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Hematopoiesis support by MSCs in LTBMC BFU-E, CFU-G, CFU-M and CFU-GM by PMF (n=3) and healthy donors (HD, n=3) expressed as number of colonies for 104 bone marrow mononuclear cells.

Supplementary Table 6 from FLT3-Mediated p38–MAPK Activation Participates in the Control of Megakaryopoiesis in Primary Myelofibrosis

Supplementary Table 2 from FLT3-Mediated p38–MAPK Activation Participates in the Control of Megakaryopoiesis in Primary Myelofibrosis

Supplementary Table 3 from FLT3-Mediated p38–MAPK Activation Participates in the Control of Megakaryopoiesis in Primary Myelofibrosis

Supplementary Figure 1 from FLT3-Mediated p38–MAPK Activation Participates in the Control of Megakaryopoiesis in Primary Myelofibrosis

Supplementary Table 1 from FLT3-Mediated p38–MAPK Activation Participates in the Control of Megakaryopoiesis in Primary Myelofibrosis

Supplementary Table 5 from FLT3-Mediated p38–MAPK Activation Participates in the Control of Megakaryopoiesis in Primary Myelofibrosis

Supplementary Methods, Table Legends 1-6 from FLT3-Mediated p38–MAPK Activation Participates in the Control of Megakaryopoiesis in Primary Myelofibrosis

Supplementary Table 4 from FLT3-Mediated p38–MAPK Activation Participates in the Control of Megakaryopoiesis in Primary Myelofibrosis

Bone marrow is a complex and dynamic microenvironment that provides essential cues to resident cells. We developed a standardized three-dimensional (3D) model to decipher mechanisms that control human cells during hematological and non-hematological processes. Our simple 3D-model is constituted of a biphasic calcium phosphate-based scaffold and human cell lines to ensure a high reproducibility. We obtained a minimal well-organized bone marrow-like structure in which various cell types and secreted extracellular matrix can be observed and characterized by

Purpose Bone marrow response to an organismal stress is made by orchestrating the interplay between hematopoietic stem/progenitor cells (HSPCs) and mesenchymal stromal cells (MSCs). Neither the cellular nor the molecular factors that regulate this process are fully understood, especially since this mechanism probably varies depending on the type of stress. Herein, we explored the differentiation and fate of MSCs and HSPCs in mice challenged with a hematopoietic stress or a mechanical stress applied separately or in combination. Methods Mice were subjected to 4 days of hypobaric hypoxia (hematopoietic challenge) and/or 7 days of hindlimb suspension (stromal challenge) and then sacrificed for blood and bone collection. Using hematological measurements, colony-forming unit assays, bone histomorphometry and array-based multiplex ELISA analysis, we evaluated challenge influences on both MSC and HSPC mobilization, differentiation (osteoblasts, osteoclasts, and mature blood cells) and fate. Results We found that hypoxia leads to HSPC mobilization and that an imbalance between bone formation and bone resorption accounts for this mobilization. Whilst suspension is also associated with an imbalance between bone formation and bone resorption, it does not induce HSPC mobilization. Then, we revealed cellular interactions by combining hematopoietic and stromal challenges together in mice. We showed that the hypoxia-driven HSPC mobilization is moderated by suspension. Moreover, when applied in a hypoxic environment, suspension offsets bone imbalance. We identified stroma cell-derived factors MIP-1α, HGF and SDF-1 as potent molecular key players sustaining interactions between hindlimb suspension and hypobaric hypoxia. Conclusion Taken together, our data highlight the benefit of combining different types of stress to better understand the interplay between MSCs and HSPCs.

International audience; Hematopoiesis and bone interact in various developmental and pathological processes. Neurogenic heterotopic ossifications (NHO) are the formation of ectopic hematopoietic bones in peri-articular muscles that develop following severe lesions of the central nervous system such as traumatic cerebral or spinal injuries or strokes. This review will focus on the hematopoietic facet of NHO. The characterization of NHO demonstrates the presence of hematopoietic marrow in which quiescent hematopoietic stem cells (HSC) are maintained by a functional stromal microenvironment, thus documenting that NHOs are neo-formed ectopic HSC niches. Similarly to adult bone marrow, the NHO permissive environment supports HSC maintenance, proliferation and differentiation through bidirectional signaling with mesenchymal stromal cells and endothelial cells, involving cell adhesion molecules, membrane-bound growth factors, hormones, and secreted matrix proteins. The participation of the nervous system, macrophages and inflammatory cytokines including oncostatin M and transforming growth factor (TGF)-β in this process, reveals how neural circuitry fine-tunes the inflammatory response to generate hematopoietic bones in injured muscles. The localization of NHOs in the peri-articular muscle environment also suggests a role of muscle mesenchymal cells and bone metabolism in development of hematopoiesis in adults. Little is known about the establishment of bone marrow niches and the regulation of HSC cycling during fetal development. Similarities between NHO and development of fetal bones make NHOs an interesting model to study the establishment of bone marrow hematopoiesis during development. Conversely, identification of stage-specific factors that specify HSC developmental state during fetal bone development will give more mechanistic insights into NHO.

