The Tetraspanin CD9 Is Involved in Primary Myelofibrosis Dysmegakaryopoiesis Through c-Myb Regulation and Stroma Interactions,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3834-3834
Author(s):  
Christophe Desterke ◽  
Costanza Bogani ◽  
Lisa Pieri ◽  
Alessandro M. Vannucchi ◽  
Bernadette Guerton ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Migration ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Actin Polymerization ◽  
Primary Myelofibrosis ◽  
Membrane Expression ◽  
Healthy Donors ◽  
Cd34 Cells

Abstract Abstract 3834 Introduction: CD9, a four transmembrane glycoprotein belonging to the tetraspanin family, is suggested to regulate cell motility and adhesion and to play a role in megakaryopoiesis. It has been reported to be a molecular marker of primary myelofibrosis (PMF) being characterized by myeloproliferation, dysmegakaryopoiesis, alterated bone marrow/spleen stroma and extramedullary haematopoiesis. CD9 mRNA has been shown to be overexpressed in CD34+ PMF HPs and its membrane expression level was correlated with platelet counts. Our recent data evidencing an alteration of CD9 expression in PMF megakaryocytes (MK) have encouraged us to investigate whether CD9 participates in the dysmegakaryopoiesis and whether it is involved in the dialogue between MK and stromal cells in PMF patients. Patients and Methods: CD34+ cells were MACS selected from the peripheral blood of PMF patients (n=67) and of unmobilized healthy donors (n=61). Functional studies were performed on MK precursor-derived from CD34+ cells cultured in MK medium with ou without monoclonal antibodies (Syb mAb) or siRNAs targeting CD9. CXCL12-induced MK migration was performed in Boyden chambers. Bone marrow mesenchymal stromal cells (MSC) from healthy donors and PMF patients were cultured in DMEM+10%FCS. Results: Our results showed that CD9 membrane expression was altered on CD34+ cells and on MK precursor-derived from PMF CD34+ cells. Binding of CD9 with Syb mAb restored the in vitro megakaryocyte differentiation process that was altered in patients as shown by an increase in: i) megakaryocytic colony formation in semisolid medium, ii) CD41 and CD62p MK differentiation marker and GATA-1 expression, iii) MK cytoplasmic maturation, iv) apoptotic MK number (reduced AKT phosphorylation and Bcl-XL expression and increased percentage of Annexin+ cells). Activation of CD9 was also associated with regulation of MAPK and AKT-GSK3β pathways whose balance is involved in MK differentiation. Treatment of PMF MK precursors by Syb modulated activation of the MAPK pathway as shown by an increased of p38, JNK and GSK3β phosphorylation and of AP-1 mRNA expression. Taking into account the structure of the tetraspanin molecular network, binding with Syb mAb might also impact the effects associated to the multimolecular complex in which CD9 is involved. This prompted us to study the effects of a molecular silencing of CD9 on the PMF MK differentiation. We showed that, in contrast to the Syb mAb, addition of CD9 siRNA to PMF megakaryocytes reduced their transcriptional program including c-Myb, a transcription factor that is involved in CD9 regulation during megakaryopoiesis. Given the role of CD9 in cell migration, we further investigated whether it could be involved in the megakaryocytic precursor migration observed in patients. We showed that silencing CD9 reduced the CXCL12-dependent megakaryocytic precursor migration as well as the CXCR4 and CXCL12 transcription and that this migration involved actin polymerization. c-Myb siRNA restored CXCR4 and CXCL12 expression and reduced actin polymerization suggesting that CD9 was involved, via c-Myb, in the CXCL12-dependent megakaryocytic precursor migration. Effect of CD9 on cell migration is often interpreted as related to modulation of integrins participating in the integrin/tetraspanin network and of their interaction with mesenchymal stromal cells (MSC). We showed that several genes involving the CD9 partner interactome were over-expressed in MSC from PMF bone marrow as compared to MSC from healthy donors. Preliminary results showing that PMF MK precursors display different behaviour in terms of cell survival and adhesion when co-cultured on bone marrow MSC from PMF patients as compared to healthy donors suggest that interactions between MKs and bone marrow MSC is involved in PMF dysmegakaryopoiesis. Addition of Syb reverses these alterations suggesting the participation of CD9 in the abnormal dialogue between MK and MSC. Conclusion: Our results show a deregulation of CD9 expression in megakaryocytes from PMF patients. They also suggest that CD9 i) participates in PMF dysmegakaryopoieis in terms of MK differentiation and survival and ii) is involved in the increased MK precursor migration through alterations of the CXCL12/CXCR4 axis. Our data further support the role of bone marrow stroma in PMF dysmegakaryopoeisis through CD9 interactions. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Endogenous TGF-beta1 Mediated Osteogenic Impairment of Medullar Mesenchymal Stromal Cells in Primary Myelofibrosis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1873-1873
Author(s):  
Christophe Martinaud ◽  
Christophe Desterke ◽  
Johanna Konopacki ◽  
Lisa Pieri ◽  
Rachel Golub ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasm ◽  
Hematopoietic Cells ◽  
Primary Myelofibrosis ◽  
Osteogenic Potential ◽  
Healthy Donors

