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

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.

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Human extramedullary bone marrow in mice: a novel in vivo model of genetically controlled hematopoietic microenvironment

Blood ◽

10.1182/blood-2011-11-389957 ◽

2012 ◽

Vol 119 (21) ◽

pp. 4971-4980 ◽

Cited By ~ 73

Author(s):

Ye Chen ◽

Rodrigo Jacamo ◽

Yue-xi Shi ◽

Rui-yu Wang ◽

Venkata Lokesh Battula ◽

...

Keyword(s):

Bone Marrow ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Critical Role ◽

Hematopoietic Cells ◽

Leukemic Cells ◽

In Vivo Model ◽

Hematopoietic Microenvironment ◽

Cell Engraftment

Abstract The interactions between hematopoietic cells and the bone marrow (BM) microenvironment play a critical role in normal and malignant hematopoiesis and drug resistance. These interactions within the BM niche are unique and could be important for developing new therapies. Here, we describe the development of extramedullary bone and bone marrow using human mesenchymal stromal cells and endothelial colony-forming cells implanted subcutaneously into immunodeficient mice. We demonstrate the engraftment of human normal and leukemic cells engraft into the human extramedullary bone marrow. When normal hematopoietic cells are engrafted into the model, only discrete areas of the BM are hypoxic, whereas leukemia engraftment results in widespread severe hypoxia, just as recently reported by us in human leukemias. Importantly, the hematopoietic cell engraftment could be altered by genetical manipulation of the bone marrow microenvironment: Extramedullary bone marrow in which hypoxia-inducible factor 1α was knocked down in mesenchymal stromal cells by lentiviral transfer of short hairpin RNA showed significant reduction (50% ± 6%; P = .0006) in human leukemic cell engraftment. These results highlight the potential of a novel in vivo model of human BM microenvironment that can be genetically modified. The model could be useful for the study of leukemia biology and for the development of novel therapeutic modalities aimed at modifying the hematopoietic microenvironment.

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TGF-Beta Signaling in Mesenchymal Stromal Cells Contributes to Myelofibrosis but Not Hematopoietic Phenotypes in Myeloproliferative Neoplasms

Blood ◽

10.1182/blood-2018-99-113745 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3053-3053

Author(s):

Juo-Chin Yao ◽

Grazia Abou Ezzi ◽

Joseph R. Krambs ◽

Eric J. Duncavage ◽

Daniel C. Link

Keyword(s):

Bone Marrow ◽

Growth Factor ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Transforming Growth Factor ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Hematopoietic Cells ◽

Dismal Prognosis ◽

Collagen Iii

Abstract The development of myelofibrosis in patients with myeloproliferative neoplasms (MPNs) is associated with a dismal prognosis. The mechanisms responsible for the progression to myelofibrosis are unclear, limiting the development of therapies to treat or prevent it. The cell of origin responsible for the increased collagen deposition is controversial, with recent studies implicating Gli1+ or leptin receptor+ mesenchymal stromal cells, monocytes, or even endothelial cells. Moreover, the signals generated by malignant hematopoietic cells in MPN that induce increased collagen expression are uncertain. There is some evidence that elevated expression of cytokines/chemokines in the bone marrow microenvironment of patients with MPN may contribute. In particular, recent studies have implicated transforming growth factor-β (TGF-β), platelet-derived growth factor and CXCL4 in the development of myelofibrosis. Here, we test the specific hypothesis that TGF-β signaling in mesenchymal stromal cells is required for the development of myelofibrosis. Moreover, we hypothesize that TGF-β signaling, by altering the expression of key niche factors by mesenchymal stromal cells, contributes to the myeloid expansion in MPN. To test this hypothesis, we abrogated TGF-β signaling in mesenchymal stem/progenitor cells (MSPCs) by deleting Tgfbr2 using a doxycycline-repressible Sp7 (osterix)-Cre transgene (Osx-Cre), which targets all mesenchymal stromal cells in the bone marrow, including CXCL12-abundant reticular (CAR) cells, osteoblasts, adipocytes, or arteriolar pericytes. We previously showed that TGF-β signaling plays a key role in the lineage specification of MSPCs during development (2017 ASH abstract #2438). In contrast, we show that post-natal deletion of Tgfbr2, by removing doxycycline at birth, is not associated with significant changes in mesenchymal stromal cells in the bone marrow. Moreover, expression of key niche factors, including Cxcl12 and stem cell factor, and basal hematopoiesis were normal in these mice. Thus, we used the post-natal Osx-Cre; Tgfbr2-deleted mice as recipients to assess the role of TGF-β signaling in mesenchymal stromal cells on the hematopoietic and myelofibrosis phenotype in Jak2V617For MPLW515Lmodels of MPN. Specifically, we transplanted hematopoietic cells from Mx1-Cre; Jak2V617Fmice (4 weeks after pIpC treatment) or hematopoietic cells transduced with MPLW515Lretrovirus into irradiated wildtype or post-natal Osx-Cre; Tgfbr2-deleted mice. Both MPN models have elevated Tgfb1 expression in the bone marrow. As reported previously, transplantation of MPLW515Ltransduced hematopoietic cells into wildtype recipients produced a rapidly fatal MPN characterized by neutrophilia, erythrocytosis, thrombocytosis, splenomegaly, and reticulin fibrosis in the bone marrow. A similar hematopoietic phenotype was observed in Osx-Cre; Tgfbr2fl/flrecipients. However, a trend to decreased reticulin fibrosis was observed in Osx-Cre; Tgfbr2fl/flcompared to wildtype recipients (reticulin histology score: 0.5 versus 1.1, respectively, n=5, p=0.23). Likewise, the degree of neutrophilia, erythrocytosis, thrombocytosis, and splenomegaly in wildtype and Osx-Cre; Tgfbr2fl/flrecipients of Jak2V617Fcells was similar. As reported previously, we did not observe overt myelofibrosis in this model (as measured by reticulin staining). However, we were able to detect increased collagen III deposition using immunofluorescence staining in 4 of 5 wildtype recipients compared to 1 of 4 Osx-Cre Tgfbr2fl/flrecipients of Jak2V617Fcells (p=0.21). In conclusion, our data suggest that TGF-β signaling in mesenchymal stromal cells contributes, but is not absolutely required, for the development of myelofibrosis. Alterations in mesenchymal stromal cells induced by increased TGF-β signaling do not appear to be a major driver of the myeloid expansion in MPN. The contribution of increased TGF-β signaling in hematopoietic cells or other bone marrow stromal cell populations to the MPN phenotype is under investigation. Disclosures No relevant conflicts of interest to declare.

