Bone Marrow Stromal Cell-conditioned Medium Research Articles

Bone marrow stromal cell-conditioned medium regenerates injured sciatic nerve by increasing expression of MPZ and NGF and decreasing apoptosis.

Despite the many benefits of mesenchymal stem cell (MSC) transplantation for tissue regeneration, there are some limitations to using them, including the high costs, applying invasive procedures, the possibility of transplant rejection, and cell malignancy. This study aimed to investigate the effect of secretions of bone marrow stromal cells (BMSCs) with the cell-free strategy on damaged sciatic nerve with an emphasis on the role of apoptosis and the expression of myelin protein zero (MPZ) and nerve growth factor (NGF) proteins. BMSCs were cultured and a 25-fold concentrated conditioned medium (CM) from the cells was provided. After creating a crush injury in the left sciatic nerve of male rats, BMSCs or CM were injected into the injured site of the nerve. Four weeks later, the expression of MPZ, NGF, Bax, and Bcl-2 proteins in the sciatic nerve and histological parameters of the sciatic nerve and gastrocnemius muscle were assessed. The results demonstrated that injection of CM decreased apoptosis and increased expression of MPZ and NGF proteins, improving remyelination and regeneration of the sciatic nerve almost as much as the transplantation of the BMSCs themselves compared to the control group. The results suggest that BMSC secretions may improve remyelination and regeneration of damaged sciatic nerve by increasing the expression of MPZ and NGF and decreasing apoptosis.

Selective homing of CAR-CIK cells to the bone marrow niche enhances control of the acute myeloid leukemia burden

AbstractAcute myeloid leukemia (AML) is a hematological malignancy derived from neoplastic myeloid progenitor cells characterized by abnormal clonal proliferation and differentiation. Although novel therapeutic strategies have recently been introduced, the prognosis of AML is still unsatisfactory. So far, the efficacy of chimeric antigen receptor (CAR)–T-cell therapy in AML has been hampered by several factors, including the poor accumulation of the blood-injected cells in the leukemia bone marrow (BM) niche in which chemotherapy-resistant leukemic stem cells reside. Thus, we hypothesized that overexpression of CXCR4, whose ligand CXCL12 is highly expressed by BM stromal cells within this niche, could improve T-cell homing to the BM and consequently enhance their intimate contact with BM-resident AML cells, facilitating disease eradication. Specifically, we engineered conventional CD33.CAR–cytokine-induced killer cells (CIKs) with the wild-type (wt) CXCR4 and the variant CXCR4R334X, responsible for leukocyte sequestration in the BM of patients with warts, hypogammaglobulinemia, immunodeficiency, and myelokathexis syndrome. Overexpression of both CXCR4wt and CXCR4mut in CD33.CAR-CIKs resulted in significant improvement of chemotaxis toward recombinant CXCL12 or BM stromal cell–conditioned medium, with no observed impairment of cytotoxic potential in vitro. Moreover, CXCR4-overexpressing CD33.CAR-CIKs showed enhanced in vivo BM homing, associated with a prolonged retention for the CXCR4R334X variant. However, only CD33.CAR-CIKs coexpressing CXCR4wt but not CXCR4mut exerted a more sustained in vivo antileukemic activity and extended animal survival, suggesting a noncanonical role for CXCR4 in modulating CAR-CIK functions independent of BM homing. Taken together, these data suggest that arming CAR-CIKs with CXCR4 may represent a promising strategy for increasing their therapeutic potential for AML.

Targeting of versican, a proteoglycan in bone marrow microenvironment by microRNAs inhibit multiple myeloma progression

Abstract Background The abnormal growth of malignant plasma cells in Multiple myeloma (MM) require bone marrow (BM) niche consisting of proteoglycans, cytokines, etc. for their growth. One of the chondroitin sulfate proteoglycan, Versican (VCAN) has gained consideration in milieu of solid tumors but there is dearth of literature in MM. Hence, involvement of VCAN in MM and its regulation by microRNAs has been studied. Materials and Methods 30 MM patients & 20 controls were recruited and BM Stromal Cells (BMSCs) were harvested by primary culture. Molecular expression of VCAN and microRNAs (miR-144, miR-199 & miR-203) were examined in study subjects and MM cell lines (RPMI8226 & U266). The involvement of VCAN in myeloma pathogenesis was studied using BMSCs conditioned medium (BMSCs-CM) and VCAN-neutralizing antibody or microRNA mimics (miR-144 and miR-199). Further, signaling pathways altered by knockdown of VCAN by antibody or microRNA mimics were identified. Results Elevated expression of VCAN was found in MM patients especially in the stroma of BM niche while microRNA expression was lower in patients and showed negative correlation with VCAN levels. VCAN being produced in stroma found at lower levels in MM cell lines. Moreover, BMSCs CM showed presence of VCAN which upon supplementing to MM cells alter parameters in favour of myeloma progression, however, this effect was neutralized by VCAN antibody or miR mimics of miR-144 and miR-199. The downstream signaling of VCAN was found to activate FAK and STAT3 which subsides by using VCAN antibody or miR mimics. Conclusion The neutralization of oncogenic effect of BMSCs CM by VCAN blockage using antibody or miR mimics affirms its plausible role in progression of MM. These findings open up new avenues for exploring VCAN as a novel therapeutic target and microRNAs as a mean to target VCAN which could be exploited in clinical settings in future.

