is functional. Moreover, expression of dominant-negative or antisense cadherin-6/2 construct in bone marrow–derived mouse stromal ST2 cells, which express only cadherin-6/2, markedly impaired their ability to support osteoclast formation in a mouse coculture model of osteoclastogenesis. Our results suggest that cadherin-6 may be a contributory molecule to the heterotypic interactions between the hemopoietic osteoclast cell lineage and osteoblast/bone marrow stromal cells required for the osteoclast differentiation. Since both osteoclasts and osteoblasts/bone marrow stromal cells are the primary cells controlling physiological bone remodeling, expression of cadherin-6 isoforms in these two cell types of different origin suggests a critical role of these molecules in the relationship of osteoclast precursors and cells of osteoblastic lineage within the bone microenvironment.     

Human diseases of telomerase dysfunction: insights into tissue aging

There are at least three human diseases that are associated with germ-line mutations of the genes encoding the two essential components of telomerase, TERT and TERC. Heterozygous mutations of these genes have been described for patients with dyskeratosis congenita, bone marrow failure and idiopathic pulmonary fibrosis. In this review, we will detail the clinical similarities and difference of these diseases and review the molecular phenotypes observed. The spectrum of mutations in TERT and TERC varies for these diseases and may in part explain the clinical differences observed. Environmental insults and genetic modifiers that accelerate telomere shortening and increase cell turnover may exaggerate the effects of telomerase haploinsufficiency, contributing to the variability of age of onset as well as tissue-specific organ pathology. A central still unanswered question is whether telomerase dysfunction and short telomeres are a much more prominent factor than previously suspected in other adult-onset, age-related diseases. Understanding the biological effects of these mutations may ultimately lead to novel treatments for these patients.     

Bone disease in children with homozygous β-thalassemia

The histological features of thalassemic bone are imperfectly known, and the roles of bone marrow hyperactivity, iron overload or vitamin D deficiency in the pathogenesis of the disease are not clearly identified. In this study we examined iliac crest biopsies from 17 transfusion-dependent children with homozygous β-thalassemia and severe radiological skeletal thalassemic changes, including widening of medullary spaces and osteoporosis. Rachitic lesions were not observed. Serum ferritin concentrations were increased in all but one subject. Iron deposits were histochemically detected in bone marrow, at the marrow-bone interface, along cement lines and mineralizing perimeters. Minor changes were present in trabecular bone, and osteomalacia was absent. By contrast, cortical bone exhibited severe changes including fissures and focal mineralization defects. Plasma 25-hydroxyvitamin D (25(OH)D) concentrations measured during the winter (December–May, 6.5 ± 4.9 ng/ml, mean ± SD, n = 6) and during the summer (June–November, 13.8 ± 8.4 ng/ml, n = 9) did not differ from those of age-matched children living in the same country. Seven patients had moderate hypocalcemia but no biological signs suggestive of vitamin D deficiency: all had normal alkaline phosphatase activity, normal or slightly elevated plasma phosphate, only two had low plasma 25(OH)D concentrations and two others supranormal values of plasma immunoreactive parathyroid hormone.These results show that iron overload and vitamin D deficiency do not seem to play an important role in the pathogenesis of thalassemic bone disease, which is characterized by cortical lesions probably related to marrow hyperactivity.     

Osteoimmunology: cytokines and the skeletal system

It has become clear that complex interactions underlie the relationship between the skeletal and immune systems. This is particularly true for the development of immune cells in the bone marrow as well as the functions of bone cells in skeletal homeostasis and pathologies. Because these two disciplines developed independently, investigators with an interest in either often do not fully appreciate the influence of the other system on the functions of the tissue that they are studying. With these issues in mind, this review will focus on several key areas that are mediated by crosstalk between the bone and immune systems. A more complete appreciation of the interactions between immune and bone cells should lead to better therapeutic strategies for diseases that affect either or both systems.     

