Primary Bone Marrow Stromal Cells Research Articles

MCP/CCR2 signaling is essential for recruitment of mesenchymal progenitor cells during the early phase of fracture healing.

ObjectiveThe purpose of this study was to investigate chemokine profiles and their functional roles in the early phase of fracture healing in mouse models.MethodsThe expression profiles of chemokines were examined during fracture healing in wild-type (WT) mice using a polymerase chain reaction array and histological staining. The functional effect of monocyte chemotactic protein-1 (MCP-1) on primary mouse bone marrow stromal cells (mBMSCs) was evaluated using an in vitro migration assay. MCP-1−/− and C-C chemokine receptor 2 (CCR2)−/− mice were fractured and evaluated by histological staining and micro-computed tomography (micro-CT). RS102895, an antagonist of CCR2, was continuously administered in WT mice before or after rib fracture and evaluated by histological staining and micro-CT. Bone graft exchange models were created in WT and MCP-1−/− mice and were evaluated by histological staining and micro-CT.Results MCP-1 and MCP-3 expression in the early phase of fracture healing were up-regulated, and high levels of MCP-1 and MCP-3 protein expression observed in the periosteum and endosteum in the same period. MCP-1, but not MCP-3, increased migration of mBMSCs in a dose-dependent manner. Fracture healing in MCP-1−/− and CCR2−/− mice was delayed compared with WT mice on day 21. Administration of RS102895 in the early, but not in the late phase, caused delayed fracture healing. Transplantation of WT-derived graft into host MCP-1−/− mice significantly increased new bone formation in the bone graft exchange models. Furthermore, marked induction of MCP-1 expression in the periosteum and endosteum was observed around the WT-derived graft in the host MCP-1−/− mouse. Conversely, transplantation of MCP-1−/− mouse-derived grafts into host WT mice markedly decreased new bone formation.ConclusionsMCP-1/CCR2 signaling in the periosteum and endosteum is essential for the recruitment of mesenchymal progenitor cells in the early phase of fracture healing.

Early-onset type 2 diabetes impairs skeletal acquisition in the male TALLYHO/JngJ mouse.

Type 2 diabetes (T2D) incidence in adolescents is rising and may interfere with peak bone mass acquisition. We tested the effects of early-onset T2D on bone mass, microarchitecture, and strength in the TALLYHO/JngJ mouse, which develops T2D by 8 weeks of age. We assessed metabolism and skeletal acquisition in male TALLYHO/JngJ and SWR/J controls (n = 8-10/group) from 4 weeks to 8 and 17 weeks of age. Tallyho mice were obese; had an approximately 2-fold higher leptin and percentage body fat; and had lower bone mineral density vs SWR at all time points (P < .03 for all). Tallyho had severe deficits in distal femur trabecular bone volume fraction (-54%), trabecular number (-27%), and connectivity density (-82%) (P < .01 for all). Bone formation was higher in Tallyho mice at 8 weeks but lower by 17 weeks of age vs SWR despite similar numbers of osteoblasts. Bone marrow adiposity was 7- to 50-fold higher in Tallyho vs SWR. In vitro, primary bone marrow stromal cell differentiation into osteoblast and adipocyte lineages was similar in SWR and Tallyho, suggesting skeletal deficits were not due to intrinsic defects in Tallyho bone-forming cells. These data suggest the Tallyho mouse might be a useful model to study the skeletal effects of adolescent T2D.

Post-transcriptional regulation in osteoblasts using localized delivery of miR-29a inhibitor from nanofibers to enhance extracellular matrix deposition

MicroRNAs are important post-transcriptional regulators of skeletal biology, and miRNA-based therapeutics have the potential to aid bone repair. However, efficient tools for delivering miRNA mimics or inhibitors to specific target tissues are limited. Polymeric nanofibers closely mimic natural extracellular matrix (ECM) morphology, and are attractive candidates for supporting delivery of cells and bone-anabolic reagents. It is hypothesized that gelatin nanofibers could be used for the localized transient delivery of miRNA-based therapeutics, using miR-29a inhibitor as a prototype to increase ECM deposition. miR-29 family members are negative regulators of ECM synthesis, targeting the mRNAs of selected collagens and osteonectin/SPARC. Inhibiting miR-29 activity may therefore increase ECM production by cells. miR-29a inhibitor-loaded gelatin nanofibers, prepared by electrospinning, demonstrated continuous release of miRNA inhibitor over 72h. Pre-osteoblastic murine MC3T3-E1 cell line seeded on miR-29a inhibitor-loaded nanofibers synthesized more osteonectin, indicating efficient inhibitor delivery. These cells also displayed increased Igf1 and Tgfb1 mRNA. Moreover, primary bone marrow stromal cells from transgenic pOBCol3.6cyan reporter mice, grown on miR-29a inhibitor scaffolds, displayed increased col3.6 cyan expression as well as collagen production. This study demonstrates that ECM mimicking nanostructured scaffolds, in conjunction with bioactive miRNA-based therapeutics, may serve as a novel platform for developing biologically active localized cell delivery systems.