PO-284 Involvement of versican, a chondroitin sulfate proteolgycan in the pathogenesis of multiple myeloma

IntroductionMultiple Myeloma (MM) is second most common haematological malignancy characterised by uncontrolled proliferation of abnormal plasma cells in bone marrow (BM). The growth of these myeloma cells is facilitated by BM niche consisting of numerous proteins, proteoglycans, cytokines and growth factors. One of the chondroitin sulfate proteoglycan, Versican (VCAN) has gained consideration in context of solid tumours where in it has been shown to promote tumour progression but there is dearth of literature in haematological malignancies including MM. Therefore, the involvement of VCAN in association with MM has been studied.Material and methods30 newly diagnosed MM patients and 20 controls were recruited. BM Mononuclear Cells (BMMNCs) were isolated from their BM aspirate. In representative samples (n=15), BM Stromal Cells (BMSCs) were harvested from BMMNCs by primary culture. The relative mRNA expression of VCAN and its four isoforms (V0, V1, V2 and V3) were investigated in BMMNCs, BMSCs and MM cell lines (RPMI8226 and U266). Conditioned medium (CM) of primary cultured BMSCs was collected and examined for presence of VCAN by ELISA. Thereafter, the effect of BMSCs CM was studied on MM cells in presence or absence of VCAN antibody. To accomplish this, PCNA along with MTT assay for proliferation, VEGF for angiogenesis and Bcl-2, PUMA for apoptosis were investigated. Further, the signalling pathways involved in the action of VCAN were also identified.Results and discussionsThe relative mRNA expression of VCAN and its isoforms were found significantly higher in MM patients in both BMMNCs and BMSCs with higher expression in BMSCs than in BMMNCs. VCAN being produced in stroma found at lower levels in MM cell lines. Furthermore, BMSCs CM showed the presence of VCAN whose effect was evaluated in MM cell lines in vitro. Upon treatment with BMSCs CM, proliferation and angiogenesis increase while apoptosis decrease in cell lines, however, effect of CM neutralised in presence of VCAN antibody. The downstream signalling of VCAN was observed to entail phosphorylation of FAK and STAT3 which subsides by treatment with VCAN antibody.ConclusionAugmented levels of VCAN and its isoforms in BM of patients especially in BMSCs imply their involvement in BM microenvironment of MM. The neutralisation of the oncogenic effect of BMSCs CM upon treatment with VCAN antibody affirms the plausible role of VCAN in pathogenesis of MM. These findings open up new avenues for exploration of VCAN as a therapeutic target for treatment of MM in future.

Bone marrow mesenchymal stem cells-conditioned medium enhances vascular remodeling after stroke in type 2 diabetic rats

Our previous studies have found that stem cells conditioned medium (CM) facilitated functional recovery after stroke in non-diabetics. However, whether bone marrow stromal cells conditioned medium (BMSCs-CM) treatment after stroke in type 2 diabetic (T2DM) rats improve functional outcomes remains unclear. T2DM rats were induced and subjected to stroke then treated with or without BMSCs-CM. Functional outcomes and blood-brain barrier (BBB) leakage were performed, the expression of Angiopoietin (Ang) 1 and tyrosine kinase (Tie) 2 were also assessed. Our results showed that BMSCs-CM treatment significantly improved functional outcomes, decreased BBB leakage and the expression of Ang1 and Tie2 were also changed after BMSCs-CM treatment in type 2 diabetes after stroke. In conclusion, enhanced expression of Ang1 and Tie2 in ischemic brain after BMSCs-CM treatment of stroke may contribute to the improved functional recovery after stroke in type 2 diabetic rats.

