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Sample records for marrow-derived osteoblast progenitor

  1. Bone marrow-derived osteoblast progenitor cells in circulating blood contribute to ectopic bone formation in mice

    SciTech Connect

    Otsuru, Satoru; Tamai, Katsuto . E-mail: tamai@gts.med.osaka-u.ac.jp; Yamazaki, Takehiko; Yoshikawa, Hideki; Kaneda, Yasufumi

    2007-03-09

    Recent studies have suggested the existence of osteoblastic cells in the circulation, but the origin and role of these cells in vivo are not clear. Here, we examined how these cells contribute to osteogenesis in a bone morphogenetic protein (BMP)-induced model of ectopic bone formation. Following lethal dose-irradiation and subsequent green fluorescent protein-transgenic bone marrow cell-transplantation (GFP-BMT) in mice, a BMP-2-containing collagen pellet was implanted into muscle. Three weeks later, a significant number of GFP-positive osteoblastic cells were present in the newly generated ectopic bone. Moreover, peripheral blood mononuclear cells (PBMNCs) from the BMP-2-implanted mouse were then shown to include osteoblast progenitor cells (OPCs) in culture. Passive transfer of the PBMNCs isolated from the BMP-2-implanted GFP-mouse to the BMP-2-implanted nude mouse led to GFP-positive osteoblast accumulation in the ectopic bone. These data provide new insight into the mechanism of ectopic bone formation involving bone marrow-derived OPCs in circulating blood.

  2. Role of endothelial nitric oxide in bone marrow-derived progenitor cell mobilization.

    PubMed

    de Resende, M Monterio; Huw, L-Y; Qian, H-S; Kauser, K

    2007-01-01

    Mobilization and recruitment of bone marrow-derived progenitor cells (BMDPCs) play an important role in postischemic tissue repair. Patients with coronary artery disease (CAD) or peripheral vascular disease (PVD) exhibit endothelial dysfunction, and as a result are likely to have a reduced number of progenitor cells mobilized in their peripheral circulation following ischemic injury. Identification of eNOS independent pathways for BMDPC mobilization may have important therapeutic value in this patient population. To identify such mechanisms we investigated the effect of granulocyte-colony stimulating factor (GCSF) and stem cell factor (SCF) in eNOS-KO mice with and without surgical hind-limb ischemia. Our results suggest that BMDPC mobilization can be achieved via activation of NO-independent pathways. PMID:17554503

  3. Quercetin protects against high glucose-induced damage in bone marrow-derived endothelial progenitor cells.

    PubMed

    Zhao, Li-Rong; Du, Yu-Jun; Chen, Lei; Liu, Zhi-Gang; Pan, Yue-Hai; Liu, Jian-Feng; Liu, Bin

    2014-10-01

    Endothelial progenitor cells (EPCs), a group of bone marrow-derived pro-angiogenic cells, contribute to vascular repair after damage. EPC dysfunction exists in diabetes and results in poor wound healing in diabetic patients with trauma or surgery. The aim of the present study was to determine the effect of quercetin, a natural flavonoid on high glucose‑induced damage in EPCs. Treatment with high glucose (40 mM) decreased cell viability and migration, and increased oxidant stress, as was evidenced by the elevated levels of reactive oxygen species (ROS), malondialdehyde (MDA) and superoxide dismutase in bone marrow-derived EPCs. Moreover, high glucose reduced the levels of endothelial nitric oxide synthase (eNOS) phosphorylation, nitric oxide (NO) production and intracellular cyclic guanosine monophosphate (cGMP). Quercetin supplement protected against high glucose‑induced impairment in cell viability, migration, oxidant stress, eNOS phosphorylation, NO production and cGMP levels. Quercetin also increased Sirt1 expression in EPCs. Inhibition of Sirt1 by a chemical antagonist sirtinol abolished the protective effect of quercetin on eNOS phosphorylation, NO production and cGMP levels following high glucose stress. To the best of our knowledge, the results provide the first evidence that quercetin protects against high glucose‑induced damage by inducing Sirt1-dependent eNOS upregulation in EPCs, and suggest that quercetin is a promising therapeutic agent for diabetic patients undergoing surgery or other invasive procedures. PMID:25197782

  4. Bone marrow-derived endothelial progenitor cells are involved in aneurysm repair in rabbits.

    PubMed

    Fang, Xinggen; Zhao, Rui; Wang, Kuizhong; Li, Zifu; Yang, Penfei; Huang, Qinghai; Xu, Yi; Hong, Bo; Liu, Jianmin

    2012-09-01

    Endothelial progenitor cells (EPC) are believed to be involved in aneurysmal repair and remodeling. The aim of this study was to test this hypothesis and, if true, explore how EPC contribute to aneurysm repair in a rabbit model of elastase-induced carotid aneurysm. Rabbits were divided randomly into an in situ carotid EPC transfusion group (ISCT group, n=5), and an intravenous EPC transfusion group (IVT group, n=5). Autologous EPC were double-labeled with Hoechst 33342 and 5,6-carboxyfluorescein diacetate succinimidyl ester before injection into the animals in either the carotid artery (ISCT group) or marginal ear veins (IVT group). Three weeks later, labeled cells in the aneurysms were observed with respect to location, adhesion, and growth to detect signs of aneurysm repair. Labeled EPC were detected within the neointima in all five aneurysms in the ISCT group and in three of the five aneurysms in the IVT group, but there was no endothelial growth in the aneurysmal neointima in either group. These results show that bone marrow-derived EPC are involved in the process of aneurysm repair in this rabbit model. PMID:22789632

  5. Paracrine effects and heterogeneity of marrow-derived stem/progenitor cells: relevance for the treatment of respiratory diseases.

    PubMed

    Conese, Massimo; Carbone, Annalucia; Castellani, Stefano; Di Gioia, Sante

    2013-01-01

    Stem cell-based treatment may represent a hope for the treatment of acute lung injury and pulmonary fibrosis, and other chronic lung diseases, such as cystic fibrosis, asthma and chronic obstructive pulmonary disease (COPD). It is well established in preclinical models that bone marrow-derived stem and progenitor cells exert beneficial effects on inflammation, immune responses and repairing of damage in virtually all lung-borne diseases. While it was initially thought that the positive outcome was due to a direct engraftment of these cells into the lung as endothelial and epithelial cells, paracrine factors are now considered the main mechanism through which stem and progenitor cells exert their therapeutic effect. This knowledge has led to the clinical use of marrow cells in pulmonary hypertension with endothelial progenitor cells (EPCs) and in COPD with mesenchymal stromal (stem) cells (MSCs). Bone marrow-derived stem cells, including hematopoietic stem/progenitor cells, MSCs, EPCs and fibrocytes, encompass a wide array of cell subsets with different capacities of engraftment and injured tissue-regenerating potential. The characterization/isolation of the stem cell subpopulations represents a major challenge to improve the efficacy of transplantation protocols used in regenerative medicine and applied to lung disorders. PMID:23652321

  6. Heme Oxygenase-1 Is Required for Angiogenic Function of Bone Marrow-Derived Progenitor Cells: Role in Therapeutic Revascularization

    PubMed Central

    Grochot-Przeczek, Anna; Kotlinowski, Jerzy; Kozakowska, Magdalena; Starowicz, Katarzyna; Jagodzinska, Jolanta; Stachurska, Anna; Volger, Oscar L.; Bukowska-Strakova, Karolina; Florczyk, Urszula; Tertil, Magdalena; Jazwa, Agnieszka; Szade, Krzysztof; Stepniewski, Jacek; Loboda, Agnieszka; Horrevoets, Anton J.G.