Modularity and integration in the hominoid scapula

In this paper, several hypotheses of morphological integration within the hominoid (ape) scapula are tested. In particular, whether the scapula represents a set of developmental tissues sharing tight correlations between constituent parts (i.e., highly integrated) or is more modularly organized (i.e., covariation is greater within regions than between) is tested. Whether the patterns of integration in the scapula have changed over phylogenetic time or in response to selective forces is also examined. Results from two different analyses (matrix correlations and edge deviance) indicate traits comprising the blade and acromion, and to a weaker degree the glenoid, correlate highly with each other. The coracoid exhibits more independence from other parts of the scapula, perhaps reflecting its distinct evolutionary developmental history. Overall, similarity in species-specific patterns of correlation was high between all taxa. Correlation matrix similarity was significantly correlated with functional similarity and morphological distance, but not with phylogenetic distance. These results are congruent with other studies of integration that suggest correlation patterns remain stable over evolutionary time. There are changes associated with phylogeny, but the tight link between functional similarity and phylogenetic distance at this level of comparison presents possible challenges to interpretation. Overall similarities in the pattern of integration in all taxa might be better interpreted as relative strengthening or weakening of trait correlations rather than broadscale changes in the pattern of relationship between developmental regions. Larger sample sizes with greater taxonomic/functional breadth, and finer scale analyses of patterns of correlation are needed to test these hypotheses further.     

DISTINCTIVE DIAGNOSTIC FEATURES OF MATERIAL ASPIRATED FROM MARROW IN METABOLIC BONE DISEASES

In a series of cases of Paget''s disease of the bone, two types of cells not previously described were observed in material aspirated from bone marrow in areas of osteitis deformans. One type was mononuclear, the other was giant, multinucleated and syncytial. They have been identified as osteoblasts and osteoclasts, respectively. The identification was based mainly on correlation with the histologic picture of osteitis deformans and of normal-growing bones as seen in section studies.Both osteoblasts and osteoclasts were recovered in aspirated bone marrow material in other instances of metabolic bone diseases associated with increased bone repair and bone resorption—in hyperparathyroidism, osteoblastic malignant lesions, rickets, hemolytic anemia in children, and in normal infants in the growth zone of bone in the tibia. They were not seen in senile and postmenopausal osteoporosis.Recognition of osteoblasts and osteoclasts in smear preparations from aspirated bone marrow material may serve as a diagnostic aid in metabolic bone diseases.The differentiation of osteoblasts from neoplastic cells is important in cases in which metastatic cancer of the bone is suspected and x-ray findings are inconclusive.     

Transplantation of genetically modified haematopoietic stem cells to induce antigen-specific tolerance as a cure for autoimmune diseases

Autoimmune diseases are incurable and are managed using therapeutic agents. Bone marrow transplantation is being trialled as a treatment for these diseases. While allogeneic bone marrow transplantation shows impressive benefit, its application is hindered by GVHD and high mortality. On the other hand, autologous bone marrow transplantation has lower mortality rate and no GVHD but is associated with higher relapse rates. Given that autoimmune diseases are a result of a failure of immune tolerance and that bone marrow-derived dendritic cells play an important role in establishing immune tolerance, the transplantation of genetically modified haematopoietic stem cells to generate molecular chimerism to induce antigen-specific tolerance offers the potential for developing a cure for autoimmune diseases. In this review, we will discuss key findings from clinical data and animal studies to provide evidence to support the above concept.     

Automated Quantification of Hematopoietic Cell – Stromal Cell Interactions in Histological Images of Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.     

Bone Marrow Stem Cells Expressing Keratinocyte Growth Factor via an Inducible Lentivirus Protects against Bleomycin-Induced Pulmonary Fibrosis

Many common diseases of the gas exchange surface of the lung have no specific treatment but cause serious morbidity and mortality. Idiopathic Pulmonary Fibrosis (IPF) is characterized by alveolar epithelial cell injury, interstitial inflammation, fibroblast proliferation and collagen accumulation within the lung parenchyma. Keratinocyte Growth Factor (KGF, also known as FGF-7) is a critical mediator of pulmonary epithelial repair through stimulation of epithelial cell proliferation. During repair, the lung not only uses resident cells after injury but also recruits circulating bone marrow-derived cells (BMDC). Several groups have used Mesenchymal Stromal Cells (MSCs) as therapeutic vectors, but little is known about the potential of Hematopoietic Stem cells (HSCs). Using an inducible lentiviral vector (Tet-On) expressing KGF, we were able to efficiently transduce both MSCs and HSCs, and demonstrated that KGF expression is induced in a regulated manner both in vitro and in vivo. We used the in vivo bleomycin-induced lung fibrosis model to assess the potential therapeutic effect of MSCs and HSCs. While both populations reduced the collagen accumulation associated with bleomycin-induced lung fibrosis, only transplantation of transduced HSCs greatly attenuated the histological damage. Using double immunohistochemistry, we show that the reduced lung damage likely occurs through endogenous type II pneumocyte proliferation induced by KGF. Taken together, our data indicates that bone marrow transplantation of lentivirus-transduced HSCs can attenuate lung damage, and shows for the first time the potential of using an inducible Tet-On system for cell based gene therapy in the lung.     