LIF/STAT3/SOCS3 Signaling Pathway in Murine Bone Marrow Stromal Cells Suppresses Osteoblast Differentiation

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine that belongs to the interleukin-6 family and is expressed by multiple tissue types. This study analyzed the effect of LIF on osteoblast differentiation using primary murine bone marrow stromal cells (BMSCs). Colony-forming unit-osteoblast formation by BMSCs was significantly suppressed by LIF treatment. To clarify the mechanism underlying the LIF suppressive effect on osteoblast differentiation, we analyzed the downstream signaling pathway of LIF. LIF/signal transducer and activator of transcription 3 (STAT3) signaling induces the expression of suppressor of cytokine signaling 3 (SOCS3). SOCS3 knockdown experiments have previously demonstrated that short-hairpin SOCS3-BMSCs reversed the LIF suppressive effect. Our results demonstrated that LIF suppresses osteoblast differentiation through the LIF/STAT3/SOCS3 signaling pathway.

Down-Regulation of CD9 by Methylation Decreased Bortezomib Sensitivity in Multiple Myeloma

Bortezomib therapy has been proven successful for the treatment of relapsed and/or refractory multiple myeloma (MM). However, both intrinsic and acquired resistance has already been observed. In this study, we explored the relationship between CD9 expression and bortezomib sensitivity in MM. We found that down-regulation of CD9 by methylation decreased bortezomib sensitivity in multiple myeloma. CD9 expression obviously increased bortezomib sensitivity through inducing apoptosis, significantly inhibiting U266 cells' adhesion to HS-5 and primary bone marrow stromal cells, but increasing U266 cells' adhesion to fibronectin. CD9 expression also significantly inhibited U266 cell migration. The mechanisms may include: the endoplasmic reticulum stress pathway, cell adhesion related signaling pathway and osteoclast differentiation related signaling pathway. Combination therapy with de-methylation reagent 5-Aza-2-deoxycytidine may prove useful to the development of novel strategies for the treatment of bortezomib-resistant MM patients.

Artesunate induces apoptosis through caspase-dependent and -independent mitochondrial pathways in human myelodysplastic syndrome SKM-1 cells

Artesunate (ART) is a semi-synthetic derivative of artemisinin extracted from Artemisia annua (sweet wormwood) that is conventionally used in anti-malarial drugs and more recently in medications that induce tumor cell apoptosis. Here, we investigated the effects and mechanistic pathways of ART in human myelodysplastic syndrome (MDS), a condition that commonly progresses to acute myeloid leukemia (AML). Human MDS SKM-1 cells, primary bone marrow (PBM) mononuclear cells from patients with refractory anemia with excess blasts (RAEB) or MDS–AML (MDS cell group), and PBM stromal cells from three patients without hematological diseases (non-MDS cell group) were cultured for 24, 48, or 72h with or without various ART concentrations. CCK-8, western blot, JC-1 fluorescence, and Annexin-V/Propidium iodide (PI) labeling were used to assess cell proliferation, protein levels, mitochondrial membrane potentials (MMPs) and apoptosis, respectively. ART administration dose- and time-dependently inhibited SKM-1 proliferation. At 24, 48, and 72h, ART IC50 values were 89.92, 4.24, and 1.28μmol/L, respectively. ART only significantly inhibited proliferation in the MDS cell group, but it has little impact on proliferation of non-MDS cells. ART decreased MMPs, and dose-dependently induced SKM-1 cell apoptosis, peaking at 82.9% when treated with 200μmol/L ART for 24h. Caspase-3 and -9 activation, poly(ADP-ribose) polymerase cleavage, decreased Bcl-2/Bax ratio and apoptosis inducing factor nuclear localization were implicated in apoptosis. Our results indicate that ART effectively induces apoptosis in SKM-1 cells through both caspase-dependent and -independent mitochondrial pathways.

MicroRNA-23a mediates post-transcriptional regulation of CXCL12 in bone marrow stromal cells

The chemokine CXCL12 regulates the interaction between hematopoietic stem and progenitor cells and bone marrow stromal cells. Although its relevance in the bone marrow niche is well recognized, the regulation of CXCL12 by microRNA is not completely understood. We transfected a library of 486 microRNA in the bone marrow stromal cell line SCP-1 and studied the expression of CXCL12. Twenty-seven microRNA were shown to downregulate expression of CXCL12. Eight microRNA (miR-23a, 130b, 135, 200b, 200c, 216, 222, and 602) interacted directly with the 3'UTR of CXCL12. Next, we determined that only miR-23a is predicted to bind to the 3'UTR and is strongly expressed in primary bone marrow stromal cells. Modulation of miR-23a changes the migratory potential of hematopoietic progenitor cells in co-culture experiments. We discovered that TGFB1 mediates its inhibitory effect on CXCL12 levels by upregulation of miR-23a. This process was partly reversed by miR-23a molecules. Finally, we determined an inverse expression of CXCL12 and miR-23a in stromal cells from patients with myelodys-plastic syndrome indicating that the interaction has a pathophysiological role. Here, we show for the first time that CXCL12-targeting miR23a regulates the functional properties of the hematopoietic niche.