The bone marrow (BM) microenvironment plays a crucial role in the development and progression of leukemia (AML). Intracellular reactive oxygen species (ROS) are involved in the regulation of the biology of leukemia-initiating cells, where the antioxidant enzyme GPx-3 could be involved as a determinant of cellular self-renewal. Little is known however about the role of the microenvironment in the control of the oxidative metabolism of AML cells. In the present study, a coculture model of BM mesenchymal stromal cells (MSCs) and AML cells (KG1a cell-line and primary BM blasts) was used to explore this metabolic pathway. MSC-contact, rather than culture with MSC-conditioned medium, decreases ROS levels and inhibits the Nrf-2 pathway through overexpression of GPx3 in AML cells. The decrease of ROS levels also inactivates p38MAPK and reduces the proliferation of AML cells. Conversely, contact with AML cells modifies MSCs in that they display an increased oxidative stress and Nrf-2 activation, together with a concomitant lowered expression of GPx-3. Altogether, these experiments suggest that a reciprocal control of oxidative metabolism is initiated by direct cell&ndash, cell contact between MSCs and AML cells. GPx-3 expression appears to play a crucial role in this cross-talk and could be involved in the regulation of leukemogenesis.

Resume Le microenvironnement medullaire, ou niche hematopoietique, est une structure complexe. A la fois spatiale et temporelle, elle est constituee d’elements cellulaires, matriciels et cytokiniques qui participent au maintien des fonctions des cellules souches hematopoietiques et a la regulation des processus de proliferation et de differenciation. Ce microenvironnement est un acteur essentiel des hemopathies. Les observations dans les modeles murins ont permis d’affirmer son role capital dans l’emergence, le maintien et la resurgence des clones hematologiques pathologiques. Dans les neoplasies myeloproliferatives, les alterations du microenvironnement sont en particulier reliees aux phenomenes inflammatoires. Une meilleure connaissance des anomalies des interactions entre ces differents elements devrait permettre de faire emerger de nouvelles pistes therapeutiques.

We investigated noninvasive procedures by hybrid imaging to assess the sites of active or inactive hematopoiesis in patients with primary myelofibrosis (PMF). To this end, we used two radionuclides, technetium 99m ((99m) Tc) and indium 111-chloride ((111) In-Cl3 ), coupled with single-photon emission tomography/computed tomography (SPECT/CT). We studied five patients with PMF and one with secondary myelofibrosis (MF). The classical pattern of lower fixation of both tracers at the axial skeleton where the myelofibrotic process occurs and the reactivation of sites of active hematopoiesis at the distal skeleton were confirmed. Coupling both radionuclides to SPECT/CT imaging allowed for more precise visualization of the sites of extramedullary hematopoiesis as those observed in the spleen and liver. Splenic high uptake of (111) In-Cl3 coupled with SPECT/CT represents a pathognomonic feature of PMF. We conclude that, the hybrid imaging procedures that we studied might constitute an alternative noninvasive method for the screening of the whole-body marrow and, by this way, to assess the impact of targeted therapies in PMF patients in whom it is well known that the distribution of the hematopoietic active areas is disturbed. Hybrid imaging could also be useful for diagnostic purposes in cases of early PMF or in suspected cases of myelofibrosis secondary to polycythemia vera or essential thrombocythemia.

Primary myelofibrosis is a myeloproliferative neoplasm that is a precursor to myeloid leukemia. Dysmegakaryopoiesis and extramedullary hematopoiesis characterize primary myelofibrosis, which is also associated with bone marrow stromal alterations marked by fibrosis, neoangiogenesis, and osteomyelosclerosis. In particular, contributions to primary myelofibrosis from mesenchymal stromal cells (MSC) have been suggested by mouse studies, but evidence in humans remains lacking. In this study, we show that bone marrow MSCs from primary myelofibrosis patients exhibit unique molecular and functional abnormalities distinct from other myeloproliferative neoplasms and these abnormalities are maintained stably ex vivo in the absence of leukemic cells. Primary myelofibrosis-MSC overexpressed heparin-binding cytokines, including proinflammatory TGFβ1 and osteogenic BMP-2, as well as glycosaminoglycans such as heparan sulfate and chondroitin sulfate. Transcriptome and functional analyses revealed alterations in MSC differentiation characterized by an increased osteogenic potential and a TGFβ1 signaling signature. Accordingly, phospho-Smad2 levels were intrinsically increased in primary myelofibrosis-MSC along with enhanced expression of the master bone regulator RUNX2, while inhibition of the endogenous TGFβ1 receptor TGFβR1 impaired osteogenic differentiation in these MSCs. Taken together, our results define the source of a critical osteogenic function in primary myelofibrosis that supports its pathophysiology, suggesting that combined targeting of both the hematopoietic and stromal cell compartments in primary myelofibrosis patients may heighten therapeutic efficacy. Cancer Res; 75(22); 4753–65. ©2015 AACR.