Osteoclast Derivation from Mouse Bone Marrow

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease.As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation.An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.     

Mutation of kri1l causes definitive hematopoiesis failure via PERK-dependent excessive autophagy induction

Dysregulation of ribosome biogenesis causes human diseases, such as Diamond-Blackfan anemia, del (5q-) syndrome and bone marrow failure. However, the mechanisms of blood disorders in these diseases remain elusive. Through genetic mapping, molecular cloning and mechanism characterization of the zebrafish mutant cas002, we reveal a novel connection between ribosomal dysfunction and excessive autophagy in the regulation of hematopoietic stem/progenitor cells (HSPCs). cas002 carries a recessive lethal mutation in kri1l gene that encodes an essential component of rRNA small subunit processome. We show that Kri1l is required for normal ribosome biogenesis, expansion of definitive HSPCs and subsequent lineage differentiation. Through live imaging and biochemical studies, we find that loss of Kri1l causes the accumulation of misfolded proteins and excessive PERK activation-dependent autophagy in HSPCs. Blocking autophagy but not inhibiting apoptosis by Bcl2 overexpression can fully rescue hematopoietic defects, but not the lethality of kri1l^cas002 embryos. Treatment with autophagy inhibitors (3-MA and Baf A1) or PERK inhibitor (GSK2656157), or knockdown of beclin1 or perk can markedly restore HSPC proliferation and definitive hematopoietic cell differentiation. These results may provide leads for effective therapeutics that benefit patients with anemia or bone marrow failure caused by ribosome disorders.     

Distinctive diagnostic features of material aspirated from marrow in metabolic bone diseases

In a series of cases of Paget's disease of the bone, two types of cells not previously described were observed in material aspirated from bone marrow in areas of osteitis deformans. One type was mononuclear, the other was giant, multinucleated and syncytial. They have been identified as osteoblasts and osteoclasts, respectively. The identification was based mainly on correlation with the histologic picture of osteitis deformans and of normal-growing bones as seen in section studies. Both osteoblasts and osteoclasts were recovered in aspirated bone marrow material in other instances of metabolic bone diseases associated with increased bone repair and bone resorption-in hyperparathyroidism, osteoblastic malignant lesions, rickets, hemolytic anemia in children, and in normal infants in the growth zone of bone in the tibia. They were not seen in senile and postmenopausal osteoporosis. Recognition of osteoblasts and osteoclasts in smear preparations from aspirated bone marrow material may serve as a diagnostic aid in metabolic bone diseases. The differentiation of osteoblasts from neoplastic cells is important in cases in which metastatic cancer of the bone is suspected and x-ray findings are inconclusive.     

Comprehensive transcriptome and immunophenotype analysis of renal and cardiac MSC-like populations supports strong congruence with bone marrow MSC despite maintenance of distinct identities