    2014-01-01

    Abstract Aims: Heme oxygenase-1 (HO-1) is a cytoprotective enzyme that can be down-regulated in diabetes. Its importance for mature endothelium has been described, but its role in proangiogenic progenitors is not well known. We investigated the effect of HO-1 on the angiogenic potential of bone marrow-derived cells (BMDCs) and on blood flow recovery in ischemic muscle of diabetic mice. Results: Lack of HO-1 decreased the number of endothelial progenitor cells (Lin−CD45−cKit-Sca-1+VEGFR-2+) in murine bone marrow, and inhibited the angiogenic potential of cultured BMDCs, affecting their survival under oxidative stress, proliferation, migration, formation of capillaries, and paracrine proangiogenic potential. Transcriptome analysis of HO-1−/− BMDCs revealed the attenuated up-regulation of proangiogenic genes in response to hypoxia. Heterozygous HO-1+/− diabetic mice subjected to hind limb ischemia exhibited reduced local expression of vascular endothelial growth factor (VEGF), placental growth factor (PlGF), stromal cell-derived factor 1 (SDF-1), VEGFR-1, VEGFR-2, and CXCR-4. This was accompanied by impaired revascularization of ischemic muscle, despite a strong mobilization of bone marrow-derived proangiogenic progenitors (Sca-1+CXCR-4+) into peripheral blood. Blood flow recovery could be rescued by local injections of conditioned media harvested from BMDCs, but not by an injection of cultured BMDCs. Innovation: This is the first report showing that HO-1 haploinsufficiency impairs tissue revascularization in diabetes and that proangiogenic in situ response, not progenitor cell mobilization, is important for blood flow recovery. Conclusions: HO-1 is necessary for a proper proangiogenic function of BMDCs. A low level of HO-1 in hyperglycemic mice decreases restoration of perfusion in ischemic muscle, which can be rescued by a local injection of conditioned media from cultured BMDCs. Antioxid. Redox Signal. 20, 1677–1692. PMID:24206054

  7. Micro-/Nano- sized hydroxyapatite directs differentiation of rat bone marrow derived mesenchymal stem cells towards an osteoblast lineage

    NASA Astrophysics Data System (ADS)

    Huang, Yan; Zhou, Gang; Zheng, Lisha; Liu, Haifeng; Niu, Xufeng; Fan, Yubo

    2012-03-01

    Regenerative medicine consisting of cells and materials provides a new way for the repair and regeneration of tissues and organs. Nano-biomaterials are highlighted due to their advantageous features compared with conventional micro-materials. The aim of this study is to investigate the effects of micro-/nano- sized hydroxyapatite (μ/n-HA) on the osteogenic differentiation of rat bone marrow derived mesenchymal stem cells (rBMSCs). μ/n-HA were prepared by a microwave synthesizer and precipitation method, respectively. Different sizes of μ/n-HA were characterized by IR, XRD, SEM, TEM and co-cultured with rBMSCs. It was shown that rBMSCs expressed higher levels of osteoblast-related markers by n-HA than μ-HA stimulation. The size of HA is an important factor for affecting the osteogenic differentiation of rBMSCs. This provides a new avenue for mechanistic studies of stem cell differentiation and a new approach to obtain more committed differentiated cells.

  8. Micro-/nano- sized hydroxyapatite directs differentiation of rat bone marrow derived mesenchymal stem cells towards an osteoblast lineage.

    PubMed

    Huang, Yan; Zhou, Gang; Zheng, Lisha; Liu, Haifeng; Niu, Xufeng; Fan, Yubo

    2012-04-01

    Regenerative medicine consisting of cells and materials provides a new way for the repair and regeneration of tissues and organs. Nano-biomaterials are highlighted due to their advantageous features compared with conventional micro-materials. The aim of this study is to investigate the effects of micro-/nano- sized hydroxyapatite (μ/n-HA) on the osteogenic differentiation of rat bone marrow derived mesenchymal stem cells (rBMSCs). μ/n-HA were prepared by a microwave synthesizer and precipitation method, respectively. Different sizes of μ/n-HA were characterized by IR, XRD, SEM, TEM and co-cultured with rBMSCs. It was shown that rBMSCs expressed higher levels of osteoblast-related markers by n-HA than μ-HA stimulation. The size of HA is an important factor for affecting the osteogenic differentiation of rBMSCs. This provides a new avenue for mechanistic studies of stem cell differentiation and a new approach to obtain more committed differentiated cells. PMID:22371072

  9. Prognostic value of circulating VEGFR2+ bone marrow-derived progenitor cells in patients with advanced cancer.

    PubMed

    Massard, Christophe; Borget, Isabelle; Le Deley, Marie Cécile; Taylor, Melissa; Gomez-Roca, Carlos; Soria, Jean Charles; Farace, Françoise

    2012-06-01

    We hypothesised that host-related markers, possibly reflecting tumour aggressiveness, such as circulating endothelial cells (CEC) and circulating VEGFR2(+) bone marrow-derived (BMD) progenitor cells, could have prognostic value in patients with advanced cancer enrolled in early anticancer drug development trials. Baseline CECs (CD45(-)CD31(+)CD146(+)7AAD(-) cells) and circulating VEGFR2(+)-BMD progenitor cells (defined as CD45(dim)CD34(+)VEGFR2(+)7AAD(-) cells) were measured by flow-cytometry in 71 and 58 patients included in phase 1 trials testing novel anti-vascular or anti-angiogenic agents. Correlations between levels of CECs, circulating VEGFR2(+)-BMD progenitor cells, clinical and biological prognostic factors (i.e. the Royal Marsden Hospital (RMH) score), and overall survival (OS) were studied. The median value of CECs was 12 CEC/ml (range 0-154/ml). The median level of VEGFR2(+)-BMD progenitor cells was 1.3% (range 0-32.5%) of circulating BMD-CD34(+) progenitors. While OS was not correlated with CEC levels, it was significantly worse in patients with high VEGFR2(+)-BMD progenitor levels (>1%) (median OS 9.0 versus 17.0 months), and with a RMH prognostic score >0 (median OS 9.0 versus 24.2 months). The prognostic value of VEGFR2(+)-BMD progenitor levels remained significant (hazard ratio (HR) = 2.3, 95% confidence interval (CI), 1.1-4.6, p = 0.02) after multivariate analysis. A composite VEGFR2(+)-BMD progenitor level/RHM score ≥ 2 was significantly associated with an increased risk of death compared to scores of 0 or 1 (median OS 9.0 versus 18.4 months, HR = 2.6 (95%CI, 1.2-5.8, p = 0.02)). High circulating VEGFR2(+)-BMD progenitor levels are associated with poor prognostics and when combined to classical clinical and biological parameters could provide a new tool for patient selection in early anticancer drug trials. PMID:22370181

  10. Differentiation of Murine Bone Marrow-Derived Smooth Muscle Progenitor Cells Is Regulated by PDGF-BB and Collagen

    PubMed Central

    Lin, Clifford; Yuan, Yifan; Courtman, David W.