Effects of Arsenic on Osteoblast Differentiation in Vitro and on Bone Mineral Density and Microstructure in Rats

Background: Arsenic is a ubiquitous toxic element and is known to contaminate drinking water in many countries. Several epidemiological studies have shown that arsenic exposure augments the risk of bone disorders. However, the detailed effect and mechanism of inorganic arsenic on osteoblast differentiation of bone marrow stromal cells and bone loss still remain unclear.Objectives: We investigated the effects and mechanism of arsenic on osteoblast differentiation in vitro and evaluated bone mineral density (BMD) and bone microstructure in rats at doses relevant to human exposure from drinking water.Methods: We used a cell model of rat primary bone marrow stromal cells (BMSCs) and a rat model of long-term exposure with arsenic-contaminated drinking water, and determined bone microstructure and BMD in rats by microcomputed tomography (μCT).Results: We observed significant attenuation of osteoblast differentiation after exposure of BMSCs to arsenic trioxide (0.5 or 1 μM). After arsenic treatment during differentiation, expression of runt-related transcription factor-2 (Runx2), bone morphogenetic protein-2 (BMP-2), and osteocalcin in BMSCs was inhibited and phosphorylation of enhanced extracellular signal-regulated kinase (ERK) was increased. These altered differentiation-related molecules could be reversed by the ERK inhibitor PD98059. Exposure of rats to arsenic trioxide (0.05 or 0.5 ppm) in drinking water for 12 weeks altered BMD and microstructure, decreased Runx2 expression, and increased ERK phosphorylation in bones. In BMSCs isolated from arsenic-treated rats, osteoblast differentiation was inhibited.Conclusions: Our results suggest that arsenic is capable of inhibiting osteoblast differentiation of BMSCs via an ERK-dependent signaling pathway and thus increasing bone loss.Citation: Wu CT, Lu TY, Chan DC, Tsai KS, Yang RS, Liu SH. 2014. Effects of arsenic on osteoblast differentiation in vitro and on bone mineral density and microstructure in rats. Environ Health Perspect 122:559–565; http://dx.doi.org/10.1289/ehp.1307832

Telomerase reverse transcriptase mediated immortalization of human bone marrow stromal cells

Primary human bone marrow stromal cells (hMSCs) were transfected with human telomerase reverse transcriptase (hTERT) gene with lipofection method. The hTERT transfected hMSCs of passage 100 underwent chondrogenesis induction with dexamethasone, transforming the growth factor β and vitamin C, osteogenesis induction with dexamethasone, β glycerophosphoric acid and vitamin C, and cardiomyocyte induction with 5-azacytidine. After 7, 14, 21 and 28 days of induction, immunocytochemistry was performed to detect the expressions of type I and II collagen and osteocalcin, and alizarin red staining was performed to detect the bone nodule formation in osteogenesis induction. Immunocytochemistry was carried out to detect the striated muscle actin expression in cardiomyocytes. The hMSCs undergoing successful transfection were positive for the hTERT. The hTERT transfected cells were grown in vitro successfully and passaged for 136 generations. Results showed that these cells could be induced to differentiate into chondrocytes, bone and myocardial cells. Introduction of exogenous hTERT into hMSCs could achieve immortalized hMSCs with the potential of multi-directional differentiation. Thus, these cells could be applied as seed cells in tissue engineering.

Plasma Elevation of Vascular Endothelial Growth Factor Leads to the Reduction of Mouse Hematopoietic and Mesenchymal Stem/Progenitor Cells in the Bone Marrow

