Search

Neurogenic heterotopic ossifications (NHOs) form in periarticular muscles following severe spinal cord (SCI) and traumatic brain injuries. The pathogenesis of NHO is poorly understood with no effective preventive treatment. The only curative treatment remains surgical resection of pathological NHOs. In a mouse model of SCI-induced NHO that involves a transection of the spinal cord combined with a muscle injury, a differential gene expression analysis revealed that genes involved in inflammation such as interleukin-1β (IL-1β) were overexpressed in muscles developing NHO. Using mice knocked-out for the gene encoding IL-1 receptor (IL1R1) and neutralizing antibodies for IL-1α and IL-1β, we show that IL-1 signaling contributes to NHO development following SCI in mice. Interestingly, other proteins involved in inflammation that were also overexpressed in muscles developing NHO, such as colony-stimulating factor-1, tumor necrosis factor or C-C chemokine ligand-2 did not promote NHO development. Finally using NHO biopsies from SCI and TBI patients, we show that IL-1β is expressed by CD68+ macrophages. IL-1α and IL-1β produced by activated human monocytes promote calcium mineralization and RUNX2 expression in fibro-adipogenic progenitors isolated from muscles surrounding NHOs. Altogether these data suggest that interleukin-1 promotes NHO development in both humans and mice. This article is protected by copyright. All rights reserved.

The endothelial adhesion protein E-selectin/CD62E is not required for leukocyte homing, unlike closely related family member P-selectin/CD62P. As transmigration through the endothelium is one of the first steps in generating a local immune response, we hypothesized that E-selectin may play additional roles in the early stages of immune activation. We found contact with E-selectin, but not P-selectin or vascular cell adhesion molecule 1 (CD106), induced phosphorylation of protein kinase B (AKT) and nuclear factor-κB in mouse bone marrow-derived macrophages (BMDMs) in vitro. This occurred within 15 min of E-selectin contact and was dependent on phosphatidylinositol-3 kinase activity. Binding to E-selectin activated downstream proteins including mammalian target of rapamycin, p70 ribosomal protein S6 kinase and eukaryotic translation initiation factor 4E-binding protein 1. Functionally, adhesion to E-selectin induced upregulation of CD86 expression and CCL2 secretion. We next asked whether contact with E-selectin impacts further BMDM stimulation. We found enhanced secretion of both interleukin (IL)-10 and CCL2, but not tumor necrosis factor or IL-6 in response to lipopolysaccharide (LPS) stimulation after adhesion to E-selectin. Importantly, adhesion to E-selectin did not polarize BMDMs to one type of response but enhanced both arginase activity and nitric oxide production following IL-4 or LPS stimulation, respectively. In cultured human monocytes, adhesion to E-selectin similarly induced phosphorylation of AKT. Finally, when E-selectin was blocked in vivo in mice, thioglycollate-elicited macrophages showed reduced CD86 expression, validating our in vitro studies. Our results imply functions for E-selectin beyond homing and suggest that E-selectin plays an early role in priming and amplifying innate immune responses.

Background Prior chemotherapy and/or underlying morbidity commonly leads to poor mobilisation of hematopoietic stem cells (HSC) for transplantation in cancer patients. Increasing the number of available HSC prior to mobilisation is a potential strategy to overcome this deficiency. Resident bone marrow (BM) macrophages are essential for maintenance of niches that support HSC and enable engraftment in transplant recipients. Here we examined potential of donor treatment with modified recombinant colony-stimulating factor 1 (CSF1) to influence the HSC niche and expand the HSC pool for autologous transplantation. Methods We administered an acute treatment regimen of CSF1 Fc fusion protein (CSF1-Fc, daily injection for 4 consecutive days) to naive C57Bl/6 mice. Treatment impacts on macrophage and HSC number, HSC function and overall hematopoiesis were assessed at both the predicted peak drug action and during post-treatment recovery. A serial treatment strategy using CSF1-Fc followed by granulocyte colony-stimulating factor (G-CSF) was used to interrogate HSC mobilisation impacts. Outcomes were assessed by in situ imaging and ex vivo standard and imaging flow cytometry with functional validation by colony formation and competitive transplantation assay. Results CSF1-Fc treatment caused a transient expansion of monocyte-macrophage cells within BM and spleen at the expense of BM B lymphopoiesis and hematopoietic stem and progenitor cell (HSPC) homeostasis. During the recovery phase after cessation of CSF1-Fc treatment, normalisation of hematopoiesis was accompanied by an increase in the total available HSPC pool. Multiple approaches confirmed that CD48−CD150+ HSC do not express the CSF1 receptor, ruling out direct action of CSF1-Fc on these cells. In the spleen, increased HSC was associated with expression of the BM HSC niche macrophage marker CD169 in red pulp macrophages, suggesting elevated spleen engraftment with CD48−CD150+ HSC was secondary to CSF1-Fc macrophage impacts. Competitive transplant assays demonstrated that pre-treatment of donors with CSF1-Fc increased the number and reconstitution potential of HSPC in blood following a HSC mobilising regimen of G-CSF treatment. Conclusion These results indicate that CSF1-Fc conditioning could represent a therapeutic strategy to overcome poor HSC mobilisation and subsequently improve HSC transplantation outcomes.

International audience; Purpose of Review: Neurogenic heterotopic ossification (NHO) is the abnormal formation of extra-skeletal bones in periarticular muscles after damage to the central nervous system (CNS) such as spinal cord injury (SCI), traumatic brain injury (TBI), stroke, or cerebral anoxia. The purpose of this review is to summarize recent developments in the understanding of NHO pathophysiology and pathogenesis. Recent animal models of NHO and recent findings investigating the communication between CNS injury, tissue inflammation, and upcoming NHO therapeutics are discussed. Recent Findings: Animal models of NHO following TBI or SCI have shown that NHO requires the combined effects of a severe CNS injury and soft tissue damage, in particular muscular inflammation and the infiltration of macrophages into damaged muscles plays a key role. In the context of a CNS injury, the inflammatory response to soft tissue damage is exaggerated and persistent with excessive signaling via substance P-, oncostatin M-, and TGF-β1-mediated pathways. Summary: This review provides an overview of the known animal models and mechanisms of NHO and current therapeutic interventions for NHO patients. While some of the inflammatory mechanisms leading to NHO are common with other forms of traumatic and genetic heterotopic ossifications (HO), NHOs uniquely involve systemic changes in response to CNS injury. Future research into these CNS-mediated mechanisms is likely to reveal new targetable pathways to prevent NHO development in patients.