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.

We investigated the effects of respiratory hypobaric hypoxia on femoral bone-defect repair in mice because hypoxia is believed to influence both mesenchymal stromal cell (MSC) and hematopoietic stem cell mobilization, a process involved in the bone-healing mechanism. To mimic conditions of non-weight-bearing limb immobilization in patients suffering from bone trauma, our hypoxic mouse model was further subjected to hind-limb unloading. A hole was drilled in the right femur of adult male C57/BL6J mice. Four days after surgery, mice were subjected to hind-limb unloading for 1 week. Seven days after surgery, mice were either housed for 4 days in a hypobaric room (FiO2 at 10%) or kept under normoxic conditions. Unsuspended control mice were housed in either hypobaric or normoxic conditions. Animals were sacrificed on postsurgery day 11 to allow for collection of both contralateral and lesioned femurs, blood, and spleen. As assessed by microtomography, delayed hypoxia enhanced bone-healing efficiency by increasing the closing of the cortical defect and the newly synthesized bone volume in the cavity by +55% and +35%, respectively. Proteome analysis and histomorphometric data suggested that bone-repair improvement likely results from the acceleration of the natural bone-healing process rather than from extended mobilization of MSC-derived osteoprogenitors. Hind-limb unloading had hardly any effect beyond delayed hypoxia-enhanced bone-healing efficiency.

In addition to its well-known effect on migration and homing of hematopoietic stem/progenitor cells (HSPCs), CXCL12 chemokine also exhibits a cell cycle and survival-promoting factor for human CD34(+) HSPCs. CXCR4 was suggested to be responsible for CXCL12-induced biological effects until the recent discovery of its second receptor, CXCR7. Until now, the participation of CXCR7 in CXCL12-induced HSPC cycling and survival is unknown. We show here that CXCL12 was capable of binding CXCR7 despite its scarce expression at CD34(+) cell surface. Blocking CXCR7 inhibited CXCL12-induced Akt activation as well as the percentage of CD34(+) cells in cycle, colony formation, and survival, demonstrating its participation in CXCL12-induced functional effects in HSPCs. At steady state, CXCR7 and β-arrestin2 co-localized near the plasma membrane of CD34(+) cells. After CXCL12 treatment, β-arrestin2 translocated to the nucleus, and this required both CXCR7 and CXCR4. Silencing β-arrestin expression decreased CXCL12-induced Akt activation in CD34(+) cells. Our results demonstrate for the first time the role of CXCR7, complementary to that played by CXCR4, in the control of HSPC cycling, survival, and colony formation induced by CXCL12. We also provide evidence for the involvement of β-arrestins as signaling hubs downstream of both CXCL12 receptors in primary human HSPCs.

Introduction/Background We previously reported the development of a new acquired neurogenic HO (NHO) mouse model, combining spinal cord transection (SCI) and chemical muscle injury. Pathological mechanisms responsible for ectopic osteogenesis after central neurological damage are still to be elucidated. In this study, we first hypothesized that peripheral nervous system (PNS) might convey pathological signals from injured spinal cord to muscles. Secondly, we sought to determine whether SCI could lead to intramuscular modifications of BMP2 signalling pathways. Material and method Twenty-one C57Bl6 mice were included in this protocol. Bilateral cardiotoxin (CTX) injection in hamstring muscles was associated with a two-stage surgical procedure, combining thoracic SCI with unilateral peripheral denervation. Volumes of HO (bone volume, BV) were measured 28 days after surgery using micro-computed tomography imaging techniques and histological analyses were made to confirm intramuscular osteogenesis. Volume comparisons were conducted between right and left hind limb of each animal, using a Wilcoxon signed rank test. Quantitative polymerase chain reaction (qPCR) was performed to explore intra muscular expression of BMP2, Alk3 and Id1. Results Nineteen mice survive the complete SCI and peripheral denervation procedure. When CTX injections were done right after surgery (n = 7), bilateral HO were detected in all animals after 28 days. Micro-CT measurements showed significantly increased BV in denervated paws (1.47 mm3 ± 0.5) compared to contralateral sides (0.56 mm3 ± 0.4), P = 0.03. When peripheral denervation and CTX injections were performed after sham SCI surgery (n = 6), bilateral HO were present in three mice at day 28. qPCR analyses showed no changes in intra muscular BMP2 expression after SCI as compared to control mice. Conclusion Peripheral denervation can be reliably added to spinal cord transection in NHO mouse model. This new experimental design confirms that neuro-inflammatory mechanisms induced by central or peripheral nervous system injury plays a key role in triggering ectopic osteogenesis.