    2016-01-01

    Smooth muscle cells (SMCs) are key regulators of vascular disease and circulating smooth muscle progenitor cells may play important roles in vascular repair or remodelling. We developed enhanced protocols to derive smooth muscle progenitors from murine bone marrow and tested whether factors that are increased in atherosclerotic plaques, namely platelet-derived growth factor—BB (PDGF-BB) and monomeric collagen, can influence the smooth muscle specific differentiation, proliferation, and survival of mouse bone marrow-derived progenitor cells. During a 21 day period of culture, bone marrow cells underwent a marked increase in expression of the SMC markers α-SMA (1.93 ± 0.15 vs. 0.0008 ± 0.0003 (ng/ng GAPDH) at 0 d), SM22-α (1.50 ± 0.27 vs. 0.005 ± 0.001 (ng/ng GAPDH) at 0 d) and SM-MHC (0.017 ± 0.004 vs. 0.001 ± 0.001 (ng/ng GAPDH) at 0 d). Bromodeoxyuridine (BrdU) incorporation experiments showed that in early culture, the smooth muscle progenitor subpopulation could be identified by high proliferative rates prior to the expression of smooth muscle specific markers. Culture of fresh bone marrow or smooth muscle progenitor cells with PDGF-BB suppressed the expression of α-SMA and SM22-α, in a rapidly reversible manner requiring PDGF receptor kinase activity. Progenitors cultured on polymerized collagen gels demonstrated expression of SMC markers, rates of proliferation and apoptosis similar to that of cells on tissue culture plastic; in contrast, cells grown on monomeric collagen gels displayed lower SMC marker expression, lower growth rates (319 ± 36 vs. 635 ± 97 cells/mm2), and increased apoptosis (5.3 ± 1.6% vs. 1.0 ± 0.5% (Annexin 5 staining)). Our data shows that the differentiation and survival of smooth muscle progenitors are critically affected by PDGF-BB and as well as the substrate collagen structure. PMID:27258003

  11. Bone Marrow Derived Eosinophil Cultures

    PubMed Central

    Lu, Thomas X.; Rothenberg, Marc E.

    2016-01-01

    Eosinophils are multifunctional effector cells implicated in the pathogenesis of a variety of diseases including asthma, eosinophil gastrointestinal disorders and helminth infection. Mouse bone marrow derived progenitor cells can be differentiated into eosinophils following IL-5 exposure. These bone marrow derived eosinophils are fully differentiated at the end of a 14 day culture based on morphology and expression of molecular markers.

  12. Particle Radiation-Induced Nontargeted Effects in Bone-Marrow-Derived Endothelial Progenitor Cells.

    PubMed

    Sasi, Sharath P; Park, Daniel; Muralidharan, Sujatha; Wage, Justin; Kiladjian, Albert; Onufrak, Jillian; Enderling, Heiko; Yan, Xinhua; Goukassian, David A

    2015-01-01

    Bone-marrow- (BM-) derived endothelial progenitor cells (EPCs) are critical for endothelial cell maintenance and repair. During future space exploration missions astronauts will be exposed to space irradiation (IR) composed of a spectrum of low-fluence protons ((1)H) and high charge and energy (HZE) nuclei (e.g., iron-(56)Fe) for extended time. How the space-type IR affects BM-EPCs is limited. In media transfer experiments in vitro we studied nontargeted effects induced by (1)H- and (56)Fe-IR conditioned medium (CM), which showed significant increase in the number of p-H2AX foci in nonirradiated EPCs between 2 and 24 h. A 2-15-fold increase in the levels of various cytokines and chemokines was observed in both types of IR-CM at 24 h. Ex vivo analysis of BM-EPCs from single, low-dose, full-body (1)H- and (56)Fe-IR mice demonstrated a cyclical (early 5-24 h and delayed 28 days) increase in apoptosis. This early increase in BM-EPC apoptosis may be the effect of direct IR exposure, whereas late increase in apoptosis could be a result of nontargeted effects (NTE) in the cells that were not traversed by IR directly. Identifying the role of specific cytokines responsible for IR-induced NTE and inhibiting such NTE may prevent long-term and cyclical loss of stem and progenitors cells in the BM milieu. PMID:26074973

  13. Phenotypic differences in white-tailed deer antlerogenic progenitor cells and marrow-derived mesenchymal stromal cells.

    PubMed

    Daley, Ethan L H; Alford, Andrea I; Miller, Joshua D; Goldstein, Steven A

    2014-05-01

    Deer antlers are bony appendages that are annually cast and rapidly regrown in a seasonal process coupled to the reproductive cycle. Due to the uniqueness of this process among mammals, we reasoned that a fundamental characterization of antler progenitor cell behavior may provide insights that could lead to improved strategies for promoting bone repair. In this study, we investigated whether white-tailed deer antlerogenic progenitor cells (APC) conform to basic criteria defining mesenchymal stromal cells (MSC). In addition, we tested the effects of the artificial glucocorticoid dexamethasone (DEX) on osteogenic and chondrogenic differentiation as well as the degree of apoptosis during the latter. Comparisons were made to animal-matched marrow-derived MSC. APC and MSC generated similar numbers of colonies. APC cultures expanded less rapidly overall but experienced population recovery at later time points. In contrast to MSC, APC did not display adipogenic in vitro differentiation capacity. Under osteogenic culture conditions, APC and MSC exhibited different patterns of alkaline phosphatase activity over time. DEX increased APC alkaline phosphatase activity only initially but consistently led to decreased activity in MSC. APC and MSC in osteogenic culture underwent different time and DEX-dependent patterns of mineralization, yet APC and MSC achieved similar levels of mineral accrual in an ectopic ossicle model. During chondrogenic differentiation, APC exhibited high levels of apoptosis without a reduction in cell density. DEX decreased proteoglycan production and increased apoptosis in chondrogenic APC cultures but had the opposite effects in MSC. Our results suggest that APC and MSC proliferation and differentiation differ in their dependence on time, factors, and milieu. Antler tip APC may be more lineage-restricted osteo/chondroprogenitors with distinctly different responses to apoptotic and glucocorticoid stimuli. PMID:24313802

  14. Altered SDF-1-mediated differentiation of bone marrow-derived endothelial progenitor cells in diabetes mellitus

    PubMed Central

    De Falco, Elena; Avitabile, Daniele; Totta, Pierangela; Straino, Stefania; Spallotta, Francesco; Cencioni, Chiara; Torella, Anna Rita; Rizzi, Roberto; Porcelli, Daniele; Zacheo, Antonella; Vito, Luca Di; Pompilio, Giulio; Napolitano, Monica; Melillo, Guido; Capogrossi, Maurizio C; Pesce, Maurizio

    2009-01-01

    In diabetic patients and animal models of diabetes mellitus (DM), circulating endothelial progenitor cell (EPC) number is lower than in normoglycaemic conditions and EPC angiogenic properties are inhibited. Stromal cell derived factor-1 (SDF-1) plays a key role in bone marrow (BM) c-kit+ stem cell mobilization into peripheral blood (PB), recruitment from PB into ischemic tissues and differentiation into endothelial cells. The aim of the present study was to examine the effect of DM in vivo and in vitro, on murine BM-derived c-kit+ cells and on their response to SDF-1. Acute hindlimb ischemia was induced in streptozotocin-treated DM and control mice; circulating c-kit+ cells exhibited a rapid increase followed by a return to control levels which was significantly faster in DM than in control mice. CXCR4 expression by BM c-kit+ cells as well as SDF-1 protein levels in the plasma and in the skeletal muscle, both before and after the induction of ischemia, were similar between normoglycaemic and DM mice. However, BM-derived c-kit+ cells from DM mice exhibited an impaired differentiation towards the endothelial phenotype in response to SDF-1; this effect was associated with diminished protein kinase phosphorylation. Interestingly, SDF-1 ability to induce differentiation of c-kit+ cells from DM mice was restored when cells were cultured under normoglycaemic conditions whereas c-kit+ cells from normoglycaemic mice failed to differentiate in response to SDF-1 when they were cultured in hyperglycaemic conditions. These results show that DM diminishes circulating c-kit+ cell number following hindlimb ischemia and inhibits SDF-1-mediated AKT phosphorylation and differentiation towards the endothelial phenotype of BM-derived c-kit+ cells. PMID:20196780

  15. Bone Marrow-Derived Endothelial Progenitor Cells Protect Against Scopolamine-Induced Alzheimer-Like Pathological Aberrations.