Abstract Primary myelofibrosis (PMF) is myeloproliferative neoplasm characterized by clonal myeloproliferation, dysmegakaryopoiesis, extramedullary hematopoiesis associated with myelofibrosis and altered stroma in bone marrow and spleen. Mesenchymal stromal cells (MSCs) are reported to play a pivotal role in fibrosis and stromal changes are considered as a reactive counterpart of the cytokine production by clonal hematopoietic cells. The present study shows that MSCs from patients demonstrate functional abnormalities that are unexpectedly maintained ex-vivo, in culture. Material and Methods: we studied MSCs and bone marrow sections from PMF patients (n=12) as compared to healthy donors (HDs) (n=6). We tested their proliferation, immunophenotype, hematopoiesis supporting capacities, differentiation abilities, in-vivo osteogenic assays, and performed secretome and transcriptome analysis. Results: We found that PMF-MSCs exhibit similar proliferative capacity and long-term hematopoiesis supporting abilities as compare to healthy donors. They overproduce interleukin 6, VEGF, RANTES, PDGF, BMP-2 and surprisingly TGF-beta1. MSCs from fibrotic PMF patients express high levels of glycosaminoglycans. Adipocytes and chondrocytes differentiation abilities were not different as compared to HDs but PMF-MSCs exhibit an increased in vitro potential. Implementation on scaffold in nude mice confirmed, in vivo, this increased osteogenic potential. We then looked into gene expression and discovered that PMF-MSCs show an original transcriptome signature related to osteogenic lineage and TGF-beta1. Indeed, osteogenic genes such as Runx2, Dlx5, Twist1, Noggin, Sclerostin, GDF5 and Serpine1 are deregulated and suggest a potential osteoprogenitor priming of PMF-MSCs. These molecular results also advocated for a TGF-beta1 impregnation that prompted us to study its impact on PMF-MSCs osteogenic differentiation. First, we then showed that Smad2 is intrinsically over-activated in PMF-MSC and that stimulation by TGF-beta1 is associated with an increase phospho-Smad2 level and an enhancement of bone master gene regulator Runx2 expression. Then, we inhibited TGF-beta1 pathway by by SB-431542 and evidenced a specific behavior of osteogenic MSCs differentiation in patients, suggesting involvement of TGF-beta1 in osteogenic impairment. Conclusion: Altogether, our results identify a signature of PMF-MSCs and suggest that they participate in PMF osteogenic dysregulation independently from in vivo local stimulation by clonal hematopoietic cells Disclosures No relevant conflicts of interest to declare.

The role of children's bone marrow mesenchymal stromal cells in the ex vivo expansion of autologous and allogeneic hematopoietic stem cells

2015 ◽  
Vol 39 (10) ◽  
pp. 1099-1110 ◽  
Author(s):  
Iordanis Pelagiadis ◽  
Eftichia Stiakaki ◽  
Christianna Choulaki ◽  
Maria Kalmanti ◽  
Helen Dimitriou
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Stem

scholarly journals Role of neoplastic monocyte-derived fibrocytes in primary myelofibrosis

2016 ◽  
Vol 213 (9) ◽  
pp. 1723-1740 ◽  
Author(s):  
Srdan Verstovsek ◽  
Taghi Manshouri ◽  
Darrell Pilling ◽  
Carlos E. Bueso-Ramos ◽  
Kate J. Newberry ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Growth Factors ◽  
Stromal Cells ◽  
Primary Myelofibrosis ◽  
Neoplastic Disease ◽  
Immunodeficient Mice ◽  
Serum Amyloid P ◽  
Xenograft Mice ◽  
Amyloid P