Resume Les cellules stromales mesenchymateuses (CMS) sont des cellules capables d’autorenouvellement et de differenciation en cellules des lignees osteoblastiques, chondrocytaires et adipocytaires a minima. Leur capacite d’adressage vers un tissu lese (homing), leur faible immunogenicite permettant d’envisager une utilisation allogenique, et l’absence de considerations ethiques liees a leur origine expliquent l’interet que suscitent ces cellules en medecine regenerative depuis maintenant deux decennies. Recemment les notions portant sur les communications qu’elles etablissent au sein des niches hematopoietiques ont ete etendues a un nombre important de types cellulaires. Une partie du benefice therapeutique lie a l’utilisation de ces cellules a pu etre expliquee par leurs proprietes trophiques, qui ont de plus ete a l’origine d’un changement du paradigme concernant leurs utilisations cliniques potentielles. Parallelement aux premiers essais cliniques il y a une dizaine d’annees, la question des risques lies a l’embolisation vasculaire apres injection de ces cellules, ainsi que celle, plus inquietante, de la possible transformation cancereuse des cellules amplifiees en culture ont suscite de vifs debats, sans que le rapport benefice-risque ne penche pour l’instant en defaveur de l’utilisation therapeutique des CSM. Au cours de cette revue nous rappellerons la definition admise pour qu’une population cellulaire puisse etre denommee CSM, les origines tissulaires de ces cellules en privilegiant celles accessibles en clinique, les essais cliniques en cours ainsi que les perspectives therapeutiques.

Identification of prevalent specific markers is crucial to stem/progenitor cell purification. Determinants such as the surface antigens CD34 and CD38 are traditionally used to analyze and purify hematopoietic stem/progenitor cells (HSCs/HPCs). However, the variable expression of these membrane antigens poses some limitations to their use in HSC/HPC purification. Techniques based on drug/stain efflux through the ATP-binding cassette (ABC)G2 pump (side population [SP] phenotype) or on detection of aldehyde dehydrogenase (ALDH) activity have been independently developed and distinguish the SP and ALDHBright (ALDHBr) cell subsets for their phenotype and proliferative capability. In this study, we developed a multiparametric flow cytometric method associating both SP and ALDH activities on human lineage negative (Lin−) bone marrow cells and sorted different cell fractions according to their SP/ALDH activity level. We find that Lin−CD34+CD38Low/− cells are found throughout the spectrum of ALDH expression and are enriched especially in ALDHBr cells when associated with SP functionality (SP/ALDHBr fraction). Furthermore, the SP marker identified G0 cells in all ALDH fractions, allowing us to sort quiescent cells regardless of ALDH activity. Moreover, we show that, within the Lin−CD34+CD38−ALDHBr population, the SP marker identifies cells with higher primitive characteristics, in terms of stemness-related gene expression and in vitro and in vivo proliferative potential, than the Lin−CD34+ CD38−ALDHBr main population cells. In conclusion, our study shows that the coexpression of SP and ALDH markers refines the Lin−CD34+CD38− hematopoietic compartment and identifies an SP/ALDHBr cell subset enriched in quiescent primitive HSCs/HPCs.

Objective Expression of tissue inhibitors of matrix metalloproteinases (TIMPs) is one way that activated platelets intervene in tissue remodeling and angiogenesis. Our study was designed to investigate their synthesis in megakaryocytes (MKs) and their storage in platelets. Materials and Methods TIMP expression in MKs derived from blood CD34 + progenitor cells of normal donors and a megakaryocytic cell line (CHRF-288-11) grown in serum-free conditions and platelets from normal donors or two patients with gray platelet syndrome was studied by immunofluorescence labeling, reverse transcription-polymerase chain reaction, and western blotting. Results Biosynthesis of TIMPs 1−4 in MKs was indicated by presence of their messenger RNAs as shown by polymerase chain reaction and of their proteins. Immunofluorescence labeling suggested a primarily granular localization of TIMPs in MKs and platelets. But when colocalization with von Willebrand factor, fibrinogen, P-selectin, and other α-granule proteins was assessed in platelets by confocal microscopy, TIMP-1, -2, and -4 were localized as distinct fluorescent patches apart from the established α-granule markers and largely independent of platelet metalloproteinases. TIMP-3 differed for it also had an α-granule location. Western blotting confirmed the presence of TIMPs 1−4 in platelets and thrombin activation resulted in their extensive release to the medium. Platelets from two patients with gray platelet syndrome, congenitally deficient in α-granules, showed sparse labeling of von Willebrand factor and fibrinogen confined to vestigial α-granules; however, localization of the TIMPs was unchanged. Conclusions TIMPs are synthesized and organized in MKs and platelets independently of other secreted proteins present in α-granule pools.