Vascular endothelial growth factor (VEGF) is reported to exhibit potent hematopoietic stem/progenitor cell (HSPC) mobilization activity. However, the detailed mechanisms of HSPC mobilization by VEGF have not been examined. In this study, we investigated the effect of VEGF on bone marrow (BM) cell and the BM environment by intravenous injection of VEGF-expressing adenovirus vector (Ad-VEGF) into mice. A colony assay using peripheral blood cells revealed that plasma elevation of VEGF leads to the mobilization of HSPCs into the circulation. Granulocyte colony-stimulating factor (G-CSF) is known to mobilize HSPCs by decreasing CXC chemokine ligand 12 (CXCL12) levels in the BM. However, we found almost no changes in the CXCL12 levels in the BM after Ad-VEGF injection, suggesting that VEGF can alter the BM microenvironment by different mechanisms from G-CSF. Furthermore, flow cytometric analysis and colony forming unit-fibroblast assay showed a reduction in the number of mesenchymal progenitor cells (MPCs), which have been reported to serve as niche cells to support HSPCs, in the BM of Ad-VEGF-injected mice. Adhesion of donor cells to the recipient BM after transplantation was also impaired in mice injected with Ad-VEGF, suggesting a decrease in the niche cell number. We also observed a dose-dependent chemoattractive effect of VEGF on primary BM stromal cells in vitro. These data suggest that VEGF alters the distribution of MPCs in the BM and can also mobilize MPCs to peripheral tissues. Taken together, our results imply that VEGF-elicited egress of HSPCs would be mediated, in part, by changing the number of MPCs in the BM.

Measuring stem cell dimensionality in tissue scaffolds

Many scaffold systems have evolved for tissue engineering and in vitro tissue models to provide a 3D (three-dimensional) microenvironment that enables cells to behave more physiologically. We hypothesized that cells would adopt morphologies with more 3D character during culture in scaffolds as compared to planar substrates. Cell shape and function are tightly linked and effects of scaffold niche properties on cell shape and dimensionality are important for directing cell function. Herein, primary human bone marrow stromal cells (hBMSCs) were cultured in 6 different scaffolds and on a planar control substrate. hBMSCs were imaged using 3D confocal microscopy, and 3D image analyses were used to assess hBMSC shape and dimensionality. A characteristic gyration tensor ellipsoid was calculated for hBMSCs in the different scaffolds which enabled hBMSC dimensionality to be classified based on shape. A “Dimensionality Matrix” was developed that showed that hBMSC shape and dimensionality were influenced by scaffold properties, and that scaffolds could drive hBMSCs into 1D, 2D or 3D shapes. In addition, the hBMSC Z-Depth was measured to determine if hBMSCs became less flat during culture in scaffolds. Z-Depth results showed that all 6 scaffolds caused an increase in cell Z-Depth compared to the 2D planar substrate. These results demonstrate that hBMSCs take on morphologies with greater 3D character in scaffolds than on a planar substrate and that scaffold properties can be adjusted to modify cell dimensionality. In addition, biomaterialists can use this measurement approach to assess and compare scaffold design modifications as they strive to create optimal cell niches that provide a 3D microenvironment.

Bio-active engineered 50 nm silica nanoparticles with bone anabolic activity: Therapeutic index, effective concentration, and cytotoxicity profile in vitro

Silica-based nanomaterials are generally considered to be excellent candidates for therapeutic applications particularly related to skeletal metabolism however the current data surrounding the safety of silica based nanomaterials is conflicting. This may be due to differences in size, shape, incorporation of composite materials, surface properties, as well as the presence of contaminants following synthesis. In this study we performed extensive in vitro safety profiling of ∼50nm spherical silica nanoparticles with OH-terminated or Polyethylene Glycol decorated surface, with and without a magnetic core, and synthesized by the Stöber method. Nineteen different cell lines representing all major organ types were used to investigate an in vitro lethal concentration (LC) and results revealed little toxicity in any cell type analyzed. To calculate an in vitro therapeutic index we quantified the effective concentration at 50% response (EC50) for nanoparticle-stimulated mineral deposition activity using primary bone marrow stromal cells (BMSCs). The EC50 for BMSCs was not substantially altered by surface or magnetic core. The calculated Inhibitory concentration 50% (IC50) for pre-osteoclasts was similar to the osteoblastic cells. These results demonstrate the pharmacological potential of certain silica-based nanomaterial formulations for use in treating bone diseases based on a favorable in vitro therapeutic index.

Lin28B/Let-7 Axis Regulates Multiple Myeloma Proliferation By Enhancing c-Myc and Ras Survival Pathways

