Treatment Of Bone Marrow Stromal Cells Research Articles

Bone Marrow Stromal Cells Sorted by Semiconducting Polymer Nanodots for Bone Repair.

The use of bone marrow stromal cells (BMSCs) for bone defect repair has shown great promise due to their differentiation potential. However, isolating the BMSCs from various cell types within the bone marrow remains challenging. To tackle this issue, we utilized semiconducting polymer dots (Pdots) as markers to select the BMSCs within a specific time frame. The therapeutic efficacy of the obtained Pdot-labeled BMSCs was assessed in a bone defect model. Initially, we evaluated the binding capacity of the Pdots with four different types of cells present in the bone marrow including BMSCs, osteoblasts, macrophages, and vascular endothelial cells, in vitro. Notably, BMSCs showed the most rapid uptake of the Pdots, being labeled within only one h of coculture, while other cells took four h to become labeled. Moreover, by colocalizing the Pdots with Prrx1, Sca-1, OSX, F480, and CD105 in the bone marrow cells of monocortical tibial defect (MTD) mice in vivo, we determined the proportions of BMSCs, macrophages, and vascular endothelial cells among all labeled cells from 1 to 8 h after the Pdots injection. It was found that BMSCs have the highest proportion (92%) among all labeled cells extracted after 1 h of Pdots injection. The therapeutic efficacy of the obtained Pdots-labeled BMSCs (1 h) was assessed in a bone defect model. Results showed that the new bone accrual was significantly increased in the treatment of Pdots-labeled BMSCs compared to the bone marrow cell-treated group. Our study revealed that BMSCs screened by the Pdots could improve bone defect repair, suggesting a promising application of the Pdots in bone healing.

Synergistic effects of electroacupuncture and bone marrow stromal cells transplantation therapy in ischemic stroke.

Animal studies and clinical trials demonstrated the effectiveness of a combination of transplanted bone marrow stromal cells (BMSC) and electroacupuncture (EA) treatment in improving neurological deficits. However, the ability of the BMSC-EA treatment to enhance brain repair processes or the neuronal plasticity of BMSC in ischemic stroke model is unclear. The purpose of this study was to investigate the neuroprotective effects and neuronal plasticity of BMSC transplantation combined with EA in ischemic stroke. A male Sprague-Dawley (SD) rat middle cerebral artery occlusion (MCAO) model was used. Intracerebral transplantation of BMSC, transfected with lentiviral vectors expressing green fluorescent protein (GFP), was performed using a stereotactic apparatus after modeling. MCAO rats were treated with BMSC injection alone or in combination with EA. After the treatment, proliferation and migration of BMSC were observed in different groups by fluorescence microscopy. Quantitative real-time PCR (qRT-PCR), Western blotting, and immunohistochemistry were performed to examine changes in the levels of neuron-specific enolase (NSE) and nestin in the injured striatum. Epifluorescence microscopy revealed that most BMSC in the cerebrum were lysed; few transplanted BMSC survived, and some living cells migrated to areas around the lesion site. NSE was overexpressed in the striatum of MCAO rats, illustrating the neurological deficits caused by cerebral ischemia-reperfusion. The combination of BMSC transplantation and EA attenuated the expression of NSE, indicating nerve injury repair. Although the qRT-PCR results showed that BMSC-EA treatment elevated nestin RNA expression, less robust responses were observed in other tests. Our results show that the combination treatment significantly improved restoration of neurological deficits in the animal stroke model. However, further studies are required to see if EA could promote the rapid differentiation of BMSC into neural stem cells in the short term.

Augmented effect of fibroblast growth factor 18 in bone morphogenetic protein 2-induced calvarial bone healing by activation of CCL2/CCR2 axis on M2 macrophage polarization

Fibroblast growth factor (FGF) signaling plays essential roles in various biological events. FGF18 is one of the ligands to be associated with osteogenesis, chondrogenesis and bone healing. The mouse critical-sized calvarial defect healing induced by the bone morphogenetic protein 2 (BMP2)-hydrogel is stabilized when FGF18 is added. Here, we aimed to investigate the role of FGF18 in the calvarial bone healing model. We first found that FGF18 + BMP2 hydrogel application to the calvarial bone defect increased the expression of anti-inflammatory markers, including those related to tissue healing M2 macrophage (M2-Mø) prior to mineralized bone formation. The depletion of macrophages with clodronate liposome hindered the FGF18 effect. We then examined how FGF18 induces M2-Mø polarization by using mouse primary bone marrow (BM) cells composed of macrophage precursors and BM stromal cells (BMSCs). In vitro studies demonstrated that FGF18 indirectly induces M2-Mø polarization by affecting BMSCs. Whole transcriptome analysis and neutralizing antibody treatment of BMSC cultured with FGF18 revealed that chemoattractant chemokine (c-c motif) ligand 2 (CCL2) is the major mediator for M2-Mø polarization. Finally, FGF18-augmented activity toward favorable bone healing with BMP2 was diminished in the calvarial defect in Ccr2-deleted mice. Altogether, we suggest a novel role of FGF18 in M2-Mø modulation via stimulation of CCL2 production in calvarial bone healing.