Cell cycle regulation plays a fundamental role in stem cell biology. A balance between quiescence and proliferation of hematopoietic stem cells in interaction with the microenvironment is critical for sustaining long-term hematopoiesis and for protection against stress. We analyzed the molecular mechanisms by which stromal cell-derived factor-1 (SDF-1) exhibited a cell cycle-promoting effect and interacted with transforming growth factor-β (TGF-β), which has negative effects on cell cycle orchestration of human hematopoietic CD34+ progenitor cells. We demonstrated that a low concentration of SDF-1 modulated the expression of key cell cycle regulators such as cyclins, cyclin-dependent kinase inhibitors, and TGF-β target genes, confirming its cell cycle-promoting effect. We showed that a cross-talk between SDF-1- and TGF-β-related signaling pathways involving phosphatidylinositol 3-kinase (PI3K)/Akt phosphorylation participated in the control of CD34+ cell cycling. We demonstrated a pivotal role of two downstream effectors of the PI3K/Akt pathway, FoxO3a and mammalian target of rapamycin, as connectors in the SDF-1-/TGF-β-induced control of the cycling/quiescence switch and proposed a model integrating a dialogue between the two molecules in cell cycle progression. Our data shed new light on the signaling pathways involved in SDF-1 cell cycle-promoting activity and suggest that the balance between SDF-1- and TGF-β-activated pathways is critical for the regulation of hematopoietic progenitor cell cycle status. Disclosure of potential conflicts of interest is found at the end of this article.

Constitutive mobilization of CD34+ cells in patients with primary myelofibrosis (PMF) has been attributed to proteolytic disruption of the CXCR4/SDF-1 axis and reduced CXCR4 expression. We document here that the number of circulating CD34+/CXCR4+ cells in PMF patients, as well as the cellular CXCR4 expression, was directly related to CXCR4 mRNA level and that reduced CXCR4 mRNA level was not due to SDF-1-induced downregulation. To address whether epigenetic regulation contributes to defective CXCR4 expression, we studied the methylation status of the CXCR4 promoter using methylation-specific polymerase chain reaction and methylation-specific sequencing in the JAK2V617F-positive HEL cell line and in CD34+ cells. We found that CD34+ cells from PMF patients, unlike those from normal subjects, presented hypermethylation of CXCR4 promoter CpG island 1. Following incubation with the demethylating agent 5-Aza-2′-deoxycytidine (5-AzaD), the percentage of PMF CD34+ cells expressing CXCR4 increased 3–10 times, whereas CXCR4 mRNA level increased approximately 4 times. 5-AzaD-treated PMF CD34+ cells displayed almost complete reversal of CpG1 island 1 hypermethylation and showed enhanced migration in vitro in response to SDF-1. These data point to abnormal methylation of the CXCR4 promoter as a mechanism contributing to constitutive migration of CD34+ cells in PMF.Disclosure of potential conflicts of interest is found at the end of this article.

Myelofibrosis with myeloid metaplasia (MMM) is a rare myeloproliferative disorder which associates clonal proliferation of multi-potent hematologic progenitors and reactive fibrosis [1]. The karyot...

International audience; Primary myelofibrosis (PMF) is a clonal myeloproliferative neoplasm whose severity and treatment complexity are attributed to the presence of bone marrow (BM) fibrosis and alterations of stroma impairing the production of normal blood cells. Despite the recently discovered mutations including the JAK2V617F mutation in about half of patients, the primitive event responsible for the clonal proliferation is still unknown. In the highly inflammatory context of PMF, the presence of fibrosis associated with a neoangiogenesis and an osteosclerosis concomitant to the myeloproliferation and to the increase number of circulating hematopoietic progenitors suggests that the crosstalk between hematopoietic and stromal cells is deregulated in the PMF BM microenvironmental niches. Within these niches, mesenchymal stromal cells (BM-MSC) play a hematopoietic supportive role in the production of growth factors and extracellular matrix which regulate the proliferation, differentiation, adhesion and migration of hematopoietic stem/progenitor cells. A transcriptome analysis of BM-MSC in PMF patients will help to characterize their molecular alterations and to understand their involvement in the hematopoietic stem/progenitor cell deregulation that features PMF.

The stromal cell-derived factor 1 (SDF-1) chemokine has various effects on hematopoietic cell functions. Its role in migration and homing of hematopoietic progenitors is currently well established. Previously it was shown that SDF-1 stimulates myeloid progenitor proliferation in synergy with cytokines. Results of this study indicate that SDF-1 alone promotes survival of purified CD34(+) cells from human unmobilized peripheral blood (PB) by counteracting apoptosis as demonstrated by its capacity to reduce DNA fragmentation, annexin-V(+) cell number, and APO2.7 detection and to modulate bcl-2 homolog protein expression. The study demonstrates that SDF-1, produced by sorted CD34(+)CD38(+) cells and over-released in response to cell damage, exerts an antiapoptotic effect on CD34(+) cells through an autocrine/paracrine regulatory loop. SDF-1 participates in the autonomous survival of circulating CD34(+) cells and its effect required activation of the phosphotidyl inositol 3 kinase (PI3-K)/Akt axis. Cell sorting based on Hoechst/pyroninY fluorescences shows that SDF-1 production is restricted to cycling CD34(+) cells. SDF-1 triggers G(0) quiescent cells in G(1) phase and, in synergy with thrombopoietin or Steel factor, makes CD34(+) cells progress through S+G(2)/M phases of cell cycle. By assessing sorted CD34(+)CD38(-) and CD34(+)CD38(+) in semisolid culture, the study demonstrates that SDF-1 promotes survival of clonogenic progenitors. In conclusion, the results are the first to indicate a role for endogenous SDF-1 in primitive hematopoiesis regulation as a survival and cell cycle priming factor for circulating CD34(+) cells. The proposal is made that SDF-1 may contribute to hematopoiesis homeostasis by participating in the autonomous survival and cycling of progenitors under physiologic conditions and by protecting them from cell aggression in stress situations.