The erythroblastic island (EBI) is a multicellular structure forming an erythropoietic niche consisting of a central macrophage surrounded by a rosette of maturing erythroblasts. Since their discovery more than 60 years ago, simultaneous quantification and visualization of EBIs remain difficult. Although flow cytometry enables high-throughput quantification of cell aggregates co-expressing macrophage and erythroblast markers, it cannot visually confirm whether the aggregates are genuine EBIs. While immunofluorescence microscopy allows visualization of EBIs, its low throughput limits its use for quantification. In the current study we employed nine-channel imaging flow cytometry (IFC) to develop a method to directly visualize and quantify EBIs in the mouse bone marrow. We found that EBI central macrophages do express F4/80, VCAM-1, and CD169, but not CD11b or Ly6G, and that CD11b+Ly6G+F4/80– granulocytes are found associated at the periphery of 40%–60% EBIs. Furthermore, we show for the first time using IFC that in vivo treatment with the hematopoietic stem cell-mobilizing cytokine granulocyte colony-stimulating factor (G-CSF) reduced EBI frequency in the bone marrow by more than 100-fold. These results indicate that mobilizing doses of G-CSF cause a collapse of EBIs in the bone marrow.

It has recently emerged that tissue-resident macrophages are key regulators of several stem cell niches orchestrating tissue formation during development, as well as postnatally, when they also organize the repair and regeneration of many tissues including the hemopoietic tissue. The fact that macrophages are also master regulators and effectors of innate immunity and inflammation allows them to coordinate hematopoietic response to infections, injuries, and inflammation. After recently reviewing the roles of phagocytes and macrophages in regulating normal and pathologic hematopoietic stem cell niches, we now focus on the key roles of macrophages in regulating erythropoiesis and iron homeostasis. We review herein the recent advances in understanding how macrophages at the center of erythroblastic islands form an erythropoietic niche that controls the terminal differentiation and maturation of erythroblasts into reticulocytes; how red pulp macrophages in the spleen control iron recycling and homeostasis; how these macrophages coordinate emergency erythropoiesis in response to blood loss, infections, and inflammation; and how persistent infections or inflammation can lead to anemia of inflammation via macrophages. Finally, we discuss the technical challenges associated with the molecular characterization of erythroid island macrophages and red pulp macrophages.

The bone marrow (BM) contains a mosaic of niches specialized in supporting different maturity stages of hematopoietic stem and progenitor cells such as hematopoietic stem cells and myeloid, lymphoid, and erythroid progenitors. Recent advances in BM imaging and conditional gene knockout mice have revealed that niches are a complex network of cells of mesenchymal, endothelial, neuronal, and hematopoietic origins, together with local physicochemical parameters. Within these complex structures, phagocytes, such as neutrophils, macrophages, and dendritic cells, all of which are of hematopoietic origin, have been found to be important in regulating several niches in the BM, including hematopoietic stem cell niches, erythropoietic niches, and niches involved in endosteal bone formation. There is also increasing evidence that these macrophages have an important role in adapting hematopoiesis, erythropoiesis, and bone formation in response to inflammatory stressors and play a key part in maintaining the integrity and function of these. Likewise, there is also accumulating evidence that subsets of monocytes, macrophages, and other phagocytes contribute to the progression and response to treatment of several lymphoid malignancies such as multiple myeloma, Hodgkin lymphoma, and non-Hodgkin lymphoma, as well as lymphoblastic leukemia, and may also play a role in myelodysplastic syndrome and myeloproliferative neoplasms associated with Noonan syndrome and aplastic anemia. In this review, the potential functions of macrophages and other phagocytes in normal and pathologic niches are discussed, as are the challenges in studying BM and other tissue-resident macrophages at the molecular level.

Current in vivo models for investigating human primary bone tumors and cancer metastasis to the bone rely on the injection of human cancer cells into the mouse skeleton. This approach does not mimic species-specific mechanisms occurring in human diseases and may preclude successful clinical translation. We have developed a protocol to engineer humanized bone within immunodeficient hosts, which can be adapted to study the interactions between human cancer cells and a humanized bone microenvironment in vivo. A researcher trained in the principles of tissue engineering will be able to execute the protocol and yield study results within 4-6 months. Additive biomanufactured scaffolds seeded and cultured with human bone-forming cells are implanted ectopically in combination with osteogenic factors into mice to generate a physiological bone 'organ', which is partially humanized. The model comprises human bone cells and secreted extracellular matrix (ECM); however, other components of the engineered tissue, such as the vasculature, are of murine origin. The model can be further humanized through the engraftment of human hematopoietic stem cells (HSCs) that can lead to human hematopoiesis within the murine host. The humanized organ bone model has been well characterized and validated and allows dissection of some of the mechanisms of the bone metastatic processes in prostate and breast cancer.