Cells resembling bone marrow mesenchymal stem cells (MSC) have been isolated from many organs but their functional relationships have not been thoroughly examined. Here we compared the immunophenotype, gene expression, multipotency and immunosuppressive potential of MSC-like colony-forming cells from adult murine bone marrow (bmMSC), kidney (kCFU-F) and heart (cCFU-F), cultured under uniform conditions. All populations showed classic MSC morphology and in vitro mesodermal multipotency. Of the two solid organ-specific CFU-F, only kCFU-F displayed suppression of T-cell alloreactivity in vitro, albeit to a lesser extent than bmMSC. Quantitative immunophenotyping using 81 phycoerythrin-conjugated CD antibodies demonstrated that all populations contained high percentages of cells expressing diagnostic MSC surface markers (Sca1, CD90.2, CD29, CD44), as well as others noted previously on murine MSC (CD24, CD49e, CD51, CD80, CD81, CD105). Illumina microarray expression profiling and bioinformatic analysis indicated a correlation of gene expression of 0.88-0.92 between pairwise comparisons. All populations expressed approximately 66% of genes in the pluripotency network (Plurinet), presumably reflecting their stem-like character. Furthermore, all populations expressed genes involved in immunomodulation, homing and tissue repair, suggesting these as conserved functions for MSC-like cells in solid organs. Despite this molecular congruence, strong biases in gene and protein expression and pathway activity were seen, suggesting organ-specific functions. Hence, tissue-derived MSC may also retain unique properties potentially rendering them more appropriate as cellular therapeutic agents for their organ of origin.     

Kinetics of gene expression and bone remodelling in the clinical phase of collagen-induced arthritis

IntroductionPathological bone changes differ considerably between inflammatory arthritic diseases and most studies have focused on bone erosion. Collagen-induced arthritis (CIA) is a model for rheumatoid arthritis, which, in addition to bone erosion, demonstrates bone formation at the time of clinical manifestations. The objective of this study was to use this model to characterise the histological and molecular changes in bone remodelling, and relate these to the clinical disease development.MethodsA histological and gene expression profiling time-course study on bone remodelling in CIA was linked to onset of clinical symptoms. Global gene expression was studied with a gene chip array system.ResultsThe main histopathological changes in bone structure and inflammation occurred during the first two weeks following the onset of clinical symptoms in the joint. Hereafter, the inflammation declined and remodelling of formed bone dominated.Global gene expression profiling showed simultaneous upregulation of genes related to bone changes and inflammation in week 0 to 2 after onset of clinical disease. Furthermore, we observed time-dependent expression of genes involved in early and late osteoblast differentiation and function, which mirrored the histopathological bone changes. The differentially expressed genes belong to the bone morphogenetic pathway (BMP) and, in addition, include the osteoblast markers integrin-binding sialoprotein (Ibsp), bone gamma-carboxyglutamate protein (Bglap1), and secreted phosphoprotein 1 (Spp1). Pregnancy-associated protein A (Pappa) and periostin (Postn), differentially expressed in the early disease phase, are proposed to participate in bone formation, and we suggest that they play a role in early bone formation in the CIA model. Comparison to human genome-wide association studies (GWAS) revealed differential expression of several genes associated with human arthritis.ConclusionsIn the CIA model, bone formation in the joint starts shortly after onset of clinical symptoms, which results in bony fusion within one to two weeks. This makes it a candidate model for investigating the relationship between inflammation and bone formation in inflammatory arthritis.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0531-7) contains supplementary material, which is available to authorized users.     

Preparation and characteristics of growth and marker properties of urinary bladder mesenchymal stem cells

Mesenchymal stem cells (MSC) are able to transdifferentiate into cells with different functional phenotypes and considered as a promising resource for regenerative therapy. MSC derived from different tissues vary in their differentiation potential and in some cases express tissue specific markers indicating a kinship between mesenchymal and parenchymal phenotypes in the same tissue. It is possible that homorganic MSC can be more effectively induced to tissue specific differentiation and preferable for cell therapy of this organ as compared with bone marrow derived cells being commonly used for this purpose. Using bladder tissue explants, we prepared primary MSC cultures from the fetal (MSC-BF) and adult syngenic BALB/c mice and characterized their abilities during long-term passaging. In contrast to the cells from adult mice, the MSC-BF cells have the ability for a sustained growth in vitro, clonogenicity and differentiation into adipose and bone cells. Similar to the bone marrow MSC, MSC-BF express the mesenchymal markers CD29, CD44, CD49f, CD90, CD105 but not the leukocyte common antigen CD45. In normal conditions, MSC-BF produce such urothelial markers as CK14 and FOXA1 although their expression level is by far lower than in the bladder tissue. The hypomethylating agent, 5-azacytidine, induces in MSC-BF the expression of the urothelial differentiation activator PPARγ and the functional urothelium markers UP1a, UP1b, UP3a, UP3b. The data obtained suggest that MSC-BF can be epigenetically reprogrammed into urothelium by the 5-azacytidine treatment, and this may offer the novel strategy for cell therapy of bladder diseases.     