    PubMed

    Safar, Marwa M; Arab, Hany H; Rizk, Sherine M; El-Maraghy, Shohda A

    2016-04-01

    Vascular endothelial dysfunction plays a key role in the pathogenesis of Alzheimer's disease (AD). Patients with AD have displayed decreased circulating endothelial progenitor cells (EPCs) which repair and maintain the endothelial function. Transplantation of EPCs has emerged as a promising approach for the management of cerebrovascular diseases including ischemic stroke, however, its impact on AD has been poorly described. Thus, the current study aimed at investigating the effects of bone marrow-derived (BM) EPCs transplantation in repeated scopolamine-induced cognitive impairment, an experimental model that replicates biomarkers of AD. Intravenously transplanted BM-EPCs migrated into the brain of rats and improved the learning and memory deficits. Meanwhile, they mitigated the deposition of amyloid plaques and associated histopathological alterations. At the molecular levels, BM-EPCs blunted the increase of hippocampal amyloid beta protein (Aβ), amyloid precursor protein (APP) and reinstated the Aβ-degrading neprilysin together with downregulation of p-tau and its upstream glycogen synthase kinase-3β (GSK-3β). They also corrected the perturbations of neurotransmitter levels including restoration of acetylcholine and associated esterase along with dopamine, GABA, and the neuroexitatory glutamate. Furthermore, BM-EPCs induced behavioral recovery via boosting of vascular endothelial growth factor (VEGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and its upstream cAMP response element binding (CREB), suppression of the proinflammatory tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), and upregulation of interleukin-10 (IL-10). BM-EPCs also augmented Nrf2 and seladin-1. Generally, these actions were analogous to those exerted by adipose tissue-derived mesenchymal stem cells (AT-MSCs) and the reference anti-Alzheimer donepezil. For the first time, these findings highlight the beneficial actions of BM-EPCs against the memory

  16. Vascular endothelial growth factor-A signaling in bone marrow-derived endothelial progenitor cells exposed to hypoxic stress.

    PubMed

    Hoffmann, Brian R; Wagner, Jordan R; Prisco, Anthony R; Janiak, Agnieszka; Greene, Andrew S

    2013-11-01

    Bone marrow-derived endothelial progenitor cells (BM-EPCs) are stimulated by vascular endothelial growth factor-A (VEGF-A) and other potent proangiogenic factors. During angiogenesis, an increase in VEGF-A expression stimulates BM-EPCs to enhance endothelial tube formation and contribute to an increase in microvessel density. Hypoxia is known to produce an enhanced angiogenic response and heightened levels of VEGF-A have been seen in oxygen deprived epithelial and endothelial cells, yet the pathways for VEGF-A signaling in BM-EPCs have not been described. This study explores the influence of hypoxia on VEGF-A signaling in rat BM-EPCs utilizing a novel proteomic strategy to directly identify interacting downstream components of the combined VEGF receptor(s) signaling pathways, gene expression analysis, and functional phenotyping. VEGF-A signaling network analysis following liquid chromatographic separation and tandem mass spectrometry revealed proteins related to inositol/calcium signaling, nitric oxide signaling, cell survival, cell migration, and inflammatory responses. Alterations in BM-EPC expression of common angiogenic genes and tube formation in response to VEGF-A during hypoxia were measured and combined with the proteomic analysis to enhance and support the signaling pathways detected. BM-EPC tube formation assays in response to VEGF-A exhibited little tube formation; however, a cell projection/migratory phenotype supported the signaling data. Additionally, a novel assay measuring BM-EPC incorporation into preformed endothelial cell tubes indicated a significant increase of incorporated BM-EPCs after pretreatment with VEGF-A during hypoxia. This study verifies known VEGF-A pathway components and reveals several unidentified mechanisms of VEGF-A signaling in BM-EPCs during hypoxia that may be important for migration to sites of vascular regeneration. PMID:24022223

  17. Vascular endothelial growth factor-A signaling in bone marrow-derived endothelial progenitor cells exposed to hypoxic stress

    PubMed Central

    Hoffmann, Brian R.; Wagner, Jordan R.; Prisco, Anthony R.; Janiak, Agnieszka

    2013-01-01

    Bone marrow-derived endothelial progenitor cells (BM-EPCs) are stimulated by vascular endothelial growth factor-A (VEGF-A) and other potent proangiogenic factors. During angiogenesis, an increase in VEGF-A expression stimulates BM-EPCs to enhance endothelial tube formation and contribute to an increase in microvessel density. Hypoxia is known to produce an enhanced angiogenic response and heightened levels of VEGF-A have been seen in oxygen deprived epithelial and endothelial cells, yet the pathways for VEGF-A signaling in BM-EPCs have not been described. This study explores the influence of hypoxia on VEGF-A signaling in rat BM-EPCs utilizing a novel proteomic strategy to directly identify interacting downstream components of the combined VEGF receptor(s) signaling pathways, gene expression analysis, and functional phenotyping. VEGF-A signaling network analysis following liquid chromatographic separation and tandem mass spectrometry revealed proteins related to inositol/calcium signaling, nitric oxide signaling, cell survival, cell migration, and inflammatory responses. Alterations in BM-EPC expression of common angiogenic genes and tube formation in response to VEGF-A during hypoxia were measured and combined with the proteomic analysis to enhance and support the signaling pathways detected. BM-EPC tube formation assays in response to VEGF-A exhibited little tube formation; however, a cell projection/migratory phenotype supported the signaling data. Additionally, a novel assay measuring BM-EPC incorporation into preformed endothelial cell tubes indicated a significant increase of incorporated BM-EPCs after pretreatment with VEGF-A during hypoxia. This study verifies known VEGF-A pathway components and reveals several unidentified mechanisms of VEGF-A signaling in BM-EPCs during hypoxia that may be important for migration to sites of vascular regeneration. PMID:24022223

  18. The role of the fibrocyte, a bone marrow-derived mesenchymal progenitor, in reactive and reparative fibroses.

    PubMed

    Bellini, Alberto; Mattoli, Sabrina

    2007-09-01

    Human fibrocytes are mesenchymal progenitors that exhibit mixed morphological and molecular characteristics of hematopoietic stem cells, monocytes and fibroblasts. They likely represent the obligate intermediate stage of differentiation into mature mesenchymal cells of a bone marrow-derived precursor of the monocyte lineage under permissive conditions. On in vitro stimulation with pro-fibrotic cytokines and growth factors, human fibrocytes produce large quantities of extracellular matrix components and further differentiate into cells identical to the contractile myofibroblasts that emerge at the tissue sites during repair processes and in some fibrotic lesions. Studies in various animal models of wound healing or fibrotic diseases have confirmed the ability of fibrocytes to differentiate into mature mesenchymal cells in vivo and have suggested a causal link between fibrocyte accumulation and ongoing tissue fibrogenesis or vascular remodeling in response to tissue damage or hypoxia. Fibrocytes synthesizing new collagen or acquiring myofibroblast markers have been detected in human hypertrophic scars, in the skin of patients affected by nephrogenic systemic fibrosis, in human atherosclerotic lesions, and in pulmonary diseases characterized by repeated cycles of inflammation and repair, like asthma. The presence of fibrocyte-like cells has been reported in human chronic pancreatitis and chronic cystitis. Similar cells also populate the stroma surrounding human benign tumors. The available data indicate that human fibrocytes serve as a source of mature mesenchymal cells during reparative processes and in fibrotic disorders or stromal reactions predominantly associated with a persistent inflammatory infiltrate or with the selective recruitment of monocytes induced by ischemic changes and tumor development. A deeper understanding of the mechanisms involved in fibrocyte differentiation in these pathological conditions may lead to the development of novel therapies for

  19. Assessment of the role of flavonoids for inducing osteoblast differentiation in isolated mouse bone marrow derived mesenchymal stem cells.