Skeletal metastases present a major clinical challenge for prostate cancer patient care, inflicting distinctive mixed osteoblastic and osteolytic lesions that cause morbidity and refractory skeletal complications. Macrophages are abundant in bone and bone marrow and can influence both osteoblast and osteoclast function in physiology and pathology. Herein, we examined the role of macrophages in prostate cancer bone lesions, particularly the osteoblastic response. First, macrophage and lymphocyte distributions were qualitatively assessed in patient's prostate cancer skeletal lesions by immunohistochemistry. Second, macrophage functional contributions to prostate tumour growth in bone were explored using an immune-competent mouse model combined with two independent approaches to achieve in vivo macrophage depletion: liposome encapsulated clodronate that depletes phagocytic cells (including macrophages and osteoclasts); and targeted depletion of CD169(+) macrophages using a suicide gene knock-in model. Immunohistochemistry and histomorphometric analysis were performed to quantitatively assess cancer-induced bone changes. In human bone metastasis specimens, CD68(+) macrophages were consistently located within the tumour mass. Osteal macrophages (osteomacs) were associated with pathological woven bone within the metastatic lesions. In contrast, lymphocytes were inconsistently present in prostate cancer skeletal lesions and when detected, had varied distributions. In the immune-competent mouse model, CD169(+) macrophage ablation significantly inhibited prostate cancer-induced woven bone formation, suggesting that CD169(+) macrophages within pathological woven bone are integral to tumour-induced bone formation. In contrast, pan-phagocytic cell, but not targeted CD169(+) macrophage depletion resulted in increased tumour mass, indicating that CD169(-) macrophage subset(s) and/or osteoclasts influenced tumour growth. In summary, these observations indicate a prominent role for macrophages in prostate cancer bone metastasis that may be therapeutically targetable to reduce the negative skeletal impacts of this malignancy, including tumour-induced bone modelling. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The unique interactions of leukemia cells with the bone marrow (BM) microenvironment (niche) are critical for disease progression and resistance to treatment. We have recently found that the vascular adhesion molecule E-selectin is a key niche component mediating acute myeloid leukemia (AML) chemoresistance, highlighting E-selectin as a promising therapeutic target. In this study, we found canonical E-selectin receptors CD44 and CD162 to be crucial for E-selectin adhesion, as mouse AML cells lacking both receptors failed to bind to E-selectin. We then developed an in vitro model to assess the chemo-sensitivity of mouse AML blasts adhering to various vascular adhesion molecules; this showed that E-selectin uniquely boosts AML cell survival to chemotherapy, but only when CD44/CD162 are present. Likewise when transplanted into recipient mice, CD44/CD162-/- AML cells were significantly more sensitive to chemotherapy compared to wildtype AML. Together these results suggest that CD44 and/or CD162 are key E-selectin receptors involved in AML chemoresistance. To validate these findings in human, we used CRISPR-Cas9 gene editing to selectively suppress CD44 or CD162 from the human AML cell line KG1a. Using our in vitro chemo-sensitivity assay, we showed that E-selectin could not promote AML survival in the absence of either CD44 or CD162, confirming our findings in mice. However interestingly, KG1a cells could still bind to E-selectin in the absence of CD44 or CD162, suggesting the involvement of several E-selectin receptors that can play different roles – either binding or signalling. To conclude, we described a novel form of niche-mediated chemoresistance that can be modelled in vitro, and identified CD44 and CD162 as key AML cell surface receptors involved. These findings highlight blockade of E-selectin or its receptors as a novel strategy to improve the treatment of AML.

Despite positive testing in animal studies, more than 80% of novel drug candidates fail to proof their efficacy when tested in humans. This is primarily due to the use of preclinical models that are not able to recapitulate the physiological or pathological processes in humans. Hence, one of the key challenges in the field of translational medicine is to “make the model organism mouse more human.” To get answers to questions that would be prognostic of outcomes in human medicine, the mouse's genome can be altered in order to create a more permissive host that allows the engraftment of human cell systems. It has been shown in the past that these strategies can improve our understanding of tumor immunology. However, the translational benefits of these platforms have still to be proven. In the 21st century, several research groups and consortia around the world take up the challenge to improve our understanding of how to humanize the animal's genetic code, its cells and, based on tissue engineering principles, its extracellular microenvironment, its tissues, or entire organs with the ultimate goal to foster the translation of new therapeutic strategies from bench to bedside. This article provides an overview of the state of the art of humanized models of tumor immunology and highlights future developments in the field such as the application of tissue engineering and regenerative medicine strategies to further enhance humanized murine model systems. Stem Cells 2015;33:1696–1704

PURPOSE OF REVIEW The nature and function of macrophages at the center of erythroblastic islands is not fully understood. This review discusses novel findings on the phenotypic and molecular characterization of erythroblastic island macrophages, and their role in regulating normal and pathological erythropoiesis. RECENT FINDINGS The phenotype to prospectively isolate erythroblastic island macrophages from mouse bone marrow has been identified. In-vivo depletion of erythroblastic island macrophages causes blockade of erythroblast maturation and delays erythropoietic recovery following chemical insults. The cytokine granulocyte colony-stimulating factor arrests medullary erythropoiesis by depleting erythroblastic island macrophages from the bone marrow. In-vivo ablation of macrophages improves anemia associated with β-thalassemia and reduces red blood cell counts in the mouse model of polycythemia vera. The role of cell adhesion molecules regulating interactions between erythroblastic island macrophages and erythroblasts has been clarified, and mechanisms of pyrenocyte engulfment by erythroblastic island macrophages have been demonstrated to involve Mer tyrosine kinase receptor. SUMMARY Prospective isolation of mouse erythroblastic island macrophages together with new genetic mouse models to specifically target erythroblastic island macrophages will enable molecular studies to better define their role in controlling erythroblast maturation. These studies have revealed the key role of erythroblastic island macrophages in regulating normal erythropoiesis and could be interesting targets to treat β-thalassemia or polycythemia vera.