Topography Influences Adherent Cell Regulation of Osteoclastogenesis

The importance of osteoclast-mediated bone resorption in the process of osseointegration has not been widely considered. In this study, cell culture was used to investigate the hypothesis that the function of implant-adherent bone marrow stromal cells (BMSCs) in osteoclastogenesis is influenced by surface topography. BMSCs isolated from femur and tibia of Sprague-Dawley rats were seeded onto 3 types of titanium surfaces (smooth, micro, and nano) and a control surface (tissue culture plastic) with or without osteogenic supplements. After 3 to 14 d, conditioned medium (CM) was collected. Subsequently, rat bone marrow–derived macrophages (BMMs) were cultured in media supplemented with soluble receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) as well as BMSC CM from each of the 4 surfaces. Gene expression levels of soluble RANKL, osteoprotegerin, tumor necrosis factor α, and M-CSF in cultured BMSCs at different time points were measured by real-time polymerase chain reaction. The number of differentiated osteoclastic cells was determined after tartrate-resistant acid phosphatase staining. Analysis of variance and t test were used for statistical analysis. The expression of prominent osteoclast-promoting factors tumor necrosis factor α and M-CSF was increased by BMSCs cultured on both micro- and nanoscale titanium topographies (P < 0.01). BMSC CM contained a heat-labile factor that increased BMMs osteoclastogenesis. CM from both micro- and nanoscale surface-adherent BMSCs increased the osteoclast number (P < 0.01). Difference in surface topography altered BMSC phenotype and influenced BMM osteoclastogenesis. Local signaling by implant-adherent cells at the implant-bone interface may indirectly control osteoclastogenesis and bone accrual around endosseous implants.

Abstract W MP87: PDGF Contributes to BMSC Treatment Induced Neurogenesis and White Matter and Axonal Remodeling in T2DM Stroke Rats

Objective: Our previous studies have found that bone-marrow-stromal cell (BMSC) treatment of stroke in Type two DM (T2DM) rats, initiated at 3 days after stroke, improved functional recovery. Neurogenesis and white matter (WM) remodeling play an important role in neurorestorative effects after stroke. In this study, we tested whether BMSCs regulate neurogenesis and WM remodeling and the underlying mechanisms of BMSC induced neurorestorative effects in T2DM stroke rats. Methods: T2DM was induced with streptozotocin injection in addition to a high fat diet. T2DM rats were subjected to 2h of middle cerebral artery occlusion (MCAo), then treated with human BMSCs (5X106) or vehicle control (n=8/group) initiated at 3 days after MCAo and rats were monitored for 28 days. Neuroblast migration, WM changes, and gene and protein expression were measured in the ischemic brain. Subventricular zone (SVZ) explant cell migration and primary cortical neuron (PCN) axonal outgrowth measurements were performed in vitro. Results: BMSC treatment in T2DM rats significantly improves functional outcome and increases WM remodeling identified by increased myelin and axonal density. BMSCs also increase the neuroblast migration protein doublecortin (DCX, 25.0±4.3% vs control: 4.5±1.1%), platelet-derived growth factor (PDGF)-AA, and bFGF expression in the ischemic border zone. Angiogenic ELISA array data are consistent with the immunostaining data, showing that BMSC treatment increases PDGF-AA (2.1 fold), PDGF-BB (2.5 fold) and bFGF (1.8 fold) in the ischemic brain. Using an in vitro cell culture model, we found that BMSCs secrete high levels of PDGF. PDGF treatment significantly increases SVZ explant cell migration (1.7 fold) and PCN axonal outgrowth (1.9 fold) compared to non-treatment control. Inhibition of PDGF with neutralized anti-PDGF antibody significantly attenuates BMSC conditioned medium induced SVZ cell migration and PCN axon outgrowth. Conclusion: BMSC treatment of stroke in T2DM increases WM remodeling and neurogenesis as well as increases PDGF expression. PDGF not only promotes neuronal migration, but also increases axonal outgrowth. Therefore, increasing PDGF likely contributes to BMSC induced neurogenesis and WM remodeling in T2DM stroke rats.