    PubMed

    Srivastava, Swati; Bankar, Rohini; Roy, Partha

    2013-06-15

    Quercetin and rutin are common flavonoids in fruit and vegetables, and have been reported to affect bone development. However, the effect of flavonoids on osteoblast differentiation remains a matter of controversy. In the present study, mouse bone marrow mesenchymal stem cells (BMMSCs) were isolated and characterized for their use in osteoblast differentiation using two flavonoids, quercetin and rutin. BMMSCs were cultured in various concentrations of quercetin and rutin during the osteoblast differentiation period of 10 days. Both quercetin and rutin were found to up regulate the osteoblast differentiation in dose dependent manner, albeit to lesser extent in case of former than that of latter. Quercetin and rutin also increased alkaline phosphatase activity by about 150 and 240% and demonstrated mineralization up to 110 and 200% respectively as compared to control (which was considered as 100%). Further, both the flavonoids were also found to increase the expression of some of the prominent markers for differentiation of osteoblast like osteopontin, osterix, RunX2, osteoprotegerin and osteocalcin. The current data suggests that certain classes of flavonoids like rutin and quercetin can be used in the cure and management of osteodegenerative disorders due to their osteoblast specific differentiation activities. PMID:23570998

  20. Clinical-scale expansion of a mixed population of bone-marrow-derived stem and progenitor cells for potential use in bone-tissue regeneration.

    PubMed

    Dennis, James E; Esterly, Kelly; Awadallah, Amad; Parrish, Christopher R; Poynter, Gregory M; Goltry, Kristin L

    2007-10-01

    Preclinical and clinical studies have demonstrated the ability of bone marrow derived stem and progenitor cells to regenerate many tissues, including bone. Methods to expand or enrich progenitors from bone marrow are common; however, these methods include many steps not amenable to clinical use. A closed automated cell production culture system was developed for clinical-scale ex vivo production of bone marrow-derived stem and progenitor cells for hematopoietic reconstitution. The current study tested the ability of this bioreactor system to produce progenitor cells, termed tissue repair cells (TRC), possessing osteogenic potential. Three TRC formulations were evaluated: (a) cells cultured without exogenous cytokines (TRC); (b) cells cultured with exogenous cytokines (TRC-C); and (c) an adherent subset of TRC-C (TRC-C(Ad)). Starting human bone marrow mononuclear cells (BM MNC) and TRC products were characterized for the expression of cell surface markers, in vitro colony forming ability, and in vivo osteogenic potential. Results showed significant expansion of mesenchymal progenitors (CD90+, CD105+, and CD166+) in each TRC formulation. In vivo bone formation, measured by histology, was highest in the TRC group, followed by TRC-C(Ad) and TRC-C. The TRC product outperformed starting BM MNC and had equivalent bone forming potential to purified MSCs at the same cell dose. Post hoc analysis revealed that the presence of CD90+, CD105+, and CD166+ correlated strongly with in vivo bone formation scores (r(2) > .95). These results demonstrate that this bioreactor system can be used to generate, in a single step, a population of progenitor cells with potent osteogenic potential. Disclosure of potential conflicts of interest is found at the end of this article. PMID:17585167

  1. Endothelial Progenitor Cell Fraction Contained in Bone Marrow-Derived Mesenchymal Stem Cell Populations Impairs Osteogenic Differentiation

    PubMed Central

    Duttenhoefer, Fabian; Lara de Freitas, Rafael; Loibl, Markus; Bittermann, Gido; Geoff Richards, R.; Alini, Mauro; Verrier, Sophie

    2015-01-01

    In bone tissue engineering (TE) endothelial cell-osteoblast cocultures are known to induce synergies of cell differentiation and activity. Bone marrow mononucleated cells (BMCs) are a rich source of mesenchymal stem cells (MSCs) able to develop an osteogenic phenotype. Endothelial progenitor cells (EPCs) are also present within BMC. In this study we investigate the effect of EPCs present in the BMC population on MSCs osteogenic differentiation. Human BMCs were isolated and separated into two populations. The MSC population was selected through plastic adhesion capacity. EPCs (CD34+ and CD133+) were removed from the BMC population and the resulting population was named depleted MSCs. Both populations were cultured over 28 days in osteogenic medium (Dex+) or medium containing platelet lysate (PL). MSC population grew faster than depleted MSCs in both media, and PL containing medium accelerated the proliferation for both populations. Cell differentiation was much higher in Dex+ medium in both cases. Real-time RT-PCR revealed upregulation of osteogenic marker genes in depleted MSCs. Higher values of ALP activity and matrix mineralization analyses confirmed these results. Our study advocates that absence of EPCs in the MSC population enables higher osteogenic gene expression and matrix mineralization and therefore may lead to advanced bone neoformation necessary for TE constructs. PMID:26491682

  2. Glucose-Dependent Insulinotropic Peptide Prevents Serum Deprivation-Induced Apoptosis in Human Bone Marrow-Derived Mesenchymal Stem Cells and Osteoblastic Cells.

    PubMed

    Berlier, J L; Kharroubi, I; Zhang, J; Dalla Valle, A; Rigutto, S; Mathieu, M; Gangji, V; Rasschaert, J

    2015-12-01

    Human bone marrow-derived mesenchymal stem cells (hBMSC) are able to differentiate into cells of connective tissue lineages, including bone and cartilage. They are therefore considered as a promising tool for the treatment of bone degenerative diseases. One of the major issues in regenerative cell therapy is the biosafety of fetal bovine serum used for cell culture. Therefore, the development of a culture medium devoid of serum but preserving hBMSC viability will be of clinical value. The glucose-dependent insulinotropic peptide (GIP) has an anti-apoptotic action in insulin-producing cells. Interestingly, GIP also exerts beneficial effects on bone turnover by acting on osteoblasts and osteoclasts. We therefore evaluated the ability of GIP to prevent cell death in osteoblastic cells cultured in serum-free conditions. In hBMSC and SaOS-2 cells, activation of the GIP receptor increased intracellular cAMP levels. Serum deprivation induced apoptosis in SaOS-2 and hBMSC that was reduced by 30 and 50 %, respectively, in the presence of GIP. The protective effect of GIP involves activation of the adenylate cyclase pathway and inhibition of caspases 3/7 activation. These findings demonstrate that GIP exerts a protective action against apoptosis in hBMSC and suggest a novel approach to preserve viability of hBMSC cultured in the absence of serum. PMID:26254594

  3. Effects of corneal stromal cell- and bone marrow-derived endothelial progenitor cell-conditioned media on the proliferation of corneal endothelial cells

    PubMed Central

    Zhu, Meng-Yu; Yao, Qin-Ke; Chen, Jun-Zhao; Shao, Chun-Yi; Yan, Chen-Xi; Ni, Ni; Fan, Xian-Qun; Gu, Ping; Fu, Yao