Bone-Forming Peptide-4 Induces Osteogenic Differentiation and VEGF Expression on Multipotent Bone Marrow Stromal Cells.

Bone morphogenetic proteins (BMPs) have been widely used as treatment for bone repair. However, clinical trials on fracture repair have challenged the effectiveness of BMPs and suggested that delivery of multipotent bone marrow stromal cells (BMSCs) might be beneficial. During bone remodeling and bone fracture repair, multipotent BMSCs differentiate into osteoblasts or chondrocytes to stimulate bone formation and regeneration. Stem cell-based therapies provide a promising approach for bone formation. Extensive research has attempted to develop adjuvants as specific stimulators of bone formation for therapeutic use in patients with bone resorption. We previously reported for the first time bone-forming peptides (BFPs) that induce osteogenesis and bone formation. BFPs are also a promising osteogenic factor for prompting bone regeneration and formation. Thus, the aim of the present study was to investigate the underlying mechanism of a new BFP-4 (FFKATEVHFRSIRST) in osteogenic differentiation and bone formation. This study reports that BFP-4 induces stronger osteogenic differentiation of BMSCs than BMP-7. BFP-4 also induces ALP activity, calcium concentration, and osteogenic factors (Runx2 and osteocalcin) in a dose dependent manner in BMSCs. Therefore, these results indicate that BFP-4 can induce osteogenic differentiation and bone formation. Thus, treatment of multipotent BMSCs with BFP-4 enhanced osteoblastic differentiation and displayed greater bone-forming ability than BMP-7 treatment. These results suggest that BFP-4-stimulated cell therapy may be an efficient and cost-effective complement to BMP-7-based clinical therapy for bone regeneration and formation.

Pivotal Role of OCL-Derived IGF1 in Drug Resistance and Bone Destruction in MM

Multiple myeloma (MM) is largely incurable, and is characterized by devastating bone destruction caused by increased osteoclast (OCL) differentiation and bone resorption in more than 85% of MM patients. OCLs in MM not only promote bone resorption but also increase MM cell growth and drug resistance. Despite recent advances in anti-myeloma treatment, development of anti-MM drug resistance is a major limitation of MM therapy. Therefore, new treatment modalities are urgently needed to overcome drug resistance and decrease bone resorption. IGF1 is a crucial factor for tumor cell growth and survival of malignant cells, especially in MM. IGFI also contributes to development of drug resistance of MM cells to anti-MM agents, including proteasome inhibitors and immunomodulatory agents, but how OCLs contribute to drug resistance is still not clearly delineated. We found that IGF1 was highly expressed in OCLs attached to bone and bone marrow myeloid cells in vivo, and the expression levels of IGF1 in OCLs from MM bearing mice is higher than in normal OCLs. Intriguingly, OCLs produced more IGF1 (0.8 ng/ml/protein) than MM cells (not detected) and bone marrow stromal cells (BMSCs) (0.4 ng/ml/protein) in vitro. In addition, IGF1 protein expression in OCLs was upregulated (1.8 fold) by treatment with conditioned media (CM) from 5TGM1 murine MM cells, TNF-α or IL-6, major paracrine factors that are increased in the bone marrow microenvironment in MM. These results suggest that OCLs are a major source of local IGF1 in the MM bone marrow microenvironment. To further characterize the role of OCL-derived IGF1, we generated a novel mouse with targeted deletion of Igf1 in OCLs (IGF1-/--OCL), and assessed the role of OCL-derived IGF1 in drug resistance of MM cells and bone destruction. Treatment of 5TGM1 cells with bortezomib (BTZ) (3 nM, 48 hours) decreased the viability of 5TGM1 cells by 50%. Importantly, the cytotoxic effects of BTZ on MM cells were decreased (by 5%) when MM cells were cocultured with OCLs from wild type (WT) mice. In contrast, coculture of MM cells with IGF1-/--OCLs or WT-OCLs treated with IGF1 neutralizing antibody (IGF1-ab) did not block BTZ's effects on MM cell death. Consistent with these results, coculture of MM cells with IGF1-/--OCLs or WT-OCLs treated with IGF1-ab resulted in BTZ-induced caspase-dependent apoptosis in MM cells. We next examined the effects of OCLs on the signaling pathways responsible for MM cell survival. WT-OCL-CM promptly induced the phosphorylation of Akt and activation of p38, ERK and NF-κB in MM cells. However, these pathways were not activated by MM cells treated with IGF1-/--OCL-CM or IGF1-ab-treated WT-OCL-CM. Since adhesion of MM cells to BMSCs via interaction of VLA-4 and VCAM-1 plays a critical role in cell adhesion-mediated drug resistance (CAMDR) in MM, we tested if treatment of human BMSCs with human OCL-CM upregulated VCAM-1 expression. We found that OCL-CM upregulated VCAM-1 expression on BMSCs (x fold). In contrast, treatment of BMSCs with OCLs treated with IGF1-ab blocked VCAM-1 induction. These data suggest that OCL-derived IGF1 can contribute to MM cell drug resistance in the bone marrow microenvironment. We then examined the role of IGF1 inhibition on osteoclastogenesis and the bone resorption capacity of OCLs. RANK ligand induced the expression of cathepsin K and NFATc1 in CD11b+ bone marrow cells from WT mice, differentiation markers of OCLs, and the formation of TRAP-positive multinucleated OCLs. However, OCLs formed by RANK ligand treatment of CD11b+ bone marrow cells from IGF1-/- mice had markedly decreased cathepsin K and NFATc1 expression and OCL formation. Next, we tested the bone resorption capacity of OCLs formed by CD11b+ bone marrow cells from IGF1-/- mice vs. WT mice. Similar numbers of OCLs were cultured with RANK ligand on bone slices for 72 hours. The bone resorption activity of Igf1-/--OCLs was significantly decreased (70%) compared with WT-OCLs. These results suggest that OCL-derived IGF1 plays a critical role in MM drug resistance and bone destruction, and that inhibition of the effect of IGF1 in OCLs should decrease MM drug resistance and bone destruction. Disclosures Roodman: Amgen trial of Denosumab versus Zoledronate: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees.