Plasma membrane rafts engaged in T cell signalling: new developments in an old concept

The receptor for advanced glycation end products (RAGE) is a single transmembrane receptor of the immunoglobulin superfamily that is mainly expressed on immune cells, neurons, activated endothelial and vascular smooth muscle cells, bone forming cells, and a variety of cancer cells. RAGE is a multifunctional receptor that binds a broad repertoire of ligands and mediates responses to cell damage and stress conditions. It activates programs responsible for acute and chronic inflammation, and is implicated in a number of pathological diseases, including diabetic complications, stroke, atheriosclerosis, arthritis, and neurodegenerative disorders. The availability of Rage knockout mice has not only advanced our knowledge on signalling pathways within these pathophysiological conditions, but also on the functional importance of the receptor in processes of cancer. Here, we will summarize molecular mechanisms through which RAGE signalling contributes to the establishment of a pro-tumourigenic microenvironment. Moreover, we will review recent findings that provide genetic evidence for an important role of RAGE in bridging inflammation and cancer.     

Expression of functional human EGF receptor on murine bone marrow cells.

The human epidermal growth factor-receptor (EGF-R) was introduced into primary mouse bone marrow cells (BMC), utilizing retrovirus mediated gene transfer. Cultivation of infected BMC in the presence of interleukin-3 (IL-3) led to the outgrowth of IL-3 dependent myeloid cells, which efficiently expressed functional EGF-R, exhibiting its two characteristic affinity states. EGF acts on these cells synergistically with IL-3 in stimulating DNA synthesis and cell proliferation even under IL-3 saturation conditions. However, EGF was not sufficient to replace the requirement for IL-3. In contrast, EGF was able to maintain proliferation of a factor-dependent hemopoietic cell line (FDC-P1) infected with the EGF-R retrovirus in the absence of IL-3, but these cells did not respond to EGF in the presence of IL-3. No influence of EGF on IL-3 induced mast cell differentiation of BMC expressing the EGF-R could be observed by histological criteria. These data show that the expression of EGF-R alone is not sufficient to induce or maintain cell proliferation in IL-3 dependent bone marrow derived cells, although it can do so in established hemopoietic cell lines.     

Subdivision of bone marrow microenvironments: purpose built homes for haematopoietic stem cells