STAT3 as a mediator of BCR-ABL1-independent resistance in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is a hematopoietic cell disorder, with an annual incidence of 1–1.5 per 100 000 in the United States. The BCR-ABL1 oncogene is necessary and sufficient to cause the leukemic phenotype. CML is typically diagnosed in the chronic phase, where the myeloid cell compartment is expanded, but cellular differentiation maintained. Without effective therapy, there is inevitable transformation to the blastic phase, which resembles an acute leukemia of myeloid or lymphoid phenotype. Current CML therapy is based on tyrosine kinase inhibitors (TKIs), which block progression from the chronic to the blastic phase in the majority of patients. The first-generation TKI, imatinib mesylate (IM), is generally well tolerated, but has a failure rate of about 25% due to resistance or intolerance. Second-generation TKIs (dasatinib; nilotinib) provide effective salvage options, but some patients will eventually progress to blastic phase and have a poor prognosis. Additionally, even in patients with profound responses (complete molecular response), recurrence is common upon discontinuation of TKI therapy, a situation referred to as persistence. TKI resistance can result from two fundamentally different mechanisms: BCR-ABL1-dependent resistance, in which kinase domain mutations restore kinase activity by disrupting TKI binding,1, 2 and BCR-ABL1-independent resistance, which can be divided into (i) extrinsic resistance, in which contact between CML cells and bone marrow-derived factors protects CML cells from TKIs,3, 4 and (ii) cell-autonomous (intrinsic) resistance, in which CML cells activate alternative signaling pathways in the absence of bone marrow-derived factors.5, 6 Importantly, BCR-ABL1-independent resistance occurs despite continued suppression of BCR-ABL1 activity. It is thought that persistence of residual leukemia in patients responding to TKIs is due to the innate BCR-ABL1 independence of CML stem cells.7 Consistent with this, we have found that primitive CML progenitor cells, including quiescent Lin− CML cells, survive despite TKI inhibition of BCR-ABL1.8 Our recent studies have implicated STAT3 in BCR-ABL1-independent resistance. In line with previous reports, we have found upregulation of pSTAT3Y705 in TKI-naive CML progenitor cells grown in HS-5 bone marrow stromal cell-conditioned medium in the presence of IM, and that pSTAT3Y705 promotes CML cell survival, implicating STAT3 as a critical mediator of extrinsic BCR-ABL1-independent resistance. Interestingly, we have also found pSTAT3Y705 activation in CML CD34+ cells from patients with intrinsic TKI resistance (clinical resistance in the absence of BCR-ABL1 mutations). This suggests that extrinsic and intrinsic BCR-ABL1-independent resistance mechanisms converge on pSTAT3Y705, a notion that was validated with STAT3-specific small hairpin RNA and dominant-negative mutants. Our observations prompted us to evaluate the effects of small-molecule STAT3 inhibitors that block STAT3 dimerization through binding to the SH2 domain. Successive STAT3 inhibitor library screens identified BP-5-087 as a highly potent and selective STAT3 inhibitor. Computational modeling and physicochemical assays confirmed binding of BP-5-087 with STAT3. In biological assays using TKI-resistant progenitors, we found that BP-5-087 combined with imatinib reduces survival and colony formation by CML progenitor and stem cells from patients with BCR-ABL1-independent resistance, consistent with a synthetically lethal pharmacologic interaction. Taken together, our data indicate that activation of pSTAT3Y705 is central to BCR-ABL1 kinase-independent TKI resistance, and that genetic, functional or pharmacologic inhibition of STAT3 reduces TKI-resistant clonogenic potential in vitro. BP-5-087 is a highly potent and selective STAT3 inhibitor. Combinations of BP5-087 with imatinib greatly reduce the survival of primary TKI-resistant stem and progenitor cells, and as such, may be considered a promising combinatorial therapy to overcome BCR-ABL1-independent TKI resistance and eliminate residual leukemia.

IL-1β induces p62/SQSTM1 and represses androgen receptor expression in prostate cancer cells.

Chronic inflammation is associated with advanced prostate cancer (PCa), although the mechanisms governing inflammation-mediated PCa progression are not fully understood. PCa progresses to an androgen independent phenotype that is incurable. We previously showed that androgen independent, androgen receptor negative (AR(-) ) PCa cell lines have high p62/SQSTM1 levels required for cell survival. We also showed that factors in the HS-5 bone marrow stromal cell (BMSC) conditioned medium can upregulate p62 in AR(+) PCa cell lines, leading us to investigate AR expression under those growth conditions. In this paper, mRNA, protein, and subcellular analyses reveal that HS-5 BMSC conditioned medium represses AR mRNA, protein, and nuclear accumulation in the C4-2 PCa cell line. Using published gene expression data, we identify the inflammatory cytokine, IL-1β, as a candidate BMSC paracrine factor to regulate AR expression and find that IL-1β is sufficient to both repress AR and upregulate p62 in multiple PCa cell lines. Immunostaining demonstrates that, while the C4-2 population shows a primarily homogeneous response to factors in HS-5 BMSC conditioned medium, IL-1β elicits a strikingly heterogeneous response; suggesting that there are other regulatory factors in the conditioned medium. Finally, while we observe concomitant AR loss and p62 upregulation in IL-1β-treated C4-2 cells, silencing of AR or p62 suggests that IL-1β regulates their protein accumulation through independent pathways. Taken together, these in vitro results suggest that IL-1β can drive PCa progression in an inflammatory microenvironment through AR repression and p62 induction to promote the development and survival of androgen independent PCa.

P62/SQSTM1 is required for cell survival of apoptosis‐resistant bone metastatic prostate cancer cell lines

Bone marrow stromal cell (BMSC) paracrine factor(s) can induce apoptosis in bone metastatic prostate cancer (PCa) cell lines. However, the PCa cells that escape BMSC-induced apoptosis can upregulate cytoprotective autophagy. C4-2, C4-2B, MDA PCa 2a, MDA PCa 2b, VCaP, PC3, or DU145 PCa cell lines were grown in BMSC conditioned medium and analyzed for mRNA and/or protein accumulation of p62 (also known as sequestome-1/SQSTM1), Microtubule-associated protein 1 light chain 3B (LC3B), or lysosomal-associated membrane protein 1 (LAMP1) using quantitative polymerase chain reaction (QPCR), Western blot, or immunofluorescence. Small interfering RNA (siRNA) was used to determine if p62 is necessary PCa cell survival. BMSC paracrine signaling upregulated p62 mRNA and protein in a subset of the PCa cell lines. The PCa cell lines that were insensitive to BMSC-induced apoptosis and autophagy induction had elevated basal p62 mRNA and protein. In the BMSC-insensitive PCa cell lines, siRNA knockdown of p62 was cytotoxic and immunostaining showed peri-nuclear clustering of autolysosomes. However, in the BMSC-sensitive PCa cell lines, p62 siRNA knockdown was not appreciably cytotoxic and did not affect autolysosome subcellular localization. A pattern emerges wherein the BMSC-sensitive PCa cell lines are known to be osteoblastic and express the androgen receptor, while the BMSC-insensitive PCa cell lines are characteristically osteolytic and do not express the androgen receptor. Furthermore, BMSC-insensitive PCa may have evolved a dependency on p62 for cell survival that could be exploited to target and kill these apoptosis-resistant PCa cells in the bone.