TBK1/Ikkε Inhibitor Amlx Blocks Multiple Myeloma Cell Growth in Vitro and In Vivo

Multiple myeloma (MM) is the most frequent cancer to involve the skeleton and remains incurable for most patients, thus novel therapies are needed. MM bone disease is characterized by osteolytic lesions that contribute significantly to patient morbidity and mortality. We showed that TBK1 signaling is a novel pathway that increases osteoclast (OCL) formation in Paget's disease, an inflammatory bone disease. Therefore, we hypothesized that TBK1 plays a similar role in MM induction of OCL. We found that MM conditioned media (MM-CM) dose-dependently increased bone marrow monocyte (BMM) expression of activated TBK1 protein and enhanced RANKL-driven OCL formation. TBK1 knockdown by shRNA transduction into BMM significantly attenuated the ability of MM-CM to increase OCL differentiation without altering OCL differentiation in control media. We found that the TBK1/IKKε inhibitor Amlexanox (Amlx) blocked normal and MM-enhanced OCL formation. Importantly, TBK1 mRNA expression in CD138+ plasma cells (PC) isolated from MM or PC leukemia patients is significantly higher as compared to PC from Monoclonal Gammopathy of Undetermined Significance (MGUS) patients.Therefore, we tested whether targeting the TBK1/ IKKε signaling pathways would also affect MM cells. We found that Amlx strongly decreased the viability of several MM cell lines and primary MM cells via induction of apoptosis. Amlx treatment of MM cell lines also induced a G1/S blockade, decreased activated ERK1/2, and increased translation of the dominant-negative C/EBPb-LIP isoform in several MM cell lines. The positive-acting C/EBPb-LAP isoform was previously shown to be a critical transcription factor for MM viability. Importantly, Amlx also enhanced the effectiveness of the proteasome inhibitors bortezomib and carfilzomib to kill MM cells in culture. Further, Amlx sensitized MM1.S cells to the induction of apoptosis by the autophagic inhibitor Bafilomycin A. Amlx dose-dependently inhibited tumor growth in a syngeneic MM mouse model in which 5TGM1 MM cells expressing secreted GLuc were injected subcutaneously into immunocompetent C57Bl/KaLwRij. Tumor growth was assessed by measuring tumor volumes and by the levels of secreted GLuc in the blood. Further, OCL formation ex vivo from bone marrow monocytes obtained from AMLX-treated mice versus controls was decreased. Amlx did not affect the viability of primary BMM, bone marrow stromal cells (BMSC), or splenocytes. Further, Amlx treatment of primary BMSC from MM patients or normal donors decreased expression of TNFα, IL-6 and RANKL, thereby decreasing BMSC support of MM survival and OCL differentiation. Amlx pretreatment of BMSC and murine pre-osteoblast MC4 cells also decreased VCAM1 expression and reduced MM cell adhesion, another mechanism for Amlx reduction of bone microenvironmental MM support.These data suggest that targeting TBK1/IKKε signaling may decrease MM bone disease by slowing MM growth, directly and indirectly, and preventing MM-induced osteolysis. DisclosuresGiuliani:Janssen Pharmaceutica: Other: Avisory Board, Research Funding; Celgene Italy: Other: Avisory Board, Research Funding; Takeda Pharmaceutical Co: Research Funding. Roodman:Amgen Denosumab: Membership on an entity's Board of Directors or advisory committees.

Solanum Muricatum Promotes Osteogenic Differentiation of Rat Bone Marrow Stromal Cells.

Solanum muricatum (SM), also known as pepino, is known for its antioxidative and anti-inflammatory effects. The aim of this study was to evaluate the effects of SM extract in promoting osteogenic differentiation and regulating the Wnt and bone morphogenetic protein (BMP) signaling pathways. Ingredients of pepino were extracted and identified. SM extracts were used to treat rat bone marrow stromal cells (BMSCs), followed by evaluating alkaline phosphatase activities and mineralization levels. The mRNA levels of osteogenic biomarkers, including OPN and Collagen I, were also evaluated with real-time polymerase chain reaction. After treatment with SM extracts, the expressions of key proteins in the Wnt and BMP signaling pathways were assessed. DKK-1 and noggin, which are Wnt and BMP inhibitors, respectively, were added with SM extracts to investigate the role of Wnt and BMP pathways in the ameliorating effects of SM extract in osteogenesis. Treatment of BMSCs with SM extract promoted osteogenesis. Meanwhile, upregulations in the Wnt and BMP pathways were also observed. However, inhibiting both pathways compromised the effects of SM extract in promoting osteogenic differentiation. SM extract promotes osteogenic differentiation in BMSCs via promoting the Wnt and BMP signaling pathways.