Primary myelofibrosis (PMF) is a fatal neoplastic disease characterized by clonal myeloproliferation and progressive bone marrow (BM) fibrosis thought to be induced by mesenchymal stromal cells stimulated by overproduced growth factors. However, tissue fibrosis in other diseases is associated with monocyte-derived fibrocytes. Therefore, we sought to determine whether fibrocytes play a role in the induction of BM fibrosis in PMF. In this study, we show that BM from patients with PMF harbors an abundance of clonal, neoplastic collagen- and fibronectin-producing fibrocytes. Immunodeficient mice transplanted with myelofibrosis patients’ BM cells developed a lethal myelofibrosis-like phenotype. Treatment of the xenograft mice with the fibrocyte inhibitor serum amyloid P (SAP; pentraxin-2) significantly prolonged survival and slowed the development of BM fibrosis. Collectively, our data suggest that neoplastic fibrocytes contribute to the induction of BM fibrosis in PMF, and inhibiting fibrocyte differentiation with SAP may interfere with this process.

Lenalidomide Modulates the Phenotype and Function of Human Mesenchymal Stromal Cells From Healthy Donors and MDS Patients.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3816-3816
Author(s):  
Manja Wobus ◽  
Gwendolin Dünnebier ◽  
Silvia Feldmann ◽  
Gerhard Ehninger ◽  
Martin Bornhauser ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Osteogenic Differentiation ◽  
Clinical Efficacy ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Morphological Changes ◽  
Adipogenic Differentiation ◽  
Healthy Controls ◽  
Healthy Donors

Abstract Abstract 3816 Poster Board III-752 Introduction Recent studies in patients with MDS have clearly demonstrated the clinical efficacy of lenalidomide. However, its exact mechanisms of action have not been elucidated yet. Myelosuppression is the most common adverse event and seems to be dependent on dose as well MDS subtype, being rather infrequent in patients other than del5q. The aim of this study was to investigate whether lenalidomide affects the bone marrow microenvironment. Therefore, we analyzed in-vitro characteristics of isolated mesenchymal stromal cells (MSCs) from MDS patients and from healthy controls. Methods Bone marrow samples were collected from healthy donors (n=5) and patients with MDS (del5q MDS n=3, RA n=2, RAEB1/2 n=3). MSCs were isolated according to the standard adhesion protocol and cultured in the presence or absence of lenalidomide. Results Lenalidomide treatment of MSCs caused no morphological changes but proliferation was slightly increased. Typical surface molecules as CD73, CD90, CD105 and CD166 were expressed in MSCs from MDS patients at comparable levels to healthy controls. Lenalidomide treatment caused an upregulation of CD29 by 17.8 ± 4.4% and of CD73 by 24 ± 5.7% (mean fluorescence intensity). Investigating the cytokine production, we found lower IL-8 mRNA and protein levels in MSCs from MDS patients (mean in MDS MSC: 138.1 pg/ml vs. mean in healthy MSC: 1177 pg/ml). Interestingly, the IL-8 production can be increased by approximately 40% under lenalidomide treatment. MDS MSCs retained the capacity for adipogenic and osteogenic differentiation as well as their supportive function towards hematopoietic cells in long term culture-initiating assays (LTC-IC). However, the LTC-IC frequency was lower on MSC which had been preincubated with lenalidomide compared to controls. Lenalidomide also slightly accelerated osteogenic differentiation because mineralization started as early as on day 5 with lenalidomide whereas in the control cells first calcium deposits were visible after 7 days. Other samples showed augmented lipid vacuoles after adipogenic differentiation under lenalidomide treatment. Conclusion In conclusion, lenalidomide modulates the phenotype of MSC and leads to an increase of their IL-8 secretion by a yet unknown mechanism. Whether these in-vitro effects are associated with the clinical efficacy of this compound in patients with MDS remains to be investigated. Disclosures: Platzbecker: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Role Of Long Pentraxin 3 (PTX3) In Wound Closure Induced By Bone Marrow-Derived Mesenchymal Stromal Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1220-1220
Author(s):  
Claudia Cappuzzello ◽  
Andrea Doni ◽  
Erica Dander ◽  
Fabio Pasqualini ◽  
Manuela Nebuloni ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Wound Closure ◽  
Conflicts Of Interest ◽  
Histological Evaluation ◽  
Pentraxin 3 ◽  
Alizarin Red ◽  
Matrix Deposition