Neurological heterotopic ossification (NHO) is the abnormal formation of bone in soft tissues as a consequence of spinal cord or traumatic brain injury. NHO causes pain, ankyloses, vascular and nerve compression and delays rehabilitation in this high-morbidity patient group. The pathological mechanisms leading to NHO remain unknown and consequently there are no therapeutic options to prevent or reduce NHO. Genetically modified mouse models of rare genetic forms of heterotopic ossification (HO) exist, but their relevance to NHO is questionable. Consequently, we developed the first model of spinal cord injury (SCI)-induced NHO in genetically unmodified mice. Formation of NHO, measured by micro-computed tomography, required the combination of both SCI and localized muscular inflammation. Our NHO model faithfully reproduced many clinical features of NHO in SCI patients and both human and mouse NHO tissues contained macrophages. Muscle-derived mesenchymal progenitors underwent osteoblast differentiation in vitro in response to serum from NHO mice without additional exogenous osteogenic stimuli. Substance P was identified as a candidate NHO systemic neuropeptide, as it was significantly elevated in the serum of NHO patients. However, antagonism of substance P receptor in our NHO model only modestly reduced the volume of NHO. In contrast, ablation of phagocytic macrophages with clodronate-loaded liposomes reduced the size of NHO by 90%, supporting the conclusion that NHO is highly dependent on inflammation and phagocytic macrophages in soft tissues. Overall, we have developed the first clinically relevant model of NHO and demonstrated that a combined insult of neurological injury and soft tissue inflammation drives NHO pathophysiology.

Granulocyte colony-stimulating factor (G-CSF) is used routinely in the clinical setting to mobilize hematopoietic stem progenitor cells (HSPCs) into the patient's blood for collection and subsequent transplantation. However, a significant proportion of patients who have previously received chemotherapy or radiotherapy and require autologous HSPC transplantation cannot mobilize the minimal threshold of mobilized HSPCs to achieve rapid and successful hematopoietic reconstitution. Although several alternatives to the G-CSF regime have been tested, few are used in the clinical setting. We have shown previously in mice that administration of prolyl 4-hydroxylase domain enzyme (PHD) inhibitors, which stabilize hypoxia-inducible factor (HIF)-1α, synergize with G-CSF in vivo to enhance mouse HSPC mobilization into blood, leading to enhanced engraftment via an HSPC-intrinsic mechanism. To evaluate whether PHD inhibitors could be used to enhance mobilization of human HSPCs, we humanized nonobese, diabetic severe combined immune-deficient Il2rg

The MYB oncogene is widely expressed in acute leukemias and is important for the continued proliferation of leukemia cells, suggesting that MYB may be a therapeutic target in these diseases. However, realization of this potential requires a significant therapeutic window for MYB inhibition, given its essential role in normal hematopoiesis, and an approach for developing an effective therapeutic. We previously showed that the interactionof c-MybwiththecoactivatorCBP/p300 is essential for its transforming activity. Here, by using cells from Booreana mice which carry a mutant allele of c-Myb, we show that this interaction is essential for in vitro transformation by the myeloid leukemia oncogenes AML1-ETO, AML1-ETO9a, MLL-ENL, and MLL-AF9. We further show that unlike cells from wild-type mice, Booreana cells transduced with AML1-ETO9a or MLL-AF9 retroviruses fail to generate leukemia upon transplantation into irradiated recipients. Finally, we have begun to explore themolecularmechanisms underlying these observations by gene expression profiling. This identified several genes previously implicated in myeloid leukemogenesis and HSC function as being regulated in a c-Myb-p300-dependent manner. These data highlight the importance of the c-Myb-p300 interaction inmyeloid leukemogenesis and suggest disruption of this interaction as a potential therapeutic strategy for acute myeloid leukemia. Refereed/Peer-reviewed

Potent functional HSCs are enriched at the endosteal bone marrow (BM), which comprises ∼10% of total BM. To identify novel niche factors that regulate HSCs, we performed a gene expression microarray seeking genes overexpressed in the endosteal relative to central BM. Prostacyclin/prostaglandin I2 (PGI2) synthase (Ptgis) was one of the highest enriched genes in the endosteal versus central BM (27-fold). PTGIS is the sole enzyme for biosynthesis of PGI2. PGI2 has no reported roles in HSC regulation in the BM and was chosen for further investigation. We found PGI2 analogue iloprost treatment of sorted BM lineage- Kit+ Sca1- (LKS+) cells in vitro for 7 days potently reduced proliferation and differentiation compared to vehicle control. BM cells pulse treated ex vivo with iloprost for 1 hour also resulted in 14-fold increased multilineage reconstitution potential compared to vehicle controls in competitive transplant assay. Iloprost administration in vivo partially rescued BM HSC reconstitution potential in mice sub-lethally (6.5 Gy) irradiated or administered pro-inflammatory granulocyte colony stimulating factor (G-CSF) for 3 days. These data suggest that PGI2 protects HSC from stress. Mechanistically, we found iloprost pulse treatment in vitro was associated with increased pSTAT3 and cell cycle progression in HSCs. Additionally, we found ∼4-fold greater proportion of untreated LKS+ HSPC homing to the BM of recipients co-administered iloprost + G-CSF compared to vehicle + G-CSF at 4 hours post-transplant. We found that G-CSF administration also decreased Scf and Cxcl12 mRNA expression in BM, which were partially rescued with iloprost co-administration. These data suggest that PGI2 protects HSC during stress by acting directly on HSCs and indirectly to preserve BM niche functions. In summary, we identified PGI2 as a novel HSC niche factor abundant in the endosteal BM.

Neurological heterotopic ossifications (NHO) are frequent complications of traumatic brain and spinal cord injuries (SCI). NHO are abnormal ossifications within periarticular muscles resulting in joint ankylosis, vessel and nerve compression. To elucidate NHO pathogenesis, we have developed the first murine model of SCI-NHO, in which NHO is induced in cardiotoxin-injured muscle only following a SCI. However, what stem cell populations within muscle differentiate into osteoblasts and directly contribute to NHO is unclear. We hypothesized that injured muscles do not repair normally after SCI, and instead osteogenic differentiation of skeletal muscle-resident stem cells takes place. We focused on the two progenitor populations known to reside in muscles: satellite cells (SCs) and fibro- adipogenic progenitors (FAPs). SCs, which specifically express the transcription factor Pax7, differentiate into myoblasts, whereas FAPs, which express are of mesenchymal origin and express Prrx1+ instead, can differentiate into fibroblasts and adipocytes in vitro. We had shown that both populations can deposit calcium under osteogenic condition in vitro. We have now generated Pax7CreER x R26-ZsGreen (Pax7ZsG) and Prrx1Cre x R26-ZsGreen (Prrx1ZsG) to specifically trace the fate of SCs and FAPs respectively in our NHO model in vivo. We demonstrate that after SCI, Pax7+ SCs fail to proliferate and regenerate myofibers with instead persistent proliferation of Prrx1+ FAPs differentiating into osteocalcin+ osteoblasts in SCI-NHO at 21 days post-surgery. The above suggest that SCI causes muscle repair failure with instead proliferation and osteogenic differentiation of FAPs residing in the muscle. In addition, SCI significantly upregulated platelet-derived growth factor receptor (PDGFR) expression in FAPs indicating dysregulation of PDGFR signalling may be involved in the NHO development.