Primary myelofibrosis is characterized by clonal myeloproliferation, dysmegakaryopoiesis, extramedullary hematopoiesis associated with myelofibrosis and altered stroma in the bone marrow and spleen. The expression of CD9, a tetraspanin known to participate in megakaryopoiesis, platelet formation, cell migration and interaction with stroma, is deregulated in patients with primary myelofibrosis and is correlated with stage of myelofibrosis. We investigated whether CD9 participates in the dysmegakaryopoiesis observed in patients and whether it is involved in the altered interplay between megakaryocytes and stromal cells. We found that CD9 expression was modulated during megakaryocyte differentiation in primary myelofibrosis and that cell surface CD9 engagement by antibody ligation improved the dysmegakaryopoiesis by restoring the balance of MAPK and PI3K signaling. When co-cultured on bone marrow mesenchymal stromal cells from patients, megakaryocytes from patients with primary myelofibrosis displayed modified behaviors in terms of adhesion, cell survival and proliferation as compared to megakaryocytes from healthy donors. These modifications were reversed after antibody ligation of cell surface CD9, suggesting the participation of CD9 in the abnormal interplay between primary myelofibrosis megakaryocytes and stroma. Furthermore, silencing of CD9 reduced CXCL12 and CXCR4 expression in primary myelofibrosis megakaryocytes as well as their CXCL12-dependent migration. Collectively, our results indicate that CD9 plays a role in the dysmegakaryopoiesis that occurs in primary myelofibrosis and affects interactions between megakaryocytes and bone marrow stromal cells. These results strengthen the “bad seed in bad soil” hypothesis that we have previously proposed, in which alterations of reciprocal interactions between hematopoietic and stromal cells participate in the pathogenesis of primary myelofibrosis.

The chemokine stromal cell-derived factor-1 (SDF-1), and its receptor, CXCR-4, have been implicated in the homing and mobilization of human CD34(+) cells. We show here that SDF-1 may also be involved in hematopoiesis, promoting the proliferation of human CD34(+) cells purified from normal adult peripheral blood (PB). CXCR-4 was expressed on PB CD34(+) cells. The amount of CXCR-4 on PB CD34(+) cells was 10 times higher when CD34(+) cells were purified following overnight incubation. CXCR-4 overexpression was correlated with a primitive PB CD34(+) cell subset defined by a CD34(high) CD38(low)CD71(low)c-Kit(low)Thy-1(+) antigenic profile. The functional significance of CXCR-4 expression was ascertained by assessing the promoting effect of SDF-1alpha on cell cycle, proliferation, and colony formation. SDF-1 alone increased the percentage of CD34(+) cells in the S+G(2)/M phases and sustained their survival. In synergy with cytokines, SDF-1 increased PB CD34(+) and CD34(high)CD38(low) cell expansion and colony formation. SDF-1 also stimulated the growth of colonies derived from primitive progenitors released from quiescence by anti-TGF-beta treatment. Thus, our results shed new light on the potential role of this chemokine in the stem cell engraftment process, which involves migration, adhesion, and proliferation. Furthermore, both adhesion-induced CXCR-4 overexpression and SDF-1 stimulating activity may be of clinical relevance for improving cell therapy settings in stem cell transplantation.

HLA-DR is one of the markers associated with hematopoietic cell differentiation, since expression of this molecule is modulated throughout hematopoiesis. We have previously described and cloned the gene encoding factor IK, which inhibits both interferon gamma (IFN-γ)-induced and constitutive HLA-DR expression. The current study demonstrates that IK gene transcripts are present in CD34+ cells purified from human umbilical cord blood. IK expression increased and was therefore inversely correlated with the gradual loss of HLA-DR during growth factor–induced CD34+ cell proliferation and differentiation. To study the possible role of IK in hematopoiesis, antisense probes were used. IK expression was specifically inhibited by an antisense oligodeoxynucleotide containing two phosphorothioate internucleotide linkages at each of the 3′ and 5′ ends and corresponding to the initiation site of IK mRNA. A control oligonucleotide was also tested in parallel. A specific decrease of IK transcripts was correlated with an increase of HLA-DR antigen expression level. In colony-forming assays, IK antisense oligonucleotide inhibited colony formation by multilineage early erythroid and granulomonocytic CD34+ progenitors. The mean colony size was decreased 70% by IK antisense oligonucleotide in comparison to controls. These results provide evidence that the IK molecule participates in the regulation of HLA-DR expression on hematopoietic cells and plays a role in growth factor–dependent CD34+ cell proliferation and differentiation by modulating HLA-DR expression.