BackgroundMicroRNAs (miRNAs) play a pivotal role in tumorigenesis, due to their ability to target mRNAs involved in the regulation of cell proliferation, survival and differentiation. Lin28B is an RNA binding protein that regulates Let-7 miRNA maturation. Lin28B and Let-7 have been described to act as oncogenes or tumor suppressor genes, respectively, as demonstrated both in solid cancer and hematologic malignancies. However, the role of the Lin28B/Let-7 axis in Multiple Myeloma (MM) has not been studied. MethodLin28B level expression in MM patients was studied using previously published gene expression profiling (GEP) datasets. Let-7 expression levels were assessed in CD138+ primary MM cells and bone marrow stromal cells (BMSCs) by using PCR, as well as in circulating exosomes using miRNA array (Nanostring® Technology). Exosomes were collected from both normal and MM peripheral blood, using ultracentrifugation; and further studied by using electron microscopy and immunogold labeling for the detection of CD63 and CD81. The knockdown of Lin28B was performed on MM cell lines (U266, MM.1S, MOLP-8) by using a lentiviral Lin28B shRNA. Gain- and loss-of function studies for Let-7 were performed using Let-7 mimic and anti-Let-7 transfection in MM cell lines (MM1S, U266) and primary BMSCs. Cell proliferation has been evaluated by using thymidine assays. Effects of Let-7 and Lin28B on signaling cascades have been evaluated by western blot. ResultsTwo independent GEP datasets (GSE16558; GSE2658) were analyzed for Lin28B expression, showing a significantly higher level in MM patients compared to healthy controls. In addition, high Lin28B levels correlated with a shorter overall survival (p = 0.0226). We next found that the Let-7 family members are significantly down-regulated in MM primary cells, particularly Let-7a and b (5 fold change, p < 0.05), as demonstrated by using qRT-PCR. Similarly, miRNA arrays showed a lower expression of Let-7-related miRNAs in circulating exosomes obtained from MM patients compared to healthy individuals. We further dissected the functional relevance of Lin28B in MM cells, by performing Lin28 knockdown (KD) in MM cell lines (U266, MOLP-8). This led to a significant decrease in MM cell proliferation associated with G1 phase cell cycle arrest. This was supported by up-regulation of Let-7 and down-regulation of c-Myc, Ras and Cyclin D1 in Lin28 KD MM cells. To further prove that Lin28B-dependent effects on MM cells are mediated by Let7, we next showed that let-7 gain- and loss-of-function studies regulate MM cell proliferation and Myc expression. Lin28B regulation in MM cells is dependent on Let-7, as demonstrated by an increase of both cell proliferation and c-Myc expression after anti-Let-7 transfection in the Lin28B KD cells. We therefore studied the regulation of Let-7 in MM cells through the interaction with BMSCs. Let-7 expression levels were significantly lower in BMSCs obtained from MM patients compared to healthy donors. Interestingly, the Let-7 expression level in MM cells was increased after co-culture with Let-7 over-expressing BMSCs, associated with a decrease of both cell proliferation and c-Myc expression. This suggests a potential transfer of Let-7 from BMSCs to MM cells. ConclusionThis work describes a new signaling pathway involving Lin28B, Let-7, Myc and Ras in MM. Let-7 expression in MM cells is also regulated through the interaction of MM cells with BMSCs, leading to cell proliferation and Myc regulation in MM. Interference with this pathway might offer therapeutic perspectives. Disclosures:Leleu:CELGENE: Honoraria; JANSSEN: Honoraria. Daley:Johnson and Johnson: Consultancy, Membership on an entity's Board of Directors or advisory committees; MPM Capital: Consultancy, Membership on an entity's Board of Directors or advisory committees; Verastem: Consultancy, Membership on an entity's Board of Directors or advisory committees; Epizyme: Consultancy, Membership on an entity's Board of Directors or advisory committees; iPierian: Consultancy, Membership on an entity's Board of Directors or advisory committees; Solasia, KK: Consultancy, Membership on an entity's Board of Directors or advisory committees. Ghobrial:Onyx: Advisoryboard Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.

Proline-Rich Tyrosine Kinase (Pyk2) Promotes Tumor Progression In Multiple Myeloma Through Modulation Of Wnt/β-Catenin Signaling Pathway