Abstract Although several studies have shown the capacity of mesenchymal stromal cells (MSCs) to repair and regenerate different tissues, the mechanisms underlying these processes are not understood. Long Pentraxin 3 (PTX3) is a multifunctional protein produced by MSCs and other cell subsets upon activation with inflammatory cytokines. PTX3 is involved in innate immunity, inflammation and extracellular matrix deposition. In the present study we analyzed the potential role of PTX3 in wound repair process induced by MSCs. PTX3 knockout MSCs (PTX3-/-MSCs) were collected from bone marrow of PTX3-/- mice. After 3-5 culture passages the expression of surface markers was analyzed by flow cytometry and their osteogenic and adipogenic differentiation capacity was detected by alizarin red O and oil red S staining, respectively. The ability of PTX3-/-MSCs to abrogate T cell proliferation was evaluated by co-culturing MSCs and PBMCs previously activated with Phytohaemagglutinin. Finally, equal number of both PTX3-/-MSCs and wild type (WT) MSCs were implanted into excisional wounds created by a biopsy punch on the back of allogenic WT and PTX3-/- mice. Wound area was measured up to 14 day and calculated using an image analysis program. The wound specimens were collected at 2, 7 and 14 days and processed for histological analysis. We demonstrated that PTX3-/-MSCs, similarly to WT MSCs, displayed typical fibroblastoid morphology, they expressed common MSC markers and were able to differentiate into adipocytes and osteoblasts. In addition, they drastically decreased the mitogen-induced proliferation of lymphocyte. Importantly, in a mouse model of wound healing, PTX3-/- MSCs showed a highly significant defect in wound closure compared to WT MSCs at each time point. Histological evaluation of skin samples treated with PTX3-/- MSCs showed a reduction of the granulation tissue and a significant increase of neutrophils (GR-1+) in the wound bed. Moreover, wounds treated with PTX3-/- MSCs were characterized by an excessive accumulation of fibrin at the 2nd day after injury. Accordingly, PTX3-/- MSCs showed a defective ability to degrade the fibrin matrix in vitro. Finally, PTX3-/- MSCs failed to close the ulcers in PTX3-/- mice. In conclusion, we demonstrated that PTX3 deficiency does not alter the phenotype and the capacity of MSCs to differentiate into mesengetic lineages; however, the production of PTX3 represents an essential requirement for MSC ability of enhancing tissue repair. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Multipotent Mesenchymal Stromal Cells: Possible Culprits in Solid Tumors?

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Pascal David Johann ◽  
Ingo Müller
Keyword(s):  
Bone Marrow ◽  
Solid Tumors ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Multipotent Mesenchymal Stromal Cells ◽  
Cell Of Origin ◽  
Mesenchymal Tumors ◽  
Experimental Approaches ◽  
The Subject

The clinical use of bone marrow derived multipotent mesenchymal stromal cells (BM-MSCs) in different settings ranging from tissue engineering to immunotherapies has prompted investigations on the properties of these cells in a variety of other tissues. Particularly the role of MSCs in solid tumors has been the subject of many experimental approaches. While a clear phenotypical distinction of tumor associated fibroblasts (TAFs) and MSCs within the tumor microenvironment is still missing, the homing of bone marrow MSCs in tumor sites has been extensively studied. Both, tumor-promoting and tumor-inhibiting effects of BM-MSCs have been described in this context. This ambiguity requires a reappraisal of the different studies and experimental methods employed. Here, we review the current literature on tumor-promoting and tumor-inhibiting effects of BM-MSCs with a particular emphasis on their interplay with components of the immune system and also highlight a potential role of MSCs as cell of origin for certain mesenchymal tumors.

Cell-cycle phases and genetic profile of bone marrow-derived mesenchymal stromal cells expanded in vitro from healthy donors

2011 ◽  
Vol 112 (7) ◽  
pp. 1817-1821 ◽  
Author(s):  
Valentina Achille ◽  
Melissa Mantelli ◽  
Giulia Arrigo ◽  
Francesca Novara ◽  
Maria Antonietta Avanzini ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Genetic Profile ◽  
Healthy Donors ◽  
Cell Cycle Phases

scholarly journals Characterization of the Hematopoietic Supporting Activity of Osteoblasts Derived from Bone Marrow Mesenchymal Stromal Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4358-4358
Author(s):  
Manal Alsheikh ◽  
Roya Pasha ◽  
Nicolas Pineault
Keyword(s):  
Bone Marrow ◽  
Osteogenic Differentiation ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Conflicts Of Interest ◽  
Soluble Factors ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
The Impact ◽  
Myeloid Progenitors