Effects of the combined treatment of bone marrow stromal cells with mild exercise and thyroid hormone on brain damage and apoptosis in a mouse focal cerebral ischemia model.

This study examined whether post-stroke bone marrow stromal cells (BMSCs) therapy combined with exercise (EX) and/or thyroid hormone (TH) could reduce brain damage in an experimental ischemic stroke in mice. Focal cerebral ischemia was induced under Laser Doppler Flowmetry (LDF) guide by 45min of middle cerebral artery occlusion (MCAO), followed by 7days of reperfusion in albino mice. BMSCs were injected into the right cerebral ventricle 24h after MCAO, followed by daily injection of T3 (20μg/100g weight S.C) and 6days of running on a treadmill. Infarct size, neurobehavioral test, TUNEL and BrdU positive cells were evaluated at 7days after MCAO. Treatment with BMSCs and mild EX alone significantly reduced the infarct volume by 23% and 44%, respectively (both, p<0.001). The BMSCs+TH, BMSCs+EX, and BMSCs+EX+TH combination therapies significantly reduced the infarct volume by 26%, 51%, and 70%, respectively (all, p<0.001). A significant improvement in the neurobehavioral functioning was observed in the EX, BMSCs+EX, and BMSCs+EX+ TH groups (p<0.001). The number of TUNEL-positive cells (a marker of apoptosis) was significantly reduced in the EX, BMSCs, BMSCs+EX, BMSCs+TH, and BMSCs+EX+TH groups (all, p<0.001). Moreover, the combination therapy considerably increased BrdU-labeled cells in the subventricular zone (SVZ) (p<0.01). Our findings indicated that the combined treatment of BMSCs with mild EX and TH more efficiently reduces the cerebral infarct size after stroke. More likely, these effects mediate via enchaining generation of new neuronal cells and the attenuation of apoptosis in ischemia stroke in young mice.

MicroRNA-183-5p Increases with Age in Bone-Derived Extracellular Vesicles, Suppresses Bone Marrow Stromal (Stem) Cell Proliferation, and Induces Stem Cell Senescence.

Microvesicle- and exosome-mediated transport of microRNAs (miRNAs) represents a novel cellular and molecular pathway for cell-cell communication. In this study, we tested the hypothesis that these extracellular vesicles (EVs) and their miRNAs might change with age, contributing to age-related stem cell dysfunction. EVs were isolated from the bone marrow interstitial fluid (supernatant) of young (3-4 months) and aged (24-28 months) mice to determine whether the size, concentration, and miRNA profile of EVs were altered with age in vivo. Results show that EVs isolated from bone marrow are CD63 and CD9 positive, and the concentration and size distribution of bone marrow EVs are similar between the young and aged mice. Bioanalyzer data indicate that EVs from both young and aged mice are highly enriched in miRNAs, and the miRNA profile of bone marrow EVs differs significantly between the young and aged mice. Specifically, the miR-183 cluster (miR-96/-182/-183) is highly expressed in aged EVs. In vitro assays demonstrate that aged EVs are endocytosed by primary bone marrow stromal cells (BMSCs), and these aged EVs inhibit the osteogenic differentiation of young BMSCs. Transfection of BMSCs with miR-183-5p mimic reduces cell proliferation and osteogenic differentiation, increases senescence, and decreases protein levels of the miR-183-5p target heme oxygenase-1 (Hmox1). In vitro assays utilizing H2O2-induced oxidative stress show that H2O2 treatment of BMSCs increases the abundance of miR-183-5p in BMSC-derived EVs, and Amplex Red assays demonstrate that H2O2 is elevated in the bone marrow microenvironment with age. Together, these data indicate that aging and oxidative stress can significantly alter the miRNA cargo of EVs in the bone marrow microenvironment, which may in turn play a role in stem cell senescence and osteogenic differentiation by reducing Hmox1 activity.

P62-ZZ Domain Inhibition Prevents MM Cell-Induced Epigenetic Changes at the Runx2 and C/EBPb Promoters