Heterotopic ossification (HO) is a pathology characterized by the formation of ectopic bone in soft tissues generally in muscles surrounding joints such hip, knee, elbow or shoulder. The etiology of this pathology is not known but it is well established that the risk of HO is increased after traumatic brain or spinal cord injuries and also after fractures or burns. Patients with HOs suffer from pain and the range of motion from limbs with ectopic bone is highly reduced. Currently, the only effective treatment for HOs is surgery but recurrences are frequent underpinning that a better understanding of its pathophysiology is required to cure patients. Hematopoietic marrow-like tissue is frequently observed on histological section of HO. Therefore, we investigated whether HOs from patients with traumatic brain or spinal cord injury represent an ectopic model of hematopoietic stem cell (HSC) niche as defined by the presence of HSC-supportive stromal cells. HOs were obtained from patients after surgical resection in Garches Hospital (France) with their informed consent. HOs were cut in small pieces and hematopoietic as well as endothelial cells were extracted from the HO marrow by gentle washing. Mononuclear cells were then recovered by ficoll gradient and were either directly analyzed by flow cytometry or purified by immunomagnetic selection for in vitro and in vivo experiments. Mesenchymal stromal cells (MSCs) were obtained by plastic adherence from HO explants. We first confirmed the presence of hematopoietic cells from different lineages in HO marrow such as neutrophils, monocytes/macrophages, B and T lymphocytes as evidenced by membrane expression of CD45, CD15, CD11/CD14, CD19, CD3/CD4 and CD3/CD8 antigens. HSC presence within HO marrow was further demonstrated by: (i) in vitro clonogenic assays, (ii) presence of quiescent CD45+CD34+CD38- cells with a side-population phenotype and, (iii) in vivo human hematopoietic reconstitution assays in immunodeficient NSG mice. We also show that human HO marrow contained functional MSCs and endothelial progenitors (EPs) as demonstrated by phenotypic and functional analyses. MSC expressing the classical mesenchymal markers CD73, CD90, CD105 and capable to in vitro and in vivo differentiate into osteoblasts, adipocytes and chondrocytes could be isolated from human HOs. These MSCs were also able to support in vitro long term human hematopoiesis and in vivo murine hematopoiesis when seeded on hydroxyapatite scaffolds and implanted into nude mice. Endothelial progenitors with a characteristic CD31+CD144+CD34+CD45-CD90+ phenotype were detected in the mononuclear cell fraction from HO marrows. After sorting, these cells were able to form colonies on plastic and vascular networks when cultured in a Matrigel and to express high levels of VCAM-1 and ICAM-1 adhesion molecules after TNFα stimulation, confirming their EP functional capability. In conclusion, we show for the first time that human HOs contain a marrow tissue with quiescent HSC that can proliferate and differentiate within a suitable and functional mesenchymal and endothelial microenvironment, acknowledging that HO marrows are ectopic HSC niches. We have recently demonstrated the involvement of macrophages in the development of neurogenic HOs in spinalized mice1. Taking into account the role of immune cells and the neurogenic origin of human HOs, our results bring new evidences for the role of interactions between bone and neuro-immune systems in the initiation and development of adult hematopoiesis. Mechanisms underlying these processes are currently studied in our laboratory. 1 Genet F et al., J Pathol. 2015 Jun;236(2):229-40 Disclosures No relevant conflicts of interest to declare.

International audience; Primary Myelofibrosis (PMF) is a chronic myeloproliferative neoplasm characterized by a clonal myeloproliferation and a myelofibrosis. The concomitant presence of neoangiogenesis and osteosclerosis suggests a deregulation of medullar stem cell niches in which hematopoietic stem cells are engaged in a constant crosstalk with their stromal environment. Despite the recently discovered mutations including the JAK2Val617F mutation, the primitive molecular event responsible for the clonal hematopoietic proliferation is still unknown. We propose that the "specificity" of the pathological process that caracterizes PMF results from alterations in the cross talk between hematopoietic and stromal cells. These alterations contribute in creating a abnormal microenvironment that participates in the maintenance of the neoplasic clone leading to a misbalance disfavouring normal hematopoiesis; in return or simultaneously, stromal cells constituting the niches are modulated by hematopoietic cells resulting in stroma dysfunctions. Therefore, PMF is a remarkable "model" in which deregulation of the stem cell niche is of utmost importance for the disease development. A better understanding of the crosstalk between stem cells and their niches should imply new therapeutic strategies targeting not only intrinsic defects in stem cells but also regulatory niche-derived signals and, consequently, hematopoietic cell proliferation.