BackgroundProline-rich tyrosine kinase (Pyk2) is a non-receptor tyrosine kinase which belongs to the focal adhesion kinase (FAK) family. It is known to facilitate TNF-α induced EMT process in solid tumors, but this has not been investigated in the field of hematologic malignancies. We therefore dissected the role of PyK2 in multiple myeloma (MM) by looking at its ability to modulate MM biology both in vitro and in vivo. MethodsLentiviral packaged small hairpin RNA (shRNA), overexpression plasmid, related scramble probe and empty vector were introduced into MM1.S (GFP+/Luc+) cell line, to generate stable Pyk2 K.D. (#A2 and #A4), Pyk2 K.I., and control cells, respectively. The efficiency of knock-down or knock-in was validated by qPCR and immunoblotting. Cell viability and cell proliferation were detected by using CellTiter-Glo® luminescent assay and thymidine uptake, respectively. Gain- and loss-of fucntion studies were also performed on MM cells in presence of primary bone marrow stromal cells isolated from MM patients (MM-BMSCs). Adhesion of Pyk2 stable cells to fibronectin was measured by using a ECM cell adhesion assay kit. The synergistic effects of Pyk2 with Bortezomib was determined through calculating the DNA synthesis of Pyk2 K.D. cells treated with Bortezomib (2.5-5µM), using Calcusyn software and Chou-Talalay method. Pyk2 K.D. stable cells were intravenously injected into SCID-Biege mice to generate xenograft model. In vivo tumor growth was observed by Bioluminescent Imaging. Pyk2 -dependent-modulation of Wnt/β-catenin pathway signaling was indentified by using immunobloting. ResultsKnockdown of Pyk2 in MM cells significantly repressed cell viability and proliferation, as well as their adhesive ability to BMSCs, compared to scramble control cells. Moreover, Pyk2 knockdown induced de-adhesion of MM cells from BMSCs thus inducing chemosensitivity of tumor cells to Bortezomib. We next corroborated our findings by studying Pyk2 knock-in MM cells, and showed that stably upregulated Pyk2 expression promoted MM cell growth as measured by either ATP quantitation or DNA synthesis. Upregulation of Pyk2 expression also stablized the adhesion of MM cells to BMSCs, leading to a drug-resistance of MM cells to Bortezomib, compared with vector control cells. Pyk2 related tumor growth was further validated by establishing a xenograft mouse model. By using bioluminescence imaging, we found a significantly lower tumor burden in mice injected with Pyk2 K.D. cells, compared to mice controls (injected with scramble cells). We next dissected the effect of Pyk2 in modulation of cellular signaling in MM cells by using immunoblotting, and demonstrated that Pyk2 played an important role in regulating β-catenin signaling. Indeed, knockdown of Pyk2 induced GSK3β-phosphorylation, leading to increased β-catening-phosphorylation, thus resulting in β-catenin degradation and inhibited translocation to nucleus. Importantly, Pyk2 K.D. cells presented with reduced expression of c-myc and cyclin D1 at protein level. Conversely, Pyk2 overexpression enhanced β-catenin expression together with c-myc and cyclin D1 up-regulation, thus confirming the role of Pyk2 in modulating Wnt/β-catenin signaling activity in MM. ConclusionThese findings indicate that Pyk2 exhibits pro-oncogenic properties in MM through modulation of Wnt/β-catenin signaling. Therefore, Pyk2 represents a novel therapeutic target in MM. Disclosures:Ghobrial:Sanofi: Research Funding; Noxxon: Research Funding; BMS: Advisory board, Advisory board Other, Research Funding; Onyx: Advisoryboard Other.

Inhibition Of Mir-21 In HS-5 Bone Marrow Stromal Cells In The Presence Of Multiple Myeloma cells Restores RANKL/OPG Ratio: A Potential Therapeutic Approach For Myeloma-Related Bone Disease

Multiple Myeloma (MM)-related osteolytic lesions of the skeleton result from asynchronous bone turnover wherein increased bone resorption by osteoclasts (OCLs) is associated to suppression of bone formation due to inhibition of osteoblasts (OBLs). In this context, the balance between osteoprotegerin (OPG) and the receptor activator for nuclear factor-κB ligand (RANKL) plays a relevant role, because RANKL signaling induces differentiation, activation and survival of OCLs, whereas OPG acts as a decoy receptor of RANKL, thus blocking bone resorption. In the MM milieu, the interaction of malignant plasma cells (PCs) and bone marrow stromal cells (BMSCs) favors the production of RANKL and reduces OPG secretion by BMSCs. The resulting severe imbalance in RANKL/OPG ratio is the main trigger of MM-related bone disease (BD). Emerging evidence indicates that microRNAs (miRNAs) play a crucial role in bone remodelling as they can act on bone effectors (OCLs, OBLs, BMSCs) and PCs. Indeed, several findings suggest that miR-29 family support OBL differentiation, while we have previously demonstrated that miR-29b is down-regulated during osteoclastogenesis and its enforced expression functionally inhibits terminally differentiated OCLs. Moreover, also miR-21 plays an important role in OCL differentiation and is up-regulated by IL-6 via STAT3 that, in turn, promotes survival and proliferation of PCs, favoring bone damage. On these bases, we investigated whether modulation of miR-21 expression in BMSCs may have an impact on MM microenviroment, focusing our attention on OPG production, taking into account that different bioinformatics resources indicate OPG mRNA as a predicted target of miR-21. To this end, we established a co-culture system where primary BMSCs and HS-5, a human BMSC cell line, were exposed to either RPMI 8226 MM cells or primary CD138+ PCs isolated from MM patients. Then, we isolated BMSCs and evaluated miR-21 and OPG expression. We observed strong up-regulation of miR-21 in BMSCs and HS-5 together with a significant reduction of OPG levels in the presence of MM cells. These preliminary data suggested that antagonizing miR-21 in BMSCs exposed to MM cells could restore RANKL/OPG ratio through the up-regulation of OPG. To address this hypothesis, we transduced HS-5 cell line with a lentiviral vector carrying anti-miR-21 sequence (a21 HS-5) or with an empty lentiviral vector carrying only the GFP gene (GFP CNT HS-5). OPG expression was determined by RT-PCR, Western Blotting and ELISA assays after 48h and 72h of exposure to RPMI 8226 cells or primary PCs. We observed a significant increase in OPG production in a21 HS-5 cells compared to controls (Fig. 1, panel A and panel B). We also evaluated RANKL expression in the same samples. Interestingly, constitutive inhibition of miR-21 in HS-5 cell line upon exposure to MM cells induced a significant RANKL down-regulation, thus restoring a physiological RANKL/OPG ratio.We hypothesized that this surprising result may be due to the activity of the protein inhibitor of activated STAT3 (PIAS3). Indeed, PIAS3 binds to STAT3 and blocks its DNA binding ability, thereby inhibiting STAT3-mediated gene activation. It is already known that PIAS3 acts as a negative regulator of the RANKL expression and it is a direct and validated target of miR-21. Therefore, the inhibition of miR-21 induces PIAS3 up-regulation and a decreased RANKL transcription. To confirm the involvement of PIAS3 in RANKL suppression in our system, we performed a WB assay, showing that in a21 HS-5 cells, PIAS3 is indeed over-expressed compared to controls. Taken together, these findings provide the first evidence that inhibition of miR-21 in MM microenviroment restores RANKL/OPG balance, which might result in prevention of skeletal- related events. These findings support the design of innovative miR-21 inhibition-based approaches against MM-related BD.Supported by the Italian Association for Cancer Research (AIRC) “Special Program Molecular Clinical Oncology - 5 per mille”, PI:P.T., n. 9980, 2010/15.” [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Thulium Ion Promotes Apoptosis of Primary Mouse Bone Marrow Stromal Cells