    2016-01-01

    AIM To explore the effects of conditioned media on the proliferation of corneal endothelial cells (CECs) and to compare the efficiency of different conditioned media (CM). METHODS Rat CECs, corneal stromal cells (CSCs), bone marrow-derived endothelial progenitor cells (BEPCs), and bone marrow-derived mesenchymal stem cells (BMSCs) were isolated and cultured in vitro. CM was collected from CSCs, BEPCs, and BMSCs. CECs were cultivated in different culture media. Cell morphology was recorded, and gene and protein expression were analyzed. RESULTS After grown in CM for 5d, CECs in each experimental group remained polygonal, in a cobblestone-like monolayer arrangement. Immunocytofluorescence revealed positive expression of Na+/K+-ATP, aquaporin 1 (AQP1), and zonula occludens 1 (ZO-1). Based on quantitative polymerase chain reaction (qPCR) analysis, Na+/K+-ATP expression in CSC-CM was notably upregulated by 1.3-fold (±0.036) (P<0.05, n=3). The expression levels of ZO-1, neuron specific enolase (NSE), Vimentin, paired homebox 6 (PAX6), and procollagen type VIII (COL8A1) were notably upregulated in each experimental group. Each CM had a positive effect on CEC proliferation, and CSC-CM had the strongest effect on proliferation. CONCLUSION CSC-CM, BEPC-CM, and BMSC-CM not only stimulated the proliferation of CECs, but also maintained the characteristic differentiated phenotypes necessary for endothelial functions. CSC-CM had the most notable effect on CEC proliferation. PMID:27158599

  4. Effects of the dichloromethane fraction of Dipsaci Radix on the osteoblastic differentiation of human alveolar bone marrow-derived mesenchymal stem cells.

    PubMed

    Kim, Beom-Su; Kim, Yoon-Chul; Zadeh, Homa; Park, Yoon-Jeong; Pi, Sung-Hee; Shin, Hyung-Shik; You, Hyung-Keun

    2011-01-01

    Dipsaci Radix is the dried root of Dipsacus asper Wall. It has been used in Korean herbal medicine to treat bone fractures. In this study, we examined the effect of the dichloromethane fraction of Dipsaci Radix (DR(DM)) on the osteoblastic differentiation of human alveolar bone marrow-derived MSCs (ABM-MSCs). The ABM-MSCs were isolated from healthy subjects and cultured in vitro, followed by phenotypic characterization. They showed a fibroblast-like morphology and expressed CD29, CD44, CD73, and CD105, but not CD34. Calcified nodules were generated in response to both dexamethasone (DEX) and DR(DM). There was a significant increase in the alkaline phosphatase (ALP) activity and protein expression of bone sialoprotein (BSP) and osteocalcin (OC) in response to DEX and DR(DM) as compared to control. These results provide evidence for the osteogenic potential of cultured ABM-MSCs in response to DR(DM). Also, an active single compound was additionally isolated from DR(DM). The single compound (hederagenin 3-O-(2-O-acetyl)-α-L-arabinopyranoside) also significantly increased ALP activity and the level of protein expression of BSP and OC. These results highlight the possible clinical applications of DR(DM) and hederagenin 3-O-(2-O-acetyl)-α-L-arabinopyranoside in bone regeneration. PMID:21228489

  5. DISTINCT PROGENITOR POPULATIONS IN SKELETAL MUSCLE ARE BONE MARROW DERIVED AND EXHIBIT DIFFERENT CELL FATES DURING VASCULAR REGENERATION

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Vascular progenitors were previously isolated from blood and bone marrow; herein, we define the presence, phenotype, potential, and origin of vascular progenitors resident within adult skeletal muscle. Two distinct populations of cells were simultaneously isolated from hindlimb muscle: the side popu...

  6. Application of perfusion culture system improves in vitro and in vivo osteogenesis of bone marrow-derived osteoblastic cells in porous ceramic materials.

    PubMed

    Wang, Yichao; Uemura, Toshimasa; Dong, Jian; Kojima, Hiroko; Tanaka, Junzo; Tateishi, Tetsuya

    2003-12-01

    Composites of bone marrow-derived osteoblasts (BMOs) and porous ceramics have been widely used as a bone graft model for bone tissue engineering. Perfusion culture has potential utility for many cell types in three-dimensional (3D) culture. Our hypothesis was that perfusion of medium would increase the cell viability and biosynthetic activity of BMOs in porous ceramic materials, which would be revealed by increased levels of alkaline phosphate (ALP) activity and osteocalcin (OCN) and enhanced bone formation in vivo. For testing in vitro, BMO/beta-tricalcium phosphate composites were cultured in a perfusion container (Minucells and Minutissue, Bad Abbach, Germany) with fresh medium delivered at a rate of 2 mL/h by a peristaltic pump. The ALP activity and OCN content of composites were measured at the end of 1, 2, 3, and 4 weeks of subculture. For testing in vivo, after subculturing for 2 weeks, the composites were subcutaneously implanted into syngeneic rats. These implants were harvested 4 or 8 weeks later. The samples then underwent a biochemical analysis of ALP activity and OCN content and were observed by light microscopy. The levels of ALP activity and OCN in the composites were significantly higher in the perfusion group than in the control group (p < 0.01), both in vitro and in vivo. Histomorphometric analysis of the hematoxylin- and eosin-stained sections revealed a higher average ratio of bone to pore in BMO/beta-TCP composites of the perfusion group after implantation: 47.64 +/- 6.16 for the perfusion group and 26.22 +/- 4.84 for control at 4 weeks (n = 6, p < 0.01); 67.97 +/- 3.58 for the perfusion group and 47.39 +/- 4.10 for control at 8 weeks (n = 6, p < 0.05). These results show that the application of a perfusion culture system during the subculture of BMOs in a porous ceramic scaffold is beneficial to their osteogenesis. After differentiation culture in vitro with the perfusion culture system, the activity of the osteoblastic cells and the

  7. Demonstration of the presence of independent pre-osteoblastic and pre-adipocytic cell populations in bone marrow-derived mesenchymal stem cells.

    PubMed

    Post, S; Abdallah, B M; Bentzon, J F; Kassem, M

    2008-07-01

    Mesenchymal stem cells (MSC) are defined as plastic-adherent, clonal cells that are common progenitors for osteoblasts and adipocytes. An inverse relationship between bone and fat has been observed in several clinical conditions and has been suggested to be caused by re-directing MSC differentiation into one particular lineage. However, this inverse relationship between bone and fat is not consistent and under certain in vivo conditions, bone and fat can change independently suggesting separate precursor cell populations. In order to test for this hypothesis, we extensively characterized two plastic-adherent clonal MSC lines (mMSC1 and mMSC2) derived from murine bone marrow. The two cell lines grew readily in culture and have undergone more than 100 population doublings with no apparent differences in their growth rates. Both cell lines were positive for the murine MSC marker Sca-1 and mMSC1 was also positive for CD13. Both cell lines were exposed to in vitro culture induction of osteogenesis and adipogenesis. mMSC1 and not mMSC2 were only able to differentiate to adipocytes evidenced by the expression of adipocyte markers (aP2, adiponectin, adipsin, PPARgamma2 and C/EBPa) and the presence of mature adipocytes visualized by Oil Red O staining. On the other hand, mMSC2 and not mMSC1 differentiated to osteoblast lineage as demonstrated by up-regulation of osteoblastic makers (CBFA1/RUNX2, Osterix, alkaline phosphatase, bone sialoprotein and osteopontin) and formation of alizarin red stained mineralized matrix in vitro. Consistent with the in vitro results, mMSC2 and not mMSC1, were able to form bone in vivo after subcutaneous implantation in immune-deficient (NOD/SCID) mice. Our data suggest that contrary to the current belief, bone marrow contains clonal subpopulations of cells that are committed to either osteoblast or adipocyte lineage. These cell populations may undergo independent changes during aging and in bone diseases and thus represent important targets for

  8. Functional analysis in vivo of engineered valved venous conduit with decellularized matrix and two bone marrow-derived progenitors in sheep.