Regulation of prostate cancer cell migration toward bone marrow stromal cell-conditioned medium by Wnt5a signaling

Bone is a major site of metastasis for several types of malignant tumor. Specific interactions between tumor cells and the bone microenvironment contribute to the tendency of tumors to metastasize to bone. Furthermore, Wnt5a participates in the progression of several types of malignant tumor. This study investigates the role of Wnt5a in the migration of the prostate cancer (PCa) cell line PC3 toward bone marrow stromal cell (BMSC)‑conditioned medium (CM). The expression of 22 genes associated with bone metastasis was measured in three PCa cell lines (LNCaP, PC3 and DU145). Subsequently, the proliferation and migration capacities of PC3 cells treated either with small interfering RNA (siRNA) against Wnt5a or with recombinant mouse (rm) Wnt5a were analyzed with alamarBlue and transwell assays. BMSC‑CM was collected to evaluate its effect on PC3 cell migration. Also, the expression of Wnt5a in BMSCs was knocked down prior to collection of the CM to evaluate its effects on the migration of PC3 cells. Significantly higher levels of Wnt5a mRNA expression were identified in the PC3 cells, compared with those in LNCaP and DU145 cells. Silencing Wnt5a expression with siRNA reduced the migration capacity of PC3 cells by 50%. The addition of rmWnt5a improved the migration capacity of PC3 cells in a concentration‑dependent manner. PC3 cells preferred to migrate toward BMSC‑CM than toward the control. CM from Wnt5a siRNA‑treated BMSCs significantly reduced PC3 cell migration. Wnt5a promotes PC3 cell migration toward BMSC‑CM, indicating that Wnt5a is a potential therapeutic target for the treatment of advanced PCa.

Matrix Metalloproteinses-13 (Collagenase-3) Secreted by Multiple Myeloma Cells Enhances the Bone Resorptive Activity of Osteoclasts.

Abstract 2853Poster Board II-829 Introduction:Osteolytic bone disease is a frequent complication of multiple myeloma (MM) that is characterized by reduced bone formation and increased bone resorption. MM cells produce several cytokines causing dysregulation of the bone remodeling process that results in excessive bone resorption mediated by activated osteoclasts (OCLs). Matrix metalloproteinases (MMPs) play a critical role in tumor invasion and progression in many solid tumors such as breast cancer but the role of MMPs in MM has have not been well elucidated. Methods and Results:We analyzed the MMP expression profiles (MMP1, 2, 3, 7, 8, 12, 13) of MM cells and found very high expression (400-fold higher) of MMP13 compared to other MMPs. Tissue array analysis for MMP-13 expression in MM tumor samples and normal control tissue indicated that MMP13 was only highly expressed in MM tumor samples. ELISA analysis of serum samples revealed a positive correlation of MMP13 expression with MM bone disease. MMP13 was not detectable in the sera of healthy donors, whereas 62% of MM patients expressed MMP13 in their serum samples, among which 87% had extensive bone disease. Co-culture of MM cells with primary bone marrow stromal cells (BMSC) resulted in a significant (P<0.001) increase (up to 14-fold) of MMP13 secretion by MM cells. RT-PCR analysis indicated that the expression of MMP13 gene in MM cells was markedly enhanced by BMSC conditioned medium, suggesting that BMSC-secreted soluble factors, instead of direct cellular interactions, induce MMP13. Functional inactivation of different cytokines with neutralizing antibodies identified IL-6 as a critical cytokine for the induction of MMP13. Electromobility shift assays showed that IL-6 mediated AP-1 activation promotes MMP13 transcription. Importantly, MMP13 significantly enhanced osteoclastic bone resorption on dentin slices induced by RANKL plus M-CSF. However, the strong induction of bone resorption was not derived from increased OCL formation since MMP13 only slightly increased OCL numbers. MMP13 significantly increased the OCL size as well as the nuclear number/OCL which increases the bone resorption activity per osteoclast. Conclusions:Our results suggest that MMP13 augments the pathogenic process of osteolytic lesions in MM by increasing the bone resorption capacity/OCL. These results suggest that targeting MMP13 may represent a novel approach to ameliorate osteolytic lesions in MM. Disclosures:Roodman:Novartis: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy; Celgene: Consultancy; Acceleron: Consultancy. Lentzsch:Celgene: Consultancy, Speakers Bureau; Pfizer: Consultancy.