Bone is one of the main target organs for the lanthanides (Ln). Biodistribution studies of Tm-based compounds in vivo showed that bone had significant uptake. But the effect of Tm(3+) on primary mouse bone marrow stromal cells (BMSCs) has not been reported. So we investigated the effect and underlying mechanisms of Tm(3+) on BMSCs. Cell viability, cell apoptosis, reactive oxygen species (ROS) level, lactate dehydrogenase (LDH) activity and mitochondrial membrane potential (MMP) were studied. The results indicated that Tm(3+) increased the viability of BMSCs at concentrations of 1×10(−7), 1×10(−6), 1×10(−5), and 1×10(−4) mol/L in a dose-dependent manner, turned to decrease the viability of BMSCs at the highest concentration of 1×10(−3) mol/L for 24, 48, and 72 h. Tm(3+) at 1×10(−3) mol/L promoted apoptosis of BMSCs, increased the ROS and LDH levels, and decreased MMP in BMSCs. Taken together, we demonstrated that Tm(3+) + at 1×10(−3) mol/L might induce cellular apoptosis through mitochondrial pathway. These resultsmay be helpful for more rational application of Tm-based compounds in the future.

In Vitro Study of a Novel Oxysterol for Osteogenic Differentiation on Rabbit Bone Marrow Stromal Cells

Bone morphogenetic proteins (BMPs) are powerful osteoinductive growth factors but are associated with exorbitant costs and undesirable side effects. Oxysterols are biocompatible cholesterol oxidation products with osteoinductive properties that may represent an alternative to BMP. In this study, the authors examine the osteogenic potential and mechanisms of actions of oxysterol 49, a novel oxysterol analogue, in primary rabbit bone marrow stromal cells. Bone marrow stromal cells were isolated from the iliac crests of New Zealand White rabbits and then treated with various concentrations of oxysterol 49 or BMP-2, either alone or in combination. Alkaline phosphatase activity and expression of osteocalcin and osteopontin were evaluated. The effect of treatment of cells with cyclopamine, a known hedgehog signaling pathway inhibitor, was also assessed. Alkaline phosphatase activity was increased in cells treated with 1 µM oxysterol 49 relative to cells treated with BMP-2. Expression of osteocalcin and osteopontin in cells treated with oxysterol 49 and BMP-2 was equivalent. Alkaline phosphatase activity was decreased with the addition of cyclopamine. Combined treatment with oxysterol 49 and BMP-2 resulted in additive increases in alkaline phosphatase activity and osteocalcin and osteopontin expression. Oxysterol 49 has osteoinductive properties that are similar to those of BMP-2 in rabbit bone marrow stromal cells. The mechanism of this activity is at least in part related to the hedgehog signaling pathway. The two growth factors demonstrate additive effects when used in combination. Further study is required to examine the potential role of oxysterol 49 as a complement or alternative to BMP-2 in bone tissue engineering.