EMBO J (2013) 32 2, 219–230 doi:10.1038/emboj.2012.308; published online November272012The identification of distinct bone marrow microenvironments that support haematopoietic stem cell (HSC) survival and function is one of the most significant recent advances in our understanding of HSC biology. While the intricate organization of these structures and their effect on HSC is on its own a fascinating biological question, solving this puzzle remains a key step in the development of more efficient stem cell therapies through improved in vitro HSC culture systems. In this issue, a study conducted by Wang and colleagues describes a novel structure within the bone marrow and provides evidence for an additional layer of complexity and compartmentalisation within the bone marrow microenvironment, suggesting the presence of specialised structures that support clonal HSC expansion.The bone marrow is a complex tissue, containing stem and progenitor cells for the haematopoietic, endothelial and osteogenic lineages. Surrounded by compact bone, the marrow is perfused by a complex network of vessels. Small arteries reach the marrow through the bone and branch into increasingly smaller arterioles. These in turn feed into intricate slow flowing sinusoidal capillaries, drained by venules, which eventually flow into a relatively large central sinus. The architecture of the bone marrow and the relationship between these structural building blocks and the haematopoietic stem cells (HSC) residing there is currently a topic of great interest. Specifically, an understanding of how elements of the endothelium, stromal cells and haematopoietic tissue interact to regulate steady state haematopoiesis is critical for the development and improvement of stem cell therapies based on HSC manipulation and administration.The close connection between vascular tissue and bone development and remodelling have been documented and studied for a number of years (Wang et al, 2007). The initial discovery that functional alterations in osteoblastic cells in spongy bones had consequences on HSC function and number attracted the interest of researchers in the HSC field (Calvi et al, 2003; Visnjic et al, 2004; Zhang et al, 2003). A growing number of studies have subsequently implicated endothelial cells and bone marrow vasculature in the support of HSC survival and function. HSC were consistently found residing in the proximity of bone marrow vessels in histological studies (Kiel et al, 2005) and it is now clear that endothelial cells can be used to support HSC culture in vitro (reviewed in Butler et al (2010)). Moreover, manipulation of the vascular bone marrow compartment can affect haematopoiesis (Ding et al, 2012). It has been proposed by multiple groups that while the osteoblastic niche favours HSC quiescence and maintenance, perivascular niches are associated with HSC expansion (Li and Li, 2006).In this issue, a study conducted by Wang et al (2012) presents an in depth histological analysis of distinct bone marrow microenvironments identified using a combination of genetic labelling and traditional immunohistochemistry. Using this approach, they document a functional unit composed of vasculature, bone and HSC, which they name ‘haemosphere''. Structurally, the haemosphere is a cyst-like protrusion of sinusoidal epithelial cells encased by an inner shell of stroma cells and, finally, mineralized bone. The most interesting aspect of haemospheres is that single or multiple, tightly packed CD45⁺ haematopoietic cells are often found enclosed within this structure. (Figure 1 depicts the proposed stages of development of a haemosphere). Using in situ SLAM marker labelling and cell cycle analysis, the authors identify the cells within the haemosphere as clonal, highly proliferative pockets of haematopoietic cells enriched for primitive HSC, suggesting that this vascular structure has a particularly important role in HSC expansion. Additionally, in lethally irradiated mice, haemospheres were shown to preferentially host the engraftment of transplanted bone marrow cells, adding further weight to the idea that they play a specialised role in the support of stem cell proliferation and expansion. The studies described here provide another step towards dissecting the composition of bone marrow microenvironments and indicate a topological location for expanding HSC clones.Open in a separate windowFigure 1Haemospheres likely initiate as an invagination of bone, mediated by bone remodelling around a vessel branch and eventually providing space for a growing clone of haematopoietic stem/progenitor cells. Larger cavities, such as the main femur diaphysis marrow space, allow mixing of haematopoietic clones. The maintenance of haemospheres depends on signalling through VEGF receptor 2.Interestingly, haemospheres were shown to be dependent on vascular endothelial growth factor (VEGF) signalling, suggesting they may be dynamic structures. This is consistent with recent findings implicating a role for angiocrine factors released by endothelial cells in vivo in the replenishment of the HSC pool and the negative effect on reconstitution efficiency when VEGF2 signalling is inhibited (Hooper et al, 2009; Butler et al, 2010). The results presented here suggest an additional layer of complexity as they indicate that VEGFR2 might mediate significant vascular and bone remodelling, that could potentially generate new microenvironments supportive of HSC.As with most novel findings, the work from Wang et al. opens a handful of new questions whose answer will considerably increase our understanding of the connection between HSC and bone marrow stroma. Most notably, which came first, haemosphere or HSC? And how plastic is this relationship? The studies here demonstrate that active remodelling of bone marrow microenvironments via VEGF signalling is necessary for haemosphere formation. However, it is still unclear how formation of these structures recruits and maintains HSC. Additionally, does the structure of the haemosphere create a unique combination of growth factors and ligands compared with other microenvironments that specifically supports HSC engraftment as well as homoeostatic proliferation? Conversely, could single HSC migrate to these perivascular sites and initiate remodelling of the local environment to their own advantage? And if this is the case, how does an HSC initiate such drastic remodelling and how can we use these factors to address clinical problems in stem cell transplantation therapy? Further studies are needed to obtain temporal information on the generation, maintenance and stability of haemospheres and to functionally compare haematopoiesis in haemospheres and in diaphyseal marrow. It is possible that the two locations may be functionally identical even though anatomically different, with the physical constraint of the encasing bone forcing haematopoietic clones to remain cohesive within haemospheres, while clones outside this environment can readily mix in the central, more fluid marrow.The findings presented by Wang et al. challenge current paradigms of the plasticity of the niche and its composition. Technological and experimental advances allowing full bone imaging and isolation of live cells from specific bone regions will be critical to obtain a complete picture of the function and dynamics of anatomically distinct bone marrow microenvironment and their relationship with HSC. In the mean time, the haemospheres are a new type of bone marrow structure that deserves further attention and guarantees further insights in our understanding of the HSC niche.