Effect of bone marrow stromal cell-conditioned medium on the glutamate uptake of peroxide-injured astrocytes

We aimed to investigate the effect of bone marrow stromal cell-conditioned medium (BCM) on glutamate uptake of peroxide (H 2O 2)-injured astrocytes. Bone marrow stromal cells (BMSC) were isolated from rat bone marrow. Confluent BMSC cultures were incubated with serum-free Dulbecco’s Modified Eagle’s Medium to create the BCM. Astrocytes were isolated from 1-day-old rats. H 2O 2-injured astrocytes were cultured in BCM (experimental group) or serum-free medium (control group). The labeled glutamate ( 3H-L-glutamate) uptake by H 2O 2-injured astrocytes with or without BCM was compared after 1 and 3 days. We found that astrocytes cultured in BCM exhibited increased glutamate uptake compared to those cultured in serum-free medium following H 2O 2-induced injury ( p < 0.01) and concluded that BCM increased the glutamate uptake capability of H 2O 2-injured rat astrocytes. The therapeutic benefits associated with BMSC transplantation following brain injury might be partly due to increased glutamate uptake by astrocytes.

Functional SDF-1 Secretion from BM Stromal Cells Is a Cell Contact-Dependent Event Mediated by Cx43 and Cx45 Gap-Junctions

Introduction: Bone marrow (BM) supporting stromal cells secrete an array of important signaling factors, in a synchronized fashion, creating a unique microenvironment required for regulation of hematopoietic stem and progenitor cells (HSPC) maintenance, localization, self-renewal and development. The chemokine SDF-1 (also termed CXCL12), whose fine tuned levels are critical for HSPC quiescence, is the only known powerful chemotactic factor for both human and murine HSPC. BM stromal cells rapidly respond to alarm situations by secreting SDF-1 to the circulation, promoting progenitor cell mobilization (Dar et al., ASH 2006) as part of host defense and repair mechanism. This well synchronized event suggests that the supporting stromal cells function as a physiological syncytium. However, the mechanism governing the synchronized SDF-1 de-novo synthesis and secretion from BM stromal cells is not known. The gap-junction (GJ) intercellular channels protein Connexins (Cx), allow the passage of secondary messengers between cells in a tissue, thus synchronizing its activity. The existence of the multi-function Cx43 and Cx45 GJ proteins in the BM compartments suggests a direct intercellular communication, coordinating their supporting capacity.Results: In order to evaluate Cx networks involvement in the regulation of SDF-1 secretion, we first checked for Cx43 expression in the BM of mice treated with G-CSF. We have previously shown that as part of G-CSF induced mobilization, SDF-1 levels are downregulated in the BM, thus promoting HSPC proliferation and mobilization (Petit et al., Nat.Immunol, 2002). Cx43 was strongly downregulated both at mRNA and protein levels in the BM of G-CSF treated mice, in correlation with reduced levels of SDF-1. Hence, we asked whether Cx directly regulates SDF-1 secretion. Treating BM stromal cells with carbenoxolone (CBX), a known Cx blocker, resulted in immediate, complete and specific blockage of SDF-1 secretion to the conditioned medium (CM). Secretion of the cytokine HGF, measured as control, was not affected. In addition, prolonged stimulation of BM stromal cells with CBX resulted in elimination of Cx43 and Cx45 transcription, as well as SDF-1 transcription. We then evaluated the direct cell-contact necessity for SDF-1 secretion. Equal numbers of BM stromal cells were seeded in different-sized culture plates allowing and restricting cell contact, in the same volume of medium. SDF-1 levels in the CM were found to be significantly higher in the contacting BM stromal cell cultures compared to the non-contacting BM stromal cell cultures, while HGF levels were not affected. Surprisingly, similar SDF-1 mRNA levels were detected in both groups. These results suggest that SDF- 1 secretion, but not transcription, is a cell contact dependent event. Next, the functional consequences of stromal Cx blockage was assessed by testing migration of human G2 cells towards CM collected from non-treated and CBX-treated BM stromal cells. G2 cells failed to migrate towards CBX-treated BM stromal cells CM. This was further supported by in-vivo homing experiments - BM mononuclear cells were intravenously injected to PBS and CBX treated C57BL/6 recipients. CBX-treated mice exhibited reduction in homing capacities to the femoral BM, in correlation with lower BM levels of SDF-1. cAMP is a small secondary messenger transmitted by Cx between cells and thus may serve as the mediator of Cx regulated SDF-1 secretion. cAMP inhibition in BM stromal cells by MDL resulted in CBX-like effect - decreased SDF-1, Cx43 and Cx45 mRNA as well as decreased SDF-1 secretion. Interestingly, cAMP elevation in BM stromal cells, by dbcAMP or forskolin, resulted in reversed regulation of SDF-1 and Cx43/Cx45. cAMP effect on SDF-1 is PKA-independent since pretreatment of BM stromal cells with H89 did not impair the cAMP elevation effects.Conclusions: Here we show, for the first time, that functional SDF-1 secretion from BM stromal cells is a cell contact-dependent event mediated by Cx43 and Cx45 gap junctions. We have identified dual regulatory roles of BM-Cx on SDF-1: a gap junction-free regulation of its transcription and a gap junctiondependent regulation of its secretion. We hypothesize that Cx43/Cx45 channels transmit cAMP, which in turn controls synchronized SDF-1 secretion and subsequent stem cell migration and development events.