The central macrophage (MΦ) in erythroblastic islands (EI) is critical to erythropoiesis by providing iron and growth factors, and mediating enucleation of the maturing erythroblasts. As MΦ are key effectors of inflammation, we investigated the effect of lipopolysaccharides (LPS) on erythropoiesis in vivo. LPS (2.5mg/kg/d for 2 days) in C57BL/6 mice caused the whitening of the bone marrow (BM) with dramatically decreased erythroblast and reticulocyte numbers. Imaging flow cytometry was used to visualize structural changes and quantify numbers of EI. LPS treatment in vivo caused a marked loss of EI in the BM. This suppressive effect of LPS on BM erythropoiesis was TLR4-dependent as it was absent in TLR4 KO mice. By qRT-PCR and flow cytometry, TLR4 is not expressed by BM erythroblasts but by myeloid cells including EI MΦ. Together with the fact that addition of 100ng/mL LPS into BFU-E assays does not inhibit BFU-E colony growth, these suggest that the suppressive effect of LPS on erythroblasts is indirectly mediated. It is known that LPS mobilizes HSC in a G-CSF-dependent manner. As MΦ express the G-CSF receptor (GCSFR), we explored LPS effects in GCSFR KO mice. Surprisingly, although HSC did not mobilize in response to LPS in GCSFR KO mice, BM erythropoiesis was still suppressed with reduced numbers of erythroblasts. Unexpectedly however, the BM from LPS-treated GCSFR KO mice was still red with high numbers of erythrocytes. The explanation of this paradox is that LPS treatment causes BM vascular leakage in GCSFR KO mice with blood plasma volume in the BM 2.9-fold higher compared to LPS-treated WT mice (measured by i.v. injection of Evans Blue). In conclusion our data show that LPS suppresses medullar erythropoiesis indirectly in a G-CSF-independent manner but GCSFR-mediated signaling is necessary to maintain the integrity of the BM vasculature in response to LPS.

Stem cells and their malignant counterparts require the support of a specific microenvironment or "niche". While various anti-cancer therapies have been broadly successful, there are growing opportunities to target the environment in which these cells reside to further improve therapeutic efficacy and outcome. This is particularly true when the aim is to target normal or malignant stem cells. The field aiming to target or use the niches that harbor, protect, and support stem cells could be designated as "nichotherapy". In this essay, we provide a few examples of nichotherapies. Some have been employed for decades, such as hematopoietic stem cell mobilization, whereas others are emerging, such as chemosensitization of leukemia stem cells by targeting their niche.

This review evaluates the latest information on the mobilisation of haemopoietic stem cells for transplantation, with the focus on what is the current best practice and how new understanding of the bone marrow stem cell niche provides new insights into optimising mobilisation regimens. The review then looks at the mobilisation of mesenchymal stromal cells, immune cells as well as malignant cells and what clinical implications there are.

Mobilization and collection of haemopoietic stem and progenitor cells (HSPC) is the cornerstone of autologous and allogeneic stem cell transplantation for a wide variety of haematological and some non-haematological malignancies. Centres providing this service face the challenge of optimizing the likelihood of successful collection of transplantable doses of cells, while maximizing the efficiency of the apheresis unit and minimizing the risk of toxicity as well as mobilization failure. Recent developments in the understanding of the molecular mechanisms of mobilization have led to the emergence of novel strategies for HSPC mobilization, which may assist in meeting these imperatives. The task for clinicians is how to incorporate the use of these strategies into practice, in the light of emerging evidence for efficacy and safety of these agents. Herein, the literature is reviewed, and a proposed algorithm for HSPC mobilization is presented.

The concept of hematopoietic stem cell (HSC) niche was formulated in 1978, but HSC niches remained unidentified for the following two decades largely owing to technical limitations. Sophisticated live microscopy techniques and genetic manipulations have identified the endosteal region of the bone marrow (BM) as a preferential site of residence for the most potent HSC - able to reconstitute in serial transplants - with osteoblasts and their progenitors as critical cellular elements of these endosteal niches. This article reviews the path to the discovery of these endosteal niches (often called 'osteoblastic' niches) for HSC, what cell types contribute to these niches with their known physical and biochemical features. In the past decade, a first wave of research uncovered many mechanisms responsible for HSC homing to, and mobilization from, the whole BM tissue. However, the recent discovery of endosteal HSC niches has initiated a second wave of research focusing on the mechanisms by which most primitive HSC lodge into and migrate out of their endosteal niches. The second part of this article reviews the current knowledge of the mechanisms of HSC lodgment into, retention in and mobilization from osteoblastic niches.