    PubMed

    Yuan, Jian-Ming; Xiong, Shao-Hu; Liu, Zhen; Wen, Yu; Dang, Rui-Shan; Shen, Man-Ru; Zhang, Yong-Zhen; Zhang, Xi; Yang, Xiang-Qun; Zhang, Chuan-Sen

    2016-07-01

    Tissue engineering has been considered a promising approach for creating grafts to replace autologous venous valves. Here, ovine bone marrow-derived endothelial progenitor cells (EPCs) and multipotent adult progenitor cells (MAPCs) were harvested and then loaded into decellularized venous matrix to create tissue-engineered (TE) valved vein. Subsequently, the ovine femoral veins containing the valve were removed and replaced by TE grafts or acellular matrix only. The morphology and function were analysed for up to 1 year by ultrasonography, angiography, H&E staining and scanning electron microscopy (SEM). The differentiation of seeded cells was traced immunofluorochemically. The results showed that decellularized venous matrix could initially and feebly attract endogenous cells, but failed afterwards and were insufficient to restore valve function. On the contrary, the seeded cells differentiated into endothelial cells (ECs) in vivo and formed a monolayer endothelium, and smooth muscle cells within the scaffold therefore produced TE grafts comparable to the native vein valve. This TE graft remained patent and sufficient after implantation into the venous circuit of the ovine lower extremity for at least 6 months. Unfortunately, cells seeded on the luminal surface and both sides of the leaflets lost their biological functions at 12 months, resulting in thrombosis formation and leading to complete occlusion of the TE grafts and impotent venous valves. These findings suggest that this TE valved venous conduit can function physiologically in vivo in the medium term. Before translating this TE venous valve into clinical practice, the durability should be improved and thrombogenicity should be suppressed. Copyright © 2016 John Wiley & Sons, Ltd. PMID:23904287

  9. ApoA-I Induced CD31 in Bone Marrow-derived Vascular Progenitor Cells Increases Adhesion: Implications for Vascular Repair

    PubMed Central

    Mythreye, Karthikeyan; Satterwhite, Lisa L.; Davidson, W. Sean; Goldschmidt-Clermont, Pascal J.

    2008-01-01

    Transgenic over expression of apolipoprotein A-I (ApoA-I) the major structural apolipoprotein of HDL appears to convey the most consistent and strongest anti atherogenic effect observed in animal models so far. We tested the hypothesis that ApoA-I mediates its cardio protective effects additionally through ApoA-I induced differentiation of bone marrow derived progenitor cells in vitro. This study demonstrates that lineage negative bone marrow cells (lin−BMCs) alter and differentiate in response to free ApoA-I. We find that lin−BMCs in culture treated with recombinant free ApoA-I at a concentration of 0.4µM are twice as large in size and have altered cell morphology compared to untreated cells; untreated cells retain the original spheroid morphology. Further, the total number of CD31 positive cells in the ApoA-I treated population consistently increased by two fold. This phenotype was significantly reduced in untreated cells and points towards a novel ApoA-I dependent differentiation. A protein lacking its best lipid-binding region (ApoA-IΔ10) did not stimulate any changes in the lin−BMCs cells indicating that ApoA-I may mediate its effects by regulating cholesterol efflux. The increased CD31 correlates with an increased ability of the lin−BMCs to adhere to both fibronectin and Mouse Brain Endothelial Cells. Our results provide the first evidence that exogenous free ApoA-I has the capacity to change the characteristics of progenitor cell populations and suggests a novel mechanism by which HDL may mediate its cardiovascular benefits. PMID:18775511

  10. TNF-TNFR2/p75 Signaling Inhibits Early and Increases Delayed Nontargeted Effects in Bone Marrow-derived Endothelial Progenitor Cells*

    PubMed Central

    Sasi, Sharath P.; Song, Jin; Park, Daniel; Enderling, Heiko; McDonald, J. Tyson; Gee, Hannah; Garrity, Brittany; Shtifman, Alexander; Yan, Xinhua; Walsh, Kenneth; Natarajan, Mohan; Kishore, Raj; Goukassian, David A.

    2014-01-01

    TNF-α, a pro-inflammatory cytokine, is highly expressed after being irradiated (IR) and is implicated in mediating radiobiological bystander responses (RBRs). Little is known about specific TNF receptors in regulating TNF-induced RBR in bone marrow-derived endothelial progenitor cells (BM-EPCs). Full body γ-IR WT BM-EPCs showed a biphasic response: slow decay of p-H2AX foci during the initial 24 h and increase between 24 h and 7 days post-IR, indicating a significant RBR in BM-EPCs in vivo. Individual TNF receptor (TNFR) signaling in RBR was evaluated in BM-EPCs from WT, TNFR1/p55KO, and TNFR2/p75KO mice, in vitro. Compared with WT, early RBR (1–5 h) were inhibited in p55KO and p75KO EPCs, whereas delayed RBR (3–5 days) were amplified in p55KO EPCs, suggesting a possible role for TNFR2/p75 signaling in delayed RBR. Neutralizing TNF in γ-IR conditioned media (CM) of WT and p55KO BM-EPCs largely abolished RBR in both cell types. ELISA protein profiling of WT and p55KO EPC γ-IR-CM over 5 days showed significant increases in several pro-inflammatory cytokines, including TNF-α, IL-1α (Interleukin-1 alpha), RANTES (regulated on activation, normal T cell expressed and secreted), and MCP-1. In vitro treatments with murine recombinant (rm) TNF-α and rmIL-1α, but not rmMCP-1 or rmRANTES, increased the formation of p-H2AX foci in nonirradiated p55KO EPCs. We conclude that TNF-TNFR2 signaling may induce RBR in naïve BM-EPCs and that blocking TNF-TNFR2 signaling may prevent delayed RBR in BM-EPCs, conceivably, in bone marrow milieu in general. PMID:24711449

  11. Oleate Abrogates Palmitate-Induced Lipotoxicity and Proinflammatory Response in Human Bone Marrow-Derived Mesenchymal Stem Cells and Osteoblastic Cells.