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Significant Engraftment of Immature Hematopoietic Cells From Patients with Low Risk Myelodysplastic Syndromes (MDS) in Immunodeficient Mice

Blood ◽

10.1182/blood.v120.21.1694.1694 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1694-1694

Author(s):

Hind Medyouf ◽

Florian Nolte ◽

Maximilian Mossner ◽

Verena Nowak ◽

Bettina Zens ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Mesenchymal Stromal Cells ◽

Myelodysplastic Syndromes ◽

Stromal Cells ◽

Hematopoietic Cells ◽

Low Risk ◽

Cell Phenotype

Abstract Abstract 1694 Introduction: Myelodysplastic syndromes are a heterogeneous group of malignant clonal hematologic disorders characterized by ineffective hematopoiesis, peripheral cytopenias and dysplastic bone marrow cells, with frequent progression to acute myeloid leukemia. Because of its heterogeneous nature, modeling of this disease has proven to be very difficult in cell culture systems as well as mice. In addition, attempts to generate a xenotransplant model in immuno-compromised mice have only achieved very low levels of engraftment that are often transient, making it very difficult to study the biology of this disease in vivo. Recent studies in mice have shown that conditional impairment of the small RNA processing enzyme Dicer in mouse osteolineages induced a stromal niche that promoted myelodysplasia, leading to the hypothesis that abnormal bone marrow stromal cells might provide a “fertile soil“ for the expansion of the malignant clone. Patients and Methods: To the date of writing, a total of 12 primary hematopoietic stem cell- and mesenchymal stroma cell (MSCs) samples selected from patients with MDS have been isolated and xenotransplanted into NOD.Cg-Prkdscid Il2rgtm1Wjl/Szj (NSG) mice: MDS 5q- (n=7), MDS RCMD (n=3), MDS RAEB I (n=1), MDS-U (n=1). Engraftment was monitored by FACS using human specific antibodies to CD45, CD34 and CD38. In addition cell cycle behavior was analyzed by Ki67/Hoechst staining. Mesenchymal stromal cells were characterized using previously described stromal markers: CD105, CD271, CD73, CD166, CD90, CD146 and CD44. To isolate genomic DNA and RNA for molecular analyses, MDS xenografts were flow sorted based on human CD45 expression. Molecular characterization of primary MDS samples and xenotransplants was carried out by serial copy number analysis using Affymetrix SNP 6.0 Arrays, metaphase cytogenetics and direct sequencing of known mutations in the transplanted MDS samples. Results: We show, that the concomitant transplantation of MDS-derived mesenchymal stromal cells with the corresponding hematopoietic patient stem/progenitor cells leads to significant and long-term engraftment (0.1 – 15% for up to 23 weeks) of cells isolated from IPSS low and intermediate risk MDS patients. In addition to the bone marrow, MDS hematopoietic cells also infiltrate other hematopoietic compartments of the mouse including the spleen. Significant engraftment of cells with progenitor (CD34+CD38+) as well as stem cell phenotype (CD34+CD38-) was observed, which is consistent with engraftment of an MDS stem cell that sustains long-term hematopoiesis. SNP array analysis confirmed the clonal origin of the engrafted cells as MDS xenografts harboring the identical genomic lesions as present in the patient disease. Conclusion: We present a robust MDS xenograft model of low risk MDS entities based on the concomitant transplantation of primary MDS hematopoietic cells with MSCs from the same patients. This model does not only allow to study the biology of this disease in vivo but also the molecular and cellular interactions between MSCs and hematopoietic MDS cells. In addition it provides a useful platform to study the effects of new experimental therapeutic agents for the treatment of MDS in molecularly defined MDS cells. Disclosures: No relevant conflicts of interest to declare.