We compared potential trafficking mechanisms used by human (h) multipotent mesenchymal stem cells (MSC) derived from bone marrow (bm) or placenta (p). Both hbmMSC and hpMSC expressed a broad range of cell surface adhesion molecules including beta1-integrins (CD29) and CD44. Array data showed that both hbmMSC and hpMSC expressed mRNA for the cell adhesion molecules CD54 (ICAM-1), E-cadherin, CD166 (ALCAM), CD56 (NCAM), CD106 (VCAM-1), CD49a, b, c, e and f (integrins alpha1, 2, 3, 4 and 6), integrin alpha11, CD51 (integrin alphaV), and CD29 (integrins beta1). Functional binding of hpMSC, but not hbmMSC to VCAM-1 was demonstrated using recombinant chimeric constructs. Neither bone marrow nor placental MSC expressed ligands to endothelial selectins such as PSGL-1 or sialyl Lewis X (sLe(x)) carbohydrates and neither were able to bind functionally to chimeric constructs of the endothelial selectins CD62E (E-selectin) and CD62P (P-selectin). Furthermore, MSC expressed a restricted range of transferases necessary for expression of sLe(x), with no detectable expression of fucosyl transferases IV or VII. Placental MSC, but not hbmMSC, expressed mRNA for the chemokine receptors CCR1 and CCR3, and both hbmMSC and hpMSC expressed mRNA for CCR7, CCR8, CCR10, CCR11, CXCR4 and CXCR6. Intracellular chemokine receptor protein expression of CCR1, CCR3, CXCR3, CXCR4 and CXCR6 was detected in both hbmMSC and hpMSC. Cell surface expression of chemokine receptors was much more restricted with only CXCR6 displaying a strong signal on hbmMSC and hpMSC. Although cell surface expression of CXCR4 was not detected, MSC migrated in response to its ligand, CXCL12 (SDF-1). Thus, hbmMSC and hpMSC have an almost identical profile for cell surface adhesion and chemokine receptor molecules at the mRNA and protein levels. However, at the functional level, hpMSC likely utilise VLA-4-mediated binding in a superior manner to hbmMSC and thus may have superior engraftment properties to hbmMSC in vivo.

Despite the fact that many hypoxia-inducible genes are important in hematopoiesis, the spatial distribution of oxygen in the bone marrow (BM) has not previously been explored in vivo. Using the hypoxia bioprobe pimonidazole, we showed by confocal laser scanning microscopy that the endosteum at the bone-BM interface is hypoxic, with constitutive expression of hypoxia-inducible transcription factor-1α (HIF-1α) protein in steady-state mice. Interestingly, at the peak of hematopoietic stem and progenitor cell (HSPC) mobilization induced by either granulocyte colony-stimulating factor or cyclophosphamide, hypoxic areas expand through the central BM. Furthermore, we found that HSPC mobilization leads to increased levels of HIF-1α protein and increased expression of vascular endothelial growth factor A (VEGF-A) mRNA throughout the BM, with an accumulation of VEGF-A protein in BM endothelial sinuses. VEGF-A is a cytokine known to induce stem cell mobilization, vasodilatation, and vascular permeability in vivo. We therefore propose that the expansion in myeloid progenitors that occurs during mobilization depletes the BM hematopoietic microenvironment of O2, leading to local hypoxia, stabilization of HIF-1α transcription factor in BM cells, increased transcription of VEGF-A, and accumulation of VEGF-A protein on BM sinuses that increases vascular permeability.Disclosure of potential conflicts of interest is found at the end of this article.

Endolyn (CD164) is a sialomucin that functions as an adhesion molecule and a negative regulator of CD34+ CD38- human haematopoietic precursor cell proliferation. The 105A5 and 103B2/9E10 CD164 monoclonal antibodies (mAbs), which act as surrogate ligands, recognize distinct glycosylation-dependent classes I and II epitopes located on domain I of the native and recombinant CD164 proteins. Here, we document five new CD164 mAbs, the 96 series, that rely on conformational integrity, but not glycosylation, of exons 2- and 3-encoded CD164 domains, thereby resembling the class III mAbs, N6B6 and 67D2. Although all the 96 series class III mAbs labelled both the 105A5+ and 103B2/9E10+ cells, cross-competition and immunoblotting studies allow them to be categorized into two distinct class III subgroups, i.e. the N6B6-like subgroup that only recognizes 80-100 kDa proteins and the 67D2-like subgroup that also recognizes a higher molecular weight (>220 kDa) form. To more closely define the reactivity patterns of mAbs to the classes I and II epitopes, the global glycosylation patterns of the soluble human (h) CD164 proteins were determined using lectin binding, high-performance liquid chromatography (HPLC) and mass spectrometry. hCD164 recombinant proteins bound to the lectins, Galanthus nivalis agglutinin, Datura stramonium agglutinin, Sambucus nigra agglutinin, Maackia amurensis agglutinin and peanut agglutinin, indicating the presence of high mannose and complex N-glycans, in addition to core 1 O-glycans (the Tn antigen) and alpha2-3 and alpha2-6 sialic acid moieties. Our HPLC and mass spectrometry results revealed both high mannose and complex N-glycosylation with various numbers of branches increasing the complexity of the glycosylation pattern. Most O-glycans were small, core 1 or 2 based. High levels of sialylation in alpha2-3 and alpha2-6 linkages, without sialyl-Lewis X, indicate that the majority of these hCD164 recombinant proteins are unable to bind to selectins in our assay system, but may interact with Siglec molecules.