Treatment of stroke with (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl) amino] diazen-1-ium-1, 2-diolate and bone marrow stromal cells upregulates angiopoietin-1/Tie2 and enhances neovascularization

Neovascularization may contribute to functional recovery after neural injury. Combination treatment of stroke with a nitric oxide donor, (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl) amino] diazen-1-ium-1, 2-diolate (DETA-NONOate) and bone marrow stromal cells promotes functional recovery. However, the mechanisms underlying functional improvement have not been elucidated. In this study, we tested the hypothesis that combination treatment upregulates angiopoietin-1 and its receptor Tie2 in the ischemic brain and bone marrow stromal cells, thereby enhancing cerebral neovascularization after stroke. Adult wild type male C57BL/6 mice were i.v. administered PBS, bone marrow stromal cells 5×105, DETA-NONOate 0.4 mg/kg or combination DETA-NONOate with bone marrow stromal cells (n=12/group) after middle cerebral artery occlusion. Combination treatment significantly upregulated angiopoietin-1/Tie2 and tight junction protein (occludin) expression, and increased the number, diameter and perimeter of blood vessels in the ischemic brain compared with vehicle control (mean±S.E., P<0.05). In vitro, DETA-NONOate significantly increased angiopoietin-1/Tie2 protein (n=6/group) and Tie2 mRNA (n=3/group) expression in bone marrow stromal cells. DETA-NONOate also significantly increased angiopoietin-1 protein (n=6/group) and mRNA (n=3/group) expression in mouse brain endothelial cells (P<0.05). Angiopoietin-1 mRNA (n=3/group) was significantly increased in mouse brain endothelial cells treated with DETA-NONOate in combination with bone marrow stromal cell–conditioned medium compared with cells treated with bone marrow stromal cell–conditioned medium or DETA-NONOate alone. Mouse brain endothelial cell capillary tube-like formation assays (n=6/group) showed that angiopoietin-1 peptide, the supernatant of bone marrow stromal cells and DETA-NONOate significantly increased capillary tube formation compared with vehicle control. Combination treatment significantly increased capillary tube formation compared with DETA-NONOate treatment alone. Inhibition of angiopoietin-1 significantly attenuated combination treatment-induced tube formation. Our data indicated that combination treatment of stroke with DETA-NONOate and bone marrow stromal cells promotes neovascularization, which is at least partially mediated by upregulation of the angiopoietin-1/Tie2 axis.

Tissue Microenvironment Modulates CXCR4 Expression and Tumor Metastasis in Neuroblastoma

Neuroblastoma (NB) is derived from intrinsic migratory neural crest cells and has a high potential for distant metastasis. Growing evidence has implicated chemokine receptors, especially CXCR4, which normally control immune and inflammatory cell migration, as having important roles in tumor progression. In this study, we investigated the expression of CXCR4 in eight different NB cell lines and found that CXCR4 expression is dynamically regulated in NB and can be modulated by different tissue stromata. In addition, we demonstrate that IL-5 and IFN-γ are released from stromal cells and act as differential mediators for CXCR4 expression. We also overexpressed CXCR4 in two NB cell lines, NUB-7 and SK-N-BE(2), and studied the role of CXCR4 in NB metastasis both in vitro and in vivo. In vitro transwell invasion assay showed that CXCR4 overexpression promoted NB cell migration preferentially toward a bone marrow stromal cell-conditioned medium. Using an in vivo xenograft model, CXCR4-overexpressing cells showed an increased incidence of metastasis, most notably bone marrow metastasis. Our studies reveal critical roles for CXCR4 in NB metastasis and provide insights into the regulatory mechanism of chemokine receptors in NB and the importance of the tissue microenvironment in modulating tumor cell behavior.