Multiple myeloma bone disease (MMBD) is a paradigm for uncoupled bone remodeling and is characterized by non-healing lytic bone lesions. Osteoblast (OB) function is highly suppressed or absent, persists in the absence of MM cells, and remains a significant cause of skeletal-related events. The persistence of OB suppression in MMBD suggests that MM cells induce repressive epigenetic changes at the Runx2 gene, the key transcription factor required for OB differentiation of bone marrow stromal cells (BMSC), which are preOB. We reported that TNFα is a major suppressor of OB in MMBD that reduces Runx2 levels by inducing Gfi1, a transcriptional repressor of Runx2. Using ChIP analysis of MM-exposed BMSC, we showed that Gfi1 directly binds the Runx2 promoter and recruits the chromatin corepressor HDAC1 to the Runx2 promoter in MM-exposed BMSC, reducing transcriptionally permissive euchromatin marks such as H3K9ac.Recently, we reported that p62 (sequestosome-1) in BMSC is critical for the formation of MM cell-induced signaling complexes that mediate OB suppression and IL-6 production. We found that XRK3F2, a novel inhibitor of the p62 ZZ domain (p62-ZZ), blunted MM cell-induced repression of Runx2 and induction of Gfi1 in BMSC, and induced new bone formation and remodeling in mice with established MMBD, but did not alter normal bone. In the current study, we further evaluated the specificity of XRK3F2's inhibition of p62-ZZ interactions and investigated the mechanism by which blocking p62-ZZ prevents MM-induced suppression of key osteogenic transcription factors in BMSC.We previously showed that TNFα and MM cell-induced IL-6 production by BMSC are both p62 dependent and independent. XRK3F2 blocked TNFα-induced NFκB signaling in BMSC and MM cells, and partially inhibited TNFα-induced IL-6 production by BMSC. We now report that XRK3F2 blunted the TNFα upregulation observed in BMSC after MM cell coculture but did not alter TNFα production in p62 knockout (p62KO) BMSC following coculture with MM cells. TNFα treatment of p62KO BMSCs resulted in minimal induction of IL-6, which was not altered by XRK3F2. Transfection of p62KO BMSC with full-length p62 constructs restored TNFα-induced IL-6 production and XRK3F2's capacity to reduce TNFα-induced IL-6. In contrast, XRK3F2 had no effect on TNFα-induced IL-6 production by p62KO BMSC transfected with p62 constructs lacking p62-ZZ.To further investigate XRK3F2's mechanism of action, we tested if XRK3F2 prevents Gfi1-induced epigenetic suppression of Runx2 by preventing Gfi1's upregulation and binding to Runx2. ChIP analysis of MM exposed BMSC treated with XRK3F2 demonstrated that XRK3F2 reduced MM-induced Gfi1 occupancy of the Runx2 promoter and prevented MM-induced reduction of H3K9ac. Since the region proximal to the Runx2 promoter contains putative C/EBPβ binding sites, we tested if MM-induced p62-ZZ signaling activates C/EBPβ, a transcription factor that regulates OB lineage maturation and IL-6 production in BMSC and plays a role in the upregulation of Gfi1 expression. MM-exposed BMSC had increased enrichment of C/EBPβ at both the Gfi1 gene and the C/EBPβ-regulated Il6 gene, and XRK3F2 reduced upregulation of MM-induced C/EBPβ binding at both the Gfi1 and Il6 genes. We conclude that XRK3F2's reduction of the MM-induced C/EBPβ binding in BMSC results in decreased Gfi1 mRNA expression, and thereby reduces MM-induced Gfi1 occupancy and HDAC1 recruitment at the Runx2 promoter. These results suggest that targeting p62-ZZ in MMBD may reverse C/EBPβ mediated Gfi1 activation, resulting in rescue of the MM cell-induced epigenetic suppression of Runx2 in BMSC, and that next-generation derivatives of XRK3F2 should impact BMSC-supported MM cell survival. DisclosuresSilbermann:Celgene: Research Funding; Amgen: Consultancy. Xie:Oxis Biotech: Consultancy, Membership on an entity's Board of Directors or advisory committees. Roodman:Eli Lilly: Research Funding; Amgen: Consultancy.

Glucagon-like peptide-1 receptor agonist Liraglutide has anabolic bone effects in ovariectomized rats without diabetes.

Recently, a number of studies have demonstrated the potential beneficial role for novel anti-diabetic GLP-1 receptor agonists (GLP-1RAs) in the skeleton metabolism in diabetic rodents and patients. In this study, we evaluated the impacts of the synthetic GLP-1RA Liraglutide on bone mass and quality in osteoporotic rats induced by ovariectomy (OVX) but without diabetes, as well as its effect on the adipogenic and osteoblastogenic differentiation of bone marrow stromal cells (BMSCs). Three months after sham surgery or bilateral OVX, eighteen 5-month old female Wistar rats were randomly divided into three groups to receive the following treatments for 2 months: (1) Sham + normal saline; (2) OVX + normal saline; and (3) OVX + Liraglutide (0.6 mg/day). As revealed by micro-CT analysis, Liraglutide improved trabecular volume, thickness and number, increased BMD, and reduced trabecular spacing in the femurs in OVX rats; similar results were observed in the lumbar vertebrae of OVX rats treated with Liraglutide. Following in vitro treatment of rat and human BMSCs with 10 nM Liraglutide, there was a significant increase in the mRNA expression of osteoblast-specific transcriptional factor Runx2 and the osteoblast markers alkaline phosphatase (ALP) and collagen α1 (Col-1), but a significant decrease in peroxisome proliferator-activated receptor γ (PPARγ). In conclusion, our results indicate that the anti-diabetic drug Liraglutide can exert a bone protective effect even in non-diabetic osteoporotic OVX rats. This protective effect is likely attributable to the impact of Liraglutide on the lineage fate determination of BMSCs.