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In Vivo Osteogenic Potential of Mesenchymal Stromal Cells Derived from Patient''s Bone Marrow on Hydroxyapatite Ceramics

Key Engineering Materials - Bioceramics 19 ◽

10.4028/0-87849-422-7.1157 ◽

2007 ◽

pp. 1157-1160

Author(s):

Asako Matsushima ◽

Noriko Kotobuki ◽

Hiroko Machida ◽

Toru Morishita ◽

Yoshinori Takakura ◽

...

Keyword(s):

Bone Marrow ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Osteogenic Potential ◽

Hydroxyapatite Ceramics

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

Blood ◽

10.1182/blood.v118.21.3834.3834 ◽

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.

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In Vivo Osteogenic Potential of Mesenchymal Stromal Cells Derived from Patient''s Bone Marrow on Hydroxyapatite Ceramics

Key Engineering Materials ◽

10.4028/www.scientific.net/kem.330-332.1157 ◽

2007 ◽

Vol 330-332 ◽

pp. 1157-1160 ◽

Cited By ~ 1

Author(s):

Asako Matsushima ◽

Noriko Kotobuki ◽

Hiroko Machida ◽

Toru Morishita ◽

Yoshinori Takakura ◽

...

Keyword(s):

Bone Marrow ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Bone Matrix ◽

Osteogenic Potential ◽

Human Mscs ◽

Hard Tissue ◽

Culture Dish ◽

Hydroxyapatite Ceramics

Since 2001, we have started tissue engineered approach for hard tissue repair using mesenchymal stromal cells (MSCs) derived from patient’s bone marrow. MSCs were culture expanded on culture dish, then applied on various ceramics including hydroxyapatite (HA) ceramics. The MSCs on the ceramics were further cultured in osteogenic media to induce osteognenic differentiation. The differentiation resulted in appearance of bone forming osteoblasts as well as bone matrix on the ceramics, thus we could fabricate the tissue engineered bone. We have reported that the tissue engineered bone is effective for treatment of large bone defect, which is difficult to repair only with artificial materials such as HA ceramics. The present study focused on osteogenic capability of cryopreserved human MSCs derived from patients who already were treated by the tissue engineered bone. The MSCs showed high alkaline phosphatase activity together with abundant bone matrix formation when cultured in osteogenic media. The MSCs also showed in vivo new bone formation when implanted at subcutaneous sites of athymic nude rats. Based on the results, we concluded that the tissue engineering approach is a reliable method to be used in hard tissue regeneration.

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Evaluation of Browning Agents on the White Adipogenesis of Bone Marrow Mesenchymal Stromal Cells: A Contribution to Fighting Obesity

Cells ◽

10.3390/cells10020403 ◽

2021 ◽

Vol 10 (2) ◽

pp. 403

Author(s):

Girolamo Di Maio ◽

Nicola Alessio ◽

Ibrahim Halil Demirsoy ◽

Gianfranco Peluso ◽

Silverio Perrotta ◽

...

Keyword(s):

Bone Marrow ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

White Adipocyte ◽

Drug Candidates ◽

Fat Depots ◽

Treatment Of Obesity ◽

White Fat

Brown-like adipocytes can be induced in white fat depots by a different environmental or drug stimuli, known as “browning” or “beiging”. These brite adipocytes express thermogenin UCP1 protein and show different metabolic advantages, such as the ability to acquire a thermogenic phenotype corresponding to standard brown adipocytes that counteracts obesity. In this research, we evaluated the effects of several browning agents during white adipocyte differentiation of bone marrow-derived mesenchymal stromal cells (MSCs). Our in vitro findings identified two compounds that may warrant further in vivo investigation as possible anti-obesity drugs. We found that rosiglitazone and sildenafil are the most promising drug candidates for a browning treatment of obesity. These drugs are already available on the market for treating diabetes and erectile dysfunction, respectively. Thus, their off-label use may be contemplated, but it must be emphasized that some severe side effects are associated with use of these drugs.

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