Targeting the CD20 and CXCR4 Pathways in Non-Hodgkin Lymphoma with Rituximab and High-Affinity CXCR4 Antagonist BKT140

Chemokine axis CXCR4/CXCL12 is critically involved in the survival and trafficking of normal and malignant B lymphocytes. Here, we investigated the effect of high-affinity CXCR4 antagonist BKT140 on lymphoma cell growth and rituximab-induced cytotoxicity in vitro and in vivo. In vitro efficacy of BKT140 alone or in combination with rituximab was determined in non-Hodgkin lymphoma (NHL) cell lines and primary samples from bone marrow aspirates of patients with NHL. In vivo efficacy was evaluated in xenograft models of localized and disseminated NHL with bone marrow involvement. Antagonizing CXCR4 with BKT140 resulted in significant inhibition of CD20+ lymphoma cell growth and in the induction of cell death, respectively. Combination of BKT140 with rituximab significantly enhanced the apoptosis against the lymphoma cells in a dose-dependent manner. Moreover, rituximab induced CXCR4 expression in lymphoma cell lines and primary lymphoma cells, suggesting the possible interaction between CD20 and CXCR4 pathways in NHL. Primary bone marrow stromal cells (BMSC) further increased CXCR4 expression and protected NHL cells from rituximab-induced apoptosis, whereas BKT140 abrogated this protective effect. Furthermore, BKT140 showed efficient antilymphoma activity in vivo in the xenograft model of disseminated NHL with bone marrow involvement. BKT140 treatment inhibited the local tumor progression and significantly reduced the number of NHL cells in the bone marrow. Combined treatment of BKT140 with rituximab further decreased the number of viable lymphoma cells in the bone marrow, achieving 93% reduction. These findings suggest the possible role of CXCR4 in NHL progression and response to rituximab and provide the scientific basis for the development of novel CXCR4-targeted therapies for refractory NHL.

Caffeine inhibits adipogenic differentiation of primary adipose-derived stem cells and bone marrow stromal cells

Caffeine consumption has been related to loss of body weight and modulates lipid metabolism. However, impacts of caffeine on adipogenic differentiation have not been well determined yet. The present study evaluated the effects of caffeine on adipogenesis using primary rat adipose-derived stem cells (ADSCs) and a mouse bone marrow stromal cell line (M2-10B4) in vitro. ADSCs and M2-10B4 were continuously exposed to caffeine (0.1–1mM) during adipogenic differentiation for 7 and 12days, respectively. Oil red O and Nile red staining showed that caffeine reduced lipid droplet and adipocyte levels in both cell types. In addition, Nile red staining and FACScan flow cytometry showed that caffeine dose-dependently decreased adipocyte differentiation from 20% to 50% of the control ADSCs and M2-10B4 cells. Caffeine decreased the expression of adipogenesis-related genes including peroxisome proliferator-activated receptor-γ, CCAAT/enhancer-binding protein-α, adipocyte lipid binding protein, lipoprotein lipase, leptin, and TNFα in a dose-dependent manner. Rather, low concentration of caffeine (0.1mM) significantly increased IL-6 expression, but unexpectedly inhibited that at a concentration more than 0.3mM. Taken together, caffeine was able to effectively inhibit adipogenic differentiation of ADSCs and M2-10B4 cells partly through its inhibition of adipogenesis-related factors.

Fabrication of 3D cell-laden hydrogel microstructures through photo-mold patterning

Native tissues are characterized by spatially organized three-dimensional (3D) microscaled units which functionally define cells–cells and cells–extracellular matrix interactions. The ability to engineer biomimetic constructs mimicking these 3D microarchitectures is subject to the control over cell distribution and organization. In the present study we introduce a novel protocol to generate 3D cell laden hydrogel micropatterns with defined size and shape. The method, named photo-mold patterning (PMP), combines hydrogel micromolding within polydimethylsiloxane (PDMS) stamps and photopolymerization through a recently introduced biocompatible ultraviolet (UVA) activated photoinitiator (VA-086). Exploiting PDMS micromolds as geometrical constraints for two methacrylated prepolymers (polyethylene glycol diacrylate and gelatin methacrylate), micrometrically resolved structures were obtained within a 3 min exposure to a low cost and commercially available UVA LED. The PMP was validated both on a continuous cell line (human umbilical vein endothelial cells expressing green fluorescent protein, HUVEC GFP) and on primary human bone marrow stromal cells (BMSCs). HUVEC GFP and BMSCs were exposed to 1.5% w/v VA-086 and UVA light (1 W, 385 nm, distance from sample = 5 cm). Photocrosslinking conditions applied during the PMP did not negatively affect cells viability or specific metabolic activity. Quantitative analyses demonstrated the potentiality of PMP to uniformly embed viable cells within 3D microgels, creating biocompatible and favorable environments for cell proliferation and spreading during a seven days' culture. PMP can thus be considered as a promising and cost effective tool for designing spatially accurate in vitro models and, in perspective, functional constructs.