Activating mutations in signaling molecules, such as JAK2-V617F, have been associated with myeloproliferative neoplasms (MPNs). Mice lacking the inhibitory adaptor protein Lnk display deregulation of thrombopoietin/thrombopoietin receptor signaling pathways and exhibit similar myeloproliferative characteristics to those found in MPN patients, suggesting a role for Lnk in the molecular pathogenesis of these diseases. Here, we showed that LNK levels are up-regulated and correlate with an increase in the JAK2-V617F mutant allele burden in MPN patients. Using megakaryocytic cells, we demonstrated that Lnk expression is regulated by the TPO-signaling pathway, thus indicating an important negative control loop in these cells. Analysis of platelets derived from MPN patients and megakaryocytic cell lines showed that Lnk can interact with JAK2-WT and V617F through its SH2 domain, but also through an unrevealed JAK2-binding site within its N-terminal region. In addition, the presence of the V617F mutation causes a tighter association with Lnk. Finally, we found that the expression level of the Lnk protein can modulate JAK2-V617F–dependent cell proliferation and that its different domains contribute to the inhibition of multilineage and megakaryocytic progenitor cell growth in vitro. Together, our results indicate that changes in Lnk expression and JAK2-V617F–binding regulate JAK2-mediated signals in MPNs.

Embryoid bodies (EBs) generated during differentiation of human embryonic stem cells (hESCs) contain vascular-like structures, suggesting that commitment of mesoderm progenitors into endothelial cells occurs spontaneously. We showed that bone morphogenetic protein 4 (BMP4), an inducer of mesoderm, accelerates the peak expression of CD133/kinase insert domain-containing receptor (KDR) and CD144/KDR. Because the CD133+KDR+ population could represent endothelial progenitors, we sorted them at day 7 and cultured them in endothelial medium. These cells were, however, unable to differentiate into endothelial cells. Under standard conditions, the CD144+KDR+ population represents up to 10% of the total cells at day 12. In culture, these cells, if sorted, give rise to a homogeneous population with a morphology typical of endothelial cells and express endothelial markers. These endothelial cells derived from the day 12 sorted population were functional, as assessed by different in vitro assays. When EBs were stimulated by BMP4, the CD144+KDR+ peak was shifted to day 7. Most of these cells, however, were CD31−, becoming CD31+ in culture. They then expressed von Willebrand factor and were functional. This suggests that, initially, the BMP4-boosted day 7, CD144+KDR+CD31− population represents immature endothelial cells that differentiate into mature endothelial cells in culture. The expression of OCT3/4, a marker of immaturity for hESCs decreases during EB differentiation, decreasing faster following BMP4 induction. We also show that BMP4 inhibits the global expression of GATA2 and RUNX1, two transcription factors involved in hemangioblast formation, at day 7 and day 12. Disclosure of potential conflicts of interest is found at the end of this article.

Primary myelofibrosis (PMF) is the rarest and the most severe Philadelphia-negative chronic myeloproliferative syndrome. By associating a clonal proliferation and a mobilization of hematopoietic stem cells from bone marrow to spleen with profound alterations of the stroma, PMF is a remarkable model in which deregulation of the stem cell niche is of utmost importance for the disease development. This paper reviews key data suggesting that an imbalance between endosteal and vascular niches participates in the development of clonal stem cell proliferation. Mechanisms by which bone marrow niches are altered with ensuing mobilization and homing of neoplastic hematopoietic stem cells in new or reinitialized niches in the spleen and liver are examined. Differences between signals delivered by both endosteal and vascular niches in the bone marrow and spleen of patients as well as the responsiveness of PMF stem cells to their specific signals are discussed. A proposal for integrating a potential role for the JAK2 mutation in their altered sensitivity is made. A better understanding of the cross talk between stem cells and their niche should imply new therapeutic strategies targeting not only intrinsic defects in stem cell signaling but also regulatory hematopoietic niche–derived signals and, consequently, stem cell proliferation.

International audience; MPL (or thrombopoietin receptor, TPO-R) 515 mutations have recently been described in 5-10% of primitive myelofibrosis (PMF) cases as decisive oncogenic events capable of triggering the disease. Here we report additional mutations located in exon 10 of MPL in PMF patients. We investigated whether these new mutations also lead to cell transformation. MPL exon 10 was systematically sequenced in 100 PMF patients. Seven different mutations were found in eight patients. We introduced each MPL mutant in Ba/F3 cells to determine whether they correspond to gain-of-function mutations. Only MPL W515 mutations induced (1) Ba/F3 proliferation independently of growth factors, (2) tumorigenesis in nude mice, (3) spontaneous activation of JAK/STAT, RAS/MAPK and PI3K transduction pathways and (4) increased S phase of cell cycle. Similar to all other myeloproliferative disorder oncogenic events identified to date, these results demonstrate that only the detected MPL W515 mutations trigger spontaneous MPL activation leading to a G(1)/S transition activation. The other mutations are devoid of significant transforming activity but may synergize with JAK2 V617F or other not yet characterized molecular events.