Objective. The twoendothelial selectins, P- and E-selectin, are critically importantfor adhesion and homing of hematopoietic progenitor cells (HPC) into the bone marrow. Little is known, however, about the roles of these two selectins in hematopoiesis. Here, we demonstrate that the most primitive HPC capable of long-term in vivo repopulation express P-selectin glycoprotein ligand-1/CD162 (PSGL-1), a receptor common to both P- and E-selectin. In addition, we demonstrate that P-selectin delays the differentiation of HPC whereas E-selectin enhances their differentiation along the monocyte/granulocyte pathway, describing different roles for these selectins in the regulation of hematopoiesis. Materials and Methods. Murine bone marrow HPC were isolated according to their expression of c-kit and PSGL-1, transplanted into lethally irradiated congenic recipients, and chimerism analyzed6monthsposttransplant.Bonemarrowlineage-negative(Lin)Sca-1c-kitcellswere then cultured on immobilized P- or E-selectin for 4 weeks in the presence of cytokines. Hematopoieticpotentialwasassessedusinginvitrophenotypingandcolony-formingassaysand in vivo spleen colony-forming unit (CFU-S) and long-term competitive repopulation assays. Results. Long-term competitive repopulating HSCs were Linc-kit bright and expressed intermediate levels of PSGL-1. Both P- and E-selectin slowed the proliferation of LinSca-1ckit cells during the first two weeks of liquid culture. After two weeks, however, cells cultured on immobilized P-selectin showed increased proliferation with increased production of both colony-forming cells (CFC) and CFU-S12 compared to the other cultures. In contrast, E-selectin enhanced the differentiation of LinSca-1c-kit cells into cells that expressed the granulocytematurationmarker,Gr-1,accompaniedbylossofCFCpotentialfromthesecultured cells. Finally, the long-term repopulation potential of these cells was not maintained following culture on either selectin. Conclusion. These results suggest that the two endothelial selectins, E-selectin and P-selectin, have very different effects on HPC. E-selectin accelerates the differentiation of maturing HPC towards granulocyte and monocyte lineages while maintaining the production of more immature CFU-S12 in ex vivo liquid suspension culture. In marked contrast, P-selectin delays the differentiation of LinSca-1c-kit cells, allowing enhanced ex vivo expansion of CFC and CFU-S12 but not HSCs. 2005 International Society for Experimental Hematology. Published by Elsevier Inc.

For decades it has been assumed that lethal irradiation in bone marrow transplantation experiments ablates the entire host hematopoietic system but preserves the host non-hematopoietic bone marrow stroma. This postulate is at the basis of transplantation chimera experiments in mice to test involvement of hematopoietic cells versus non-hematopoietic stroma. As BM-Macs support HSC niche homeostasis, we examined whether host-derived BM-Macs persist lethal irradiation and play a role in HSC engraftment. Recipient MacGreen mice (expressing GFP in myeloid cells under the control of Csf1r promoter) were lethally irradiated (11.5Gy) and transplanted with sorted syngeneic B6.SJL CD45.1+ Lin-Kit+Sca1+ sorted cells. Flow cytometry analyses of BM 2-30 weeks (wk) post-transplant confirmed more than 99% donor chimerism of monocytes and granulocytes validating ablation of recipient HSC. In contrast, GFP+CD11b+F4/80+CD169+VCAM-1+ERHR3+Ly6Gneg recipient BM-Macs were detected throughout the time-course. A 5.9 fold expansion of these recipient BM-Macs occurred between wk 2 and 5 ( from 45,000 to 270,000 cells/femur) post-transplant which coincided with increased number of phenotypic donor HSC (GFP-Lin-Kit+Sca1+CD48-CD150+). Host BM-Mac proliferation was cell autonomous in the absence of host HSC and granulocytes. Recipient Macs in spleen displayed different frequency and longevity kinetics that correlated with transient post-Tx splenic extramedullary haematopoiesis. In situ, GFP+F480+ recipient BM-Macs were enriched in perivascular microenvironments within both central BM and endosteal regions. These GFP+ host-derived macrophages persisted long-term forming 8.4% of BM macrophages 7months post-transplant. To evaluate the importance of these host-derived radiation-resistant macrophages, we selectively depleted recipient BM-Macs using CD169-DTR mice transplanted with syngeneic ubiquitous GFP+ HSC. Depletion of recipient CD169+ Macs abated engraftment of donor phenotypic HSC by 70% at 5 wk post-transplant and reduced BM reconstitution potential in competitive secondary transplants. In conclusion BM contains a myeloablation-resistant self-repopulating Mac subset that is necessary for efficient and/or sustained HSC BM engraftment following transplantation. Therefore interpretation of transplantation chimera experiments must take into account the persistence of this subset of host macrophages that support donor HSC engraftment.

Objective. Administration of granulocyte colony-stimulating factor (G-CSF) results in the mobilization of hematopoietic progenitor and stem cells from the bone marrow into the peripheral blood. Although the mechanisms leading to the mobilization of primitive hematopoietic cells is not fully understood, it has been noted that the yield of mobilization in humans is correlated to the down-regulation of c-KIT/CD117 expression on mobilized cells. We sought to determine the mechanisms responsible for the reduced expression of c-KIT on mobilized hematopoietic progenitor cells. Materials and Methods. Mice were mobilized with G-CSF and primitive hematopoietic cells were collected from bone marrow and blood to analyze c-KIT expression. Using cell lines expressing mouse and human c-KIT and a recombinant protein comprising the entire extracellular domain of human c-KIT, we analyzed by flow cytometry and immunoblotting the proteolytic cleavage of c-KIT by proteases released in bone marrow extracellular fluids extracted from mobilized mice. Results. Administration of G-CSF into mice results in the reduction of c-KIT expression on primitive hematopoietic cells in bone marrow and peripheral blood. Bone marrow extracellular fluids isolated from G-CSF-mobilized mice contain serine proteases that cleave c-KIT into discrete fragments. Proteases capable of cleaving c-KIT include neutrophil elastase, cathepsin G, proteinase-3 and matrix metalloproteinase-9. Conclusions. In addition to transcriptional controls, exocytosis, and ligand-induced internalization, the direct proteolytic cleavage of c-KIT by neutrophil and macrophage proteases represents a novel pathway to regulate the levels of c-KIT expression at the surface of hematopoietic cells and may be responsible in part for the down-regulation of c-KIT expression on mobilized hematopoietic progenitors in vivo.

Advances in tissue-engineering have resulted in a versatile tool-box to specifically design a tailored microenvironment for hematopoietic stem cells (HSCs) in order to study diseases that develop within this setting. However, most current in vivo models fail to recapitulate the biological processes seen in humans. Here we describe a highly reproducible method to engineer humanized bone constructs that are able to recapitulate the morphological features and biological functions of the HSC niches. Ectopic implantation of biodegradable composite scaffolds cultured for 4 weeks with human mesenchymal progenitor cells and loaded with rhBMP-7 resulted in the development of a chimeric bone organ including a large number of human mesenchymal cells which were shown to be metabolically active and capable of establishing a humanized microenvironment supportive of the homing and maintenance of human HSCs. A syngeneic mouse-to-mouse transplantation assay was used to prove the functionality of the tissue-engineered ossicles. We predict that the ability to tissue engineer a morphologically intact and functional large-volume bone organ with a humanized bone marrow compartment will help to further elucidate physiological or pathological interactions between human HSCs and their native niches.