Increased extracellular and intracellular Ca2+ lead to adipocyte accumulation in bone marrow stromal cells by different mechanisms

Mesenchymal stem cells found in bone marrow stromal cells (BMSCs) are the common progenitors for both adipocyte and osteoblast. An increase in marrow adipogenesis is associated with age-related osteopenia and anemia. Both extracellular and intracellular Ca2+ ([Ca2+]o and [Ca2+]i) are versatile signaling molecules that are involved in the regulation of cell functions, including proliferation and differentiation. We have recently reported that upon treatment of BMSCs with insulin and dexamethasone, both high [Ca2+]o and high [Ca2+]i enhanced adipocyte accumulation, which suggested that increases in [Ca2+]o caused by bone resorption may accelerate adipocyte accumulation in aging and diabetic patients. In this study, we used primary mouse BMSCs to investigate the mechanisms by which high [Ca2+]o and high [Ca2+]i may enhance adipocyte accumulation. In the process of adipocyte accumulation, two important keys are adipocyte differentiation and the proliferation of BMSCs, which have the potential to differentiate into adipocytes. Use of MTT assay and real-time RT-PCR revealed that high [Ca2+]i (ionomycin)-dependent adipocyte accumulation is caused by enhanced proliferation of BMSCs but not enhanced differentiation into adipocytes. Using fura-2 fluorescence-based approaches, we showed that high [Ca2+]o (addition of CaCl2) leads to increases in [Ca2+]i. Flow cytometric methods revealed that high [Ca2+]o suppressed the phosphorylation of ERK independently of intracellular Ca2+. The inhibition of ERK by U0126 and PD0325901 enhanced the differentiation of BMSCs into adipocytes. These data suggest that increased extracellular Ca2+ provides the differentiation of BMSCs into adipocytes by the suppression of ERK activity independently of increased intracellular Ca2+, which results in BMSC proliferation.

The Morphofunctional Effect of the Transplantation of Bone Marrow Stromal Cells and Predegenerated Peripheral Nerve in Chronic Paraplegic Rat Model via Spinal Cord Transection.

Functional recovery following spinal cord injury (SCI) is limited by poor axonal and cellular regeneration as well as the failure to replace damaged myelin. Employed separately, both the transplantation of the predegenerated peripheral nerve (PPN) and the transplantation of bone marrow stromal cells (BMSCs) have been shown to promote the regrowth and remyelination of the damaged central axons in SCI models of hemisection, transection, and contusion injury. With the aim to test the effects of the combined transplantation of PPN and BMSC on regrowth, remyelination, and locomotor function in an adult rat model of spinal cord (SC) transection, 39 Fischer 344 rats underwent SC transection at T9 level. Four weeks later they were randomly assigned to traumatic spinal cord injury (TSCI) without treatment, TSCI + Fibrin Glue (FG), TSCI + FG + PPN, and TSCI + FG + PPN + BMSCs. Eight weeks after, transplantation was carried out on immunofluorescence and electron microscope studies. The results showed greater axonal regrowth and remyelination in experimental groups TSCI + FG + PPN and TSCI + FG + PPN + BMSCs analyzed with GAP-43, neuritin, and myelin basic protein. It is concluded that the combined treatment of PPN and BMSCs is a favorable strategy for axonal regrowth and remyelination in a chronic SC transection model.

Antagonistic Effect of Small Molecule Inhibitors of Wnt/β-Catenin in Multiple Myeloma

AbstractAbstract 5012Development and progression of multiple myeloma is dependent on the bone marrow (BM) microenvironment. Bone marrow stromal cells (BMSC) secrete Wnt ligands that activate the Wnt signaling pathway. The canonical Wnt pathway, which is mediated through the key transcriptional effector β-catenin (β-cat), is commonly deregulated in many cancers. Cells with β-cat-regulated transcription (CRT) are protected against apoptosis; conversely inhibition of CRT may inhibit cell proliferation. In this study we tested the efficacy of recently described inhibitors of CRT (iCRTs) for their selective antagonistic effect on Wnt-β-cat in MM cells. Although, earlier studies have documented Wnt signaling in human MM cells, in order to test the chemosensitivity of iCRTs in myeloma cells, we first confirmed the expression of β-cat in human MM cell types. An immunofluorescence detection of β-cat in U266 cells showed nuclear localization in > 70% of the cells, a similar trend of nuclear β-cat was observed in MM1 and patient derived BMMC cells. This observation is consistent with the Western blot analysis of the total protein from three cell types. The above data on the expression of nuclear β-cat in MM cell lines and cells from patient sample (n=16) provides the rationale for using these cells to test the efficacy of iCRTs (Oxazole-iCRT-3 and Thiazole-iCRT-5), that are designed to target β-cat signaling. Wnt reporter plasmid STF16-transfected MM cells treated with iCRTs at the doses of 10, 25, and 50 μM showed a dose dependent decrease (2–3 fold) in the Wnt/β-cat reporter activity, with a significant decline at a maximum dose of 50 μM (p<0. 001). Given the role of Wnt and the upstream antagonist DKK1 in normal bone formation and MM development, we examined whether inhibition of Wnt-β-cat response by iCRTs indirectly affects the protein expression of DKK1. As expected, our data from Western blot analysis of MM cells treated with iCRTs showed no significant change in the expression of DKK1, thereby confirming the effect of iCRTs on β-cat itself. While bone marrow angiogenesis is a hallmark in multiple myeloma, it is also correlated with the severity of the disease. Since we observed an elevated level of VEGF in the serum samples ranging from 300 pg/ml to 500 pq/ml in patients (n=16) with late stage MM, we tested the effect of iCRTs on VEGF level in MM cells in the cell culture medium obtained from the co-culture of bone marrow stromal cells (BMSC) with human U266 cells. Treatment of BMSC and U266 in co-culture with iCRT-3 and iCRT-5 at a dose of 50μM of each showed a decrease in the VEGF level and down regulated VEGF at the mRNA level by >3 fold determined by qRT-PCR analysis. To determine whether the effect of iCRTs on the VEGF activity is specific to its ability to antagonize b-cat activity, we used culture medium collected from cells transiently transfected with siRNA for β-cat. siRNA mediated down regulation of β-cat showed a decrease in the VEGF levels, thereby confirming that the effect of iCRTs is indeed mediated by their inhibitory effect on b-cat. Although several aspect of our key findings are yet to be confirmed in preclinical in vivo models for MM, this part of the study provide evidence that indicate a VEGF-dependent increase in cell proliferation and migration of MM cells that can be antagonized by specific inhibitors of nuclear b-cat, thereby underscoring the importance of developing iCRTs as a novel class of Wnt-directed therapeutics in human MM. Disclosures:No relevant conflicts of interest to declare.