Abstract Osteoblasts (OST) found within the endosteal niche are important regulators of Hematopoietic Stem and Progenitor Cells (HSPC) under steady state and during hematopoietic reconstitution. OST are derived from mesenchymal stromal cells (MSC) following osteogenic differentiation. MSC and OST secrete a wide array of soluble factors that sustain hematopoiesis. Recently, we showed that media conditioned with OST derived from MSC (referred as M-OST) after 6 days of osteogenic differentiation were superior to MSC conditioned media (CM) for the expansion of cord blood (CB) progenitors, and CB cells expanded with M-OST CM supported a more robust engraftment of platelets in NSG mice after transplantation. These findings raised the possibility that M-OST could be superior to MSC for the ex vivoexpansion HSPC. In this study, we set out to test the hypothesis that the growth modulatory activity of M-OST would vary as a function of their maturation status. The objectives were to first monitor the impact of M-OST differentiation and maturation status on the expression of soluble factors that promote HSPC expansion and in second, to investigate the capacity of M-OST CMs prepared from M-OST at distinct stages of differentiation to support the expansion and differentiation of HSPCs in culture. M-OST at distinct stages of differentiation were derived by culturing bone marrow MSC in osteogenic medium for various length of time (3 to 21 days). All CB CD34+ enriched (92±7% purity) cell cultures were done with serum free media conditioned or not with MSC or M-OST and supplemented with cytokines SCF, TPO and FL. We first confirmed the progressive differentiation and maturation of M-OST as a function of osteogenic culture length, which was evident by the induction of the osteogenic transcription factors Osterix, Msx2 and Runx2 mRNAs, the gradual increase in osteopontin and alkaline phosphatase positive cells and quantitative increases in calcium deposit. Next, we investigated the expression in MSC and M-OSTs of genes known to collaborate for the expansion of HSPCs by Q-PCR. Transcript copy numbers for IGFBP-2 increased swiftly during osteogenic differentiation, peaking at day-3 (˃100-fold vs MSC, n=2) and returning below MSC level by day-21. In contrast, ANGPTL members (ANGPTL-1, -2, -3 and -5) remained superior in M-OSTs throughout osteogenic differentiation with expression levels peaking around day 6 (n=2). Next, we tested the capacity of media conditioned with primitive (day-3, -6), semi-mature (day-10, -14) and mature M-OST (day-21) to support the growth of CB cells. All M-OST CMs increased (p˂0.03) the growth of total nucleated cells (TNC) after 6 days of culture compared to non-conditioned medium used as control (mean 2.0-fold, n=4). Moreover, there was a positive correlation between cell growth and M-OST maturation status though differences between the different M-OST CMs tested were not significant. The capacity of M-OST CMs to increase (mean 2-fold, n=4) the expansion of CD34+ cells was also shared by all M-OST CMs (p˂0.05), as supported by significant increases with immature day-3 (mean ± SD of 18 ± 6, p˂0.02) and mature day-21 M-OST CMs (14 ± 5, p˂0.05) vs. control (8 ± 3, n=4). Conversely, expansions of TNC and CD34+ cells in MSC CM cultures were in-between that of control and M-OST CMs cultures. Interestingly, M-OST CMs also modulated the expansion of the HSPC compartment. Indeed, while the expansion of multipotent progenitors defined as CD34+CD45RA+ was promoted in control culture (ratio of 4.5 for CD34+CD45RA+/CD34+CD45RA- cells), M-OST CMs supported greater expansion of the more primitive CD34+CD45RA- HSPC subpopulation reducing the ratio to 3.3±0.4 for M-OST cultures (cumulative mean of 10 cultures, n=2). Moreover, the expansions of CD34+CD38- cells and of the long term HSC-enriched subpopulation (CD34+CD38-CD45RA-Thy1+) in M-OST CM cultures were respectively 2.7- and 2.8-fold greater than those measured in control cultures (n=2-4). Finally, the impact of M-OST CMs on the expansion of myeloid progenitors was investigated using a colony forming assay; expansion of myeloid progenitors were superior in all M-OST CM cultures (1.6±0.2 fold, n=2). In conclusion, our results demonstrate that M-OST rapidly acquire the expression of growth factors known to promote HSPC expansion. Moreover, the capacity of M-OST CMs to support the expansion of HSPCs appears to be a property shared by M-OST at various stages of maturation. Disclosures No relevant conflicts of interest to declare.

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