Bone marrow stromal cells mediate protection through stimulation of PI3-K/Akt and MAPK signaling in neurons

Application of adult bone marrow stromal cells (BMSC) improves functional outcome in animal models of cerebral ischemia, traumatic brain injury, and spinal cord injury. Accumulating evidence suggests that such functional recovery after BMSC treatment is mediated by enhanced trophic support of the injured neurons and improved neuronal plasticity rather than tissue replacement by bone marrow-derived stem cells. Therefore, the aim of the present study was to explore the potential of non-hematopoietic BMSC to stimulate signaling pathways in neurons that mediate trophic effects and neuroprotection. In primary embryonic rat neurons, BMSC conditioned medium (CM) attenuated staurosporine (STS) or amyloid-beta peptide-induced apoptosis in a concentration-dependent manner. The neuroprotective effect of CM required several hours of pretreatment and was abolished by heating over 90 °C. Immunoblot analyses revealed that CM enhanced Erk1/2 and Akt phosphorylation in neurons, and the specific MEK1 inhibitor PD98059 or the phosphoinositide-3 kinase (PI3-K) inhibitor Ly294002 abolished the neuroprotective effect of CM. Further, double-conditioned medium (DCM) obtained from BMSC previously stimulated by medium from STS-challenged neurons showed a more potent anti-apoptotic effect compared to the single-conditioned medium. Overall, these findings demonstrate that BMSC trigger endogenous survival signaling pathways in neurons that mediate protection against apoptotic insults. Moreover, the interaction between stressed neurons and BMSC further amplifies the observed neuroprotective effect.

Bone Marrow Stromal Cells Can Provide a Local Environment That Favors Migration and Formation of Tubular Structures of Endothelial Cells

Findings suggest that mesenchymal progenitor cells can support the process of blood vessel formation, which may be relevant during granulation tissue formation at defect sites. The aim of this study was to investigate possible mechanisms of the angiogenic process that can be stimulated by mesenchymal progenitor cells. In the in vivo-like model of the chick embryo chorioallantoic membrane assay, we observed blood vessel ingrowth into collagen sponges containing conditioned medium from undifferentiated bone marrow stromal cells. In the Boyden chamber assay, the conditioned medium was chemotactic for human umbilical vascular endothelial cells and human uterus microvascular endothelial cells, and when cells were placed on Matrigel-coated culture dishes, formation of tubular structures was enhanced. The presence of vascular endothelial growth factor-neutralizing antibodies did not affect the outcome of the two in vitro assays. Bone marrow stromal cell-conditioned medium had no effect on proliferation of endothelial cells, as determined by measuring [3H]thymidine incorporation, and on matrix metalloproteinase 2 expression, as evaluated by reverse transcription-polymerase chain reaction and gelatin zymography. These data indicate that mesenchymal progenitor cells can provide a local environment that supports the ingrowth of blood vessels into a defect site.

Osteoprotegerin (OPG) Produced by Bone Marrow Stromal Cells Protects Breast Cancer Cells from TRAIL-Induced Apoptosis

Advanced breast cancer is often associated with metastatic bone disease, causing a number of serious complications for the patients such as hypercalceamia, pain, nerve compression and fractures. The formation of bone metastases depends on complex interactions between tumour cells and the cells of the bone microenvironment, but the precise molecular mechanisms involved in the development of tumour-induced bone disease have not been identified. We have investigated the ability of bone marrow stromal cells (BMSC) isolated from breast cancer patients to generate osteoprotegerin (OPG), a molecule involved both in bone turnover and cell survival. The potential survival effects of OPG are mediated through binding to a member of the TNF super family, TNF-related Apoptosis Inducing Ligand (TRAIL), preventing association between TRAIL and its death-inducing receptors present on a number of tumour cell types. In the present report we show that bone marrow stromal cells isolated from breast cancer patients produce OPG when grown in culture. The levels of OPG present in BMSC conditioned medium is sufficient to protect breast cancer cells from undergoing TRAIL induced apoptosis. Our data suggest that bone-derived OPG may increase survival of breast cancer cells that reach the bone microenvironment as part of the metastatic process.

Chemokine receptor CCR2 is expressed by human multiple myeloma cells and mediates migration to bone marrow stromal cell-produced monocyte chemotactic proteins MCP-1, -2 and -3.

The restricted bone marrow (BM) localisation of multiple myeloma (MM) cells most likely results from a specific homing influenced by chemotactic factors, combined with the proper signals for growth and survival provided by the BM microenvironment. In analogy to the migration and homing of normal lymphocytes, one can hypothesise that the BM homing of MM cells is mediated by a multistep process, involving the concerted action of adhesion molecules and chemokines. In this study, we report that primary MM cells and myeloma derived cell lines (Karpas, LP-1 and MM5.1) express the chemokine receptor CCR2. In addition, we found that the monocyte chemotactic proteins (MCPs) MCP-1, -2 and -3, three chemokines acting as prominent ligands for CCR2, are produced by stromal cells, cultured from normal and MM BM samples. Conditioned medium (CM) from BM stromal cells, as well as MCP-1, -2 and -3, act as chemoattractants for human MM cells. Moreover, a blocking antibody against CCR2, as well as a combination of neutralizing antibodies against MCP-1, -2 and -3, significantly reduced the migration of human MM cells to BM stromal cell CM. The results obtained in this study indicate the involvement of CCR2 and the MCPs in the BM homing of human MM cells.