Cyclic AMP signaling in bone marrow stromal cells has reciprocal effects on the ability of mesenchymal stem cells to differentiate into mature osteoblasts versus mature adipocytes

Stimulatory G protein-mediated cAMP signaling is intimately involved in skeletal homeostasis. However, limited information is available on the role of the cAMP signaling in regulating the differentiation of mesenchymal stem cells into mature osteoblasts and adipocytes. To investigate this, we treated primary mouse bone marrow stromal cells (BMSCs) with forskolin to stimulate cAMP signaling and determined the effect on osteoblast and adipocyte differentiation. Exposure of differentiating osteoblasts to forskolin markedly inhibited progression to the late stages of osteoblast differentiation, and this effect was replicated by continuous exposure to PTH. Strikingly, forskolin activation of cAMP signaling in BMSCs conditioned mesenchymal stem cells (MSCs) to undergo increased osteogenic differentiation and decreased adipogenic differentiation. PTH treatment of BMSCs also enhanced subsequent osteogenesis, but promoted an increased adipogenesis as well. Thus, activation of cAMP signaling alters the lineage commitment of MSCs, favoring osteogenesis at the expense of adipogenesis.

Impact of ageing on biological features of bone marrow stromal cells (BMSC) in cell transplantation therapy for CNS disorders: Functional enhancement by granulocyte‐colony stimulating factor (G‐CSF)

This study was designed to clarify the effects of donor age on biological features of bone marrow stromal cells (BMSC), one of the candidates for cell transplantation therapy for CNS disorders, because many aged patients might require such therapy. This study was also aimed to test whether ex vivo treatments with granulocyte-colony stimulating factor (G-CSF) could modify biological properties of BMSC from aged donors and enhance its therapeutic effects in an animal model of traumatic brain injury. The BMSC were harvested from young (6-week-old) and aged (100-week-old) rats. The ageing significantly increased the senescence-associated β-galactosidase (SA-β-gal) activity of the cultured BMSC, and decreased their proliferative capacity and production of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). As the next step, the rats were subjected to brain freezing injury by applying liquid nitrogen onto the neocortex through the thinned skull. The 6-week BMSC, 100-week BMSC, G-CSF-treated 100-week BMSC or vehicle were stereotactically injected into the ipsilateral striatum at 7 days post-injury. Transplantation of the 6-week BMSC, but not 100-week BMSC, significantly improved locomotor function. However, treatment of the 100-week BMSC with 0.1 µmol of G-CSF significantly improved their proliferation activity and growth factor production, and recovered therapeutic effects in the injured brain. In conclusion, donor age may largely determine biological aspects of BMSC. G-CSF may contribute to improve the outcome of BMSC transplantation therapy for CNS disorders in aged patients.

Selectin-Mediated Recruitment of Bone Marrow Stromal Cells in the Postischemic Cerebral Microvasculature

The therapeutic potential of bone marrow stromal cells (BMSCs) has been demonstrated in different models of stroke. Although it is well established that BMSCs selectively migrate to the site of brain injury, the mechanisms underlying this process are poorly understood. This study addresses the hypothesis that selectins mediate the recruitment of BMSCs into the postischemic cerebral microvasculature. Focal ischemic stroke was induced by middle cerebral artery occlusion and reperfusion. Cell recruitment was monitored using either fluorescent- or radiolabeled BMSCs detected by intravital microscopy or tissue radioactivity. Mice were treated with either a blocking antibody against P- or E-selectin or with the nonselective selectin antagonist, fucoidin. The role of CD44 in cell recruitment was evaluated using BMSCs from CD44 knockout mice. Middle cerebral artery occlusion and reperfusion was associated with a significantly increased adhesion of BMSCs in cerebral venules compared with sham mice. Immunoneutralization of either E- or P-selectin blocked the middle cerebral artery occlusion and reperfusion-induced recruitment of adherent BMSCs. An attenuated recruitment response in the postischemic hemisphere was also noted after fucoidin treatment or administration of CD44-deficient BMSCs. Cerebral vascular endothelium assume a proadhesive phenotype after ischemic stroke that favors the recruitment of BMSCs, which use both P- and E-selectin to home into the infarct site. CD44 may serve as the critical ligand for selectin-mediated BMSC recruitment.