Objective. Hematopoietic stem and progenitor cells normally reside in the bone marrow but can be mobilized into the peripheral blood following treatment with granulocyte colony-stimulating factor (G-CSF) or myelosuppressive chemotherapy. Although the number of transplants performed with mobilized blood currently exceeds those performed with bone marrow, little is known of the molecular mechanisms responsible for this phenomenon. We sought to determine whether mobilization induced by G-CSF or chemotherapy was triggered by common or distinct mechanisms. Methods. Balb/c mice were mobilized with either G-CSF alone, cyclophosphamide alone, or the combination of both agents. Spleens, peripheral blood, bone marrow extracellular fluids, and cells were taken at different time points and analyzed for the expression of VCAM-1, the number of peripheral blood progenitor cells, concentration of neutrophil proteases, and number of granulocytes. Results. Administration of either G-CSF or the myelosuppressive agent cyclophosphamide results in a sharp reduction of VCAM-1/CD106 expression in the bone marrow that coincides with the accumulation of granulocytic precursors and release of active neutrophil proteases neutrophil elastase and cathepsin G that directly cleave VCAM-1/CD106 in vitro. These events follow precisely the kinetics of hematopoietic progenitor cell mobilization into the peripheral blood. Conclusion. We have identified a commonality of events during mobilization induced by either G-CSF or chemotherapy, which include the accumulation in the bone marrow of active neutrophil proteases that directly cleave VCAM-1 and lead to the sharp reduction of VCAM-1 expression in this tissue.

The International Society of Experimental Hematology holds its annual meeting every northern summer. This year the meeting comprised of eight plenary sessions with distinguished invited speakers on in vivo imaging and tracking of hematopoietic stem cells (HSCs), HSC niches, epigenetic regulations of stem cells and regulation of stem cell fate, leukemogenesis, and mesenchymal stem cells. The small size of the meeting (300 attendees) permitted excellent discussion and face-to-face contacs between students, junior scientists and experts. Owing to the large number of keynote speakers, this report focuses on the most novel, unpublished data presented during the meeting.

Anti-cancer chemotherapy drugs challenge hematopoietic tissues to regenerate, but commonly produce long-term sequelae. Deficits in hematopoietic stem or stromal cell function have been described, but the mechanisms mediating chemotherapy-induced hematopoietic dysfunction remain unclear. Administration of multiple cycles of cisplatin chemotherapy causes significant sensory neuropathy. Here, we demonstrate that chemotherapy-induced nerve injury in the bone marrow is a critical lesion impairing hematopoietic regeneration. We show using various pharmacological and genetic models that the selective loss of adrenergic innervation in the BM alters its regeneration following genotoxic insult. Sympathetic nerves in the marrow promote the survival of stem cell niche constituents that initiate recovery. Neuroprotection by deletion of Trp53 in sympathetic neurons or neuro-regeneration using 4-methylcatechol or glial-derived neurotrophic factor (GDNF) administration can restore hematopoietic recovery. Thus, these results shed light on the potential benefit of adrenergic nerve protection to shield hematopoietic niches from injury.

Harvest of granulocyte colony-stimulating factor (G-CSF)-mobilized hematopoietic stem cells (HSCs) begins at day 5 of G-CSF administration, when most donors have achieved maximal mobilization. This is based on surrogate markers for HSC mobilization, such as CD34(+) cells and colony-forming activity in blood. However, CD34(+) cells or colony-forming units in culture (CFU-C) are heterogeneous cell populations with hugely divergent long-term repopulation potential on transplantation. HSC behavior is influenced by the vascular bed in the vicinity of which they reside. We hypothesized that G-CSF may mobilize sequentially cells proximal and more distal to bone marrow venous sinuses where HSCs enter the blood. We addressed this question with functional serial transplantation assays using blood and bone marrow after specific time points of G-CSF treatment in mice. We found that in mice, blood collected after only 48 hours of G-CSF administration was as enriched in serially reconstituting HSCs as blood collected at 5 days of G-CSF treatment. Similarly, mobilized Lin(-)CD34(+) cells were relatively enriched in more primitive Lin(-)CD34(+)CD38(-) cells at day 2 of G-CSF treatment compared with later points in half of human donors tested (n = 6). This suggests that in both humans and mice, hematopoietic progenitor and stem cells do not mobilize uniformly according to their maturation stage, with most potent HSCs mobilizing as early as day 2 of G-CSF.

Hematopoietic stem cell (HSC) regulation is highly dependent on interactions with the marrow microenvironment. Controversy exists on N-cadherin's role in support of HSCs. Specifically, it is unknown whether microenvironmental N-cadherin is required for normal marrow microarchitecture and for hematopoiesis. To determine whether osteoblastic N-cadherin is required for HSC regulation, we used a genetic murine model in which deletion of Cdh2, the gene encoding N-cadherin, has been targeted to cells of the osteoblastic lineage. Targeted deletion of N-cadherin resulted in an age-dependent bone phenotype, ultimately characterized by decreased mineralized bone, but no difference in steady-state HSC numbers or function at any time tested, and normal recovery from myeloablative injury. Intermittent parathyroid hormone (PTH) treatment is well established as anabolic to bone and to increase marrow HSCs through microenvironmental interactions. Lack of osteoblastic N-cadherin did not block the bone anabolic or the HSC effects of PTH treatment. This report demonstrates that osteoblastic N-cadherin is not required for regulation of steady-state hematopoiesis, HSC response to myeloablation, or for rapid expansion of HSCs through intermittent treatment with PTH.