Ephrin B1 Regulates Bone Marrow Stromal Cell Differentiation and Bone Formation by Influencing TAZ Transactivation via Complex Formation with NHERF1

Mutations of ephrin B1 in humans result in craniofrontonasal syndrome. Because little is known of the role and mechanism of action of ephrin B1 in bone, we examined the function of osteoblast-produced ephrin B1 in vivo and identified the molecular mechanism by which ephrin B1 reverse signaling regulates bone formation. Targeted deletion of the ephrin B1 gene in type 1alpha2 collagen-producing cells resulted in severe calvarial defects, decreased bone size, bone mineral density, and trabecular bone volume, caused by impairment in osterix expression and osteoblast differentiation. Coimmunoprecipitation of the TAZ complex with TAZ-specific antibody revealed a protein complex containing ephrin B1, PTPN13, NHERF1, and TAZ in bone marrow stromal (BMS) cells. Activation of ephrin B1 reverse signaling with soluble EphB2-Fc led to a time-dependent increase in TAZ dephosphorylation and shuttling from cytoplasm to nucleus. Treatment of BMS cells with exogenous EphB2-Fc resulted in a 4-fold increase in osterix expression as determined by Western blotting. Disruption of TAZ expression using specific lentivirus small hairpin RNA (shRNA) decreased TAZ mRNA by 80% and ephrin B1 reverse signaling-mediated increases in osterix mRNA by 75%. Knockdown of NHERF1 expression reduced basal levels of osterix expression by 90% and abolished ephrin B1-mediated induction of osterix expression. We conclude that locally produced ephrin B1 mediates its effects on osteoblast differentiation by a novel molecular mechanism in which activation of reverse signaling leads to dephosphorylation of TAZ and subsequent release of TAZ from the ephrin B1/NHERF1/TAZ complex to translocate to the nucleus to induce expression of the osterix gene and perhaps other osteoblast differentiation genes. Our findings provide strong evidence that ephrin B1 reverse signaling in osteoblasts is critical for BMS cell differentiation and bone formation.

Electroacupuncture-Enhanced Differentiation of Bone Marrow Stromal Cells into Neuronal Cells

Bone marrow stromal cells (BMSCs) can be differentiated into neuronal cells and are used to treat spinal cord injury (SCI). This study investigated whether electroacupuncture enhances BMSC's effects on SCI in rats. The effects of transplantation of phosphate-buffered saline or BMSC, electroacupuncture, and a combination of BMSC transplantation and electroacupuncture on SCI were evaluated using a combined behavioral score (CBS). Expressions of neuronal marker neuron-specific enolase (NSE) and gliocyte-specific marker glial fibrillary acidic protein (GFAP) of transplanted BMSC were detected using immunohistochemistry to assess the effect of electroacupuncture on differentiation of BMSC into neuronal cells. The combination of BMSC transplantation and electroacupuncture significantly alleviated CBS in rats with SCI compared with the separate treatment of BMSC or electroacupuncture. In addition, electroacupuncture increased the NSE- and GFAP-positive transplanted BMSCs in spinal cord. Combined treatment showed a better effect, and the mechanisms may be partially caused by enhanced differentiation of BMSC into neuronal cells. Future studies are needed to confirm this.

Roles of activated astrocytes in bone marrow stromal cell proliferation and differentiation

Local microenvironment plays an important role in determining the fate choice of stem cells in the central nervous system (CNS). Astrocytes, a major component of local microenvironment in the CNS, have been demonstrated to influence the proliferation and neural differentiation of stem cells including neural stem/progenitor cells, embryonic stem cells and bone marrow stromal cells (BMSCs). However, it has remained to be ascertained if inflammation-activated astrocytes can affect the behavior of BMSCs. To this end, astrocyte-conditioned medium (ACM) was prepared in this study for treatment of BMSCs. The ACM derived from Wistar rat astrocytes stimulated by lipopolysaccharide for 12, 36 or 72 h, respectively, served as inflammatory ACM (12 h ACM, 36 h ACM and 72 h ACM), while that from unstimulated astrocytes was used as normal control astrocyte-conditioned medium (N-ACM). The results showed that the proliferation and neural differentiation of BMSCs grown in inflammatory ACM were significantly increased compared with those grown in N-ACM. The efficiency of BMSCs exposed to 36 h ACM was significantly greater than that of those exposed to 12 or 72 h ACM. Following neutralization of interleukin-6 (IL-6) of the ACM, both the proliferation and astrocytic differentiation of BMSCs were decreased; on the other hand, the neuronal differentiation was significantly increased. The present findings suggest that inflammation-activated astrocytes can facilitate the proliferation and neural differentiation of BMSCs and activated astrocytes at different phase after CNS injuries might have distinct effects on BMSCs. Moreover, astrocyte-derived IL-6 participates in the proliferation and neural differentiation of BMSCs.