    PubMed

    Gillet, C; Spruyt, D; Rigutto, S; Dalla Valle, A; Berlier, J; Louis, C; Debier, C; Gaspard, N; Malaisse, W J; Gangji, V; Rasschaert, J

    2015-11-01

    Osteoporosis is a metabolic bone disease associated with unequilibrated bone remodeling resulting from decreased bone formation and/or increased bone resorption, leading to progressive bone loss. In osteoporotic patients, low bone mass is associated with an increase of bone marrow fat resulting from accumulation of adipocytes within the bone marrow. Marrow adipocytes are active secretory cells, releasing cytokines, adipokines and free fatty acids (FA) that influence the bone marrow microenvironment and alter the biology of neighboring cells. Therefore, we examined the effect of palmitate (Palm) and oleate (Ole), 2 highly prevalent FA in human organism and diet, on the function and survival of human mesenchymal stem cells (MSC) and MSC-derived osteoblastic cells. The saturated FA Palm exerted a cytotoxic action via initiation of endoplasmic reticulum stress and activation of the nuclear factor κB (NF-κB) and ERK pathways. In addition, Palm induced a proinflammatory response, as determined by the up-regulation of Toll-like receptor 4 expression as well as the increase of IL-6 and IL-8 expression and secretion. Moreover, we showed that MSC-derived osteoblastic cells were more sensitive to lipotoxicity than undifferentiated MSC. The monounsaturated FA Ole fully neutralized Palm-induced lipotoxicity by impairing activation of the pathways triggered by the saturated FA. Moreover, Ole promoted Palm detoxification by fostering its esterification into triglycerides and storage in lipid droplets. Altogether, our data showed that physiological concentrations of Palm and Ole differently modulated cell death and function in bone cells. We therefore propose that FA could influence skeletal health. PMID:26327577

  12. Effect of AGM and fetal liver-derived stromal cell lines on globin expression in adult baboon (P. anubis) bone marrow-derived erythroid progenitors.

    PubMed

    Lavelle, Donald; Vaitkus, Kestutis; Ruiz, Maria Armila; Ibanez, Vinzon; Kouznetsova, Tatiana; Saunthararajah, Yogen; Mahmud, Nadim; DeSimone, Joseph

    2012-01-01

    This study was performed to investigate the hypothesis that the erythroid micro-environment plays a role in regulation of globin gene expression during adult erythroid differentiation. Adult baboon bone marrow and human cord blood CD34+ progenitors were grown in methylcellulose, liquid media, and in co-culture with stromal cell lines derived from different developmental stages in identical media supporting erythroid differentiation to examine the effect of the micro-environment on globin gene expression. Adult progenitors express high levels of γ-globin in liquid and methylcellulose media but low, physiological levels in stromal cell co-cultures. In contrast, γ-globin expression remained high in cord blood progenitors in stromal cell line co-cultures. Differences in γ-globin gene expression between adult progenitors in stromal cell line co-cultures and liquid media required cell-cell contact and were associated with differences in rate of differentiation and γ-globin promoter DNA methylation. We conclude that γ-globin expression in adult-derived erythroid cells can be influenced by the micro-environment, suggesting new potential targets for HbF induction. PMID:22693559

  13. Effect of AGM and Fetal Liver-Derived Stromal Cell Lines on Globin Expression in Adult Baboon (P. anubis) Bone Marrow-Derived Erythroid Progenitors

    PubMed Central

    Lavelle, Donald; Vaitkus, Kestutis; Ruiz, Maria Armila; Ibanez, Vinzon; Kouznetsova, Tatiana; Saunthararajah, Yogen; Mahmud, Nadim; DeSimone, Joseph

    2012-01-01

    This study was performed to investigate the hypothesis that the erythroid micro-environment plays a role in regulation of globin gene expression during adult erythroid differentiation. Adult baboon bone marrow and human cord blood CD34+ progenitors were grown in methylcellulose, liquid media, and in co-culture with stromal cell lines derived from different developmental stages in identical media supporting erythroid differentiation to examine the effect of the micro-environment on globin gene expression. Adult progenitors express high levels of γ-globin in liquid and methylcellulose media but low, physiological levels in stromal cell co-cultures. In contrast, γ-globin expression remained high in cord blood progenitors in stromal cell line co-cultures. Differences in γ-globin gene expression between adult progenitors in stromal cell line co-cultures and liquid media required cell-cell contact and were associated with differences in rate of differentiation and γ-globin promoter DNA methylation. We conclude that γ-globin expression in adult-derived erythroid cells can be influenced by the micro-environment, suggesting new potential targets for HbF induction. PMID:22693559

  14. Estrogen receptor-α signaling in osteoblast progenitors stimulates cortical bone accrual

    PubMed Central

    Almeida, Maria; Iyer, Srividhya; Martin-Millan, Marta; Bartell, Shoshana M.; Han, Li; Ambrogini, Elena; Onal, Melda; Xiong, Jinhu; Weinstein, Robert S.; Jilka, Robert L.; O’Brien, Charles A.; Manolagas, Stavros C.

    2012-01-01

    The detection of estrogen receptor-α (ERα) in osteoblasts and osteoclasts over 20 years ago suggested that direct effects of estrogens on both of these cell types are responsible for their beneficial effects on the skeleton, but the role of ERα in osteoblast lineage cells has remained elusive. In addition, estrogen activation of ERα in osteoclasts can only account for the protective effect of estrogens on the cancellous, but not the cortical, bone compartment that represents 80% of the entire skeleton. Here, we deleted ERα at different stages of differentiation in murine osteoblast lineage cells. We found that ERα in osteoblast progenitors expressing Osterix1 (Osx1) potentiates Wnt/β-catenin signaling, thereby increasing proliferation and differentiation of periosteal cells. Further, this signaling pathway was required for optimal cortical bone accrual at the periosteum in mice. Notably, this function did not require estrogens. The osteoblast progenitor ERα mediated a protective effect of estrogens against endocortical, but not cancellous, bone resorption. ERα in mature osteoblasts or osteocytes did not influence cancellous or cortical bone mass. Hence, the ERα in both osteoblast progenitors and osteoclasts functions to optimize bone mass but at distinct bone compartments and in response to different cues. PMID:23221342

  15. Demonstration of early functional compromise of bone marrow derived hematopoietic progenitor cells during bovine neonatal pancytopenia through in vitro culture of bone marrow biopsies

    PubMed Central

    2012-01-01

    Background Bovine neonatal pancytopenia (BNP) is a syndrome characterised by thrombocytopenia associated with marked bone marrow destruction in calves, widely reported since 2007 in several European countries and since 2011 in New Zealand. The disease is epidemiologically associated with the use of an inactivated bovine virus diarrhoea (BVD) vaccine and is currently considered to be caused by absorption of colostral antibody produced by some vaccinated cows (“BNP dams”). Alloantibodies capable of binding to the leukocyte surface have been detected in BNP dams and antibodies recognising bovine MHC class I and β-2-microglobulin have been detected in vaccinated cattle. In this study, calves were challenged with pooled colostrum collected from BNP dams or from non-BNP dams and their bone marrow hematopoietic progenitor cells (HPC) cultured in vitro from sternal biopsies taken at 24 hours and 6 days post-challenge. Results Clonogenic assay demonstrated that CFU-GEMM (colony forming unit-granulocyte/erythroid/macrophage/megakaryocyte; pluripotential progenitor cell) colony development was compromised from HPCs harvested as early as 24 hour post-challenge. By 6 days post challenge, HPCs harvested from challenged calves failed to develop CFU-E (erythroid) colonies and the development of both CFU-GEMM and CFU-GM (granulocyte/macrophage) was markedly reduced. Conclusion This study suggests that the bone marrow pathology and clinical signs associated with BNP are related to an insult which compromises the pluripotential progenitor cell within the first 24 hours of life but that this does not initially include all cell types. PMID:23110710

  16. Spine Fusion Using Cell Matrix Composites Enriched in Bone Marrow-Derived Cells

    PubMed Central

    Nitto, Hironori; Matsukura, Yoichi; Boehm, Cynthia; Valdevit, Antonio; Kambic, Helen; Davros, William; Powell, Kimerly; Easley, Kirk

    2005-01-01

    Bone marrow-derived cells including osteoblastic progenitors can be concentrated rapidly from bone marrow aspirates using the surface of selected implantable matrices for selective cell attachment. Concentration of cells in this way to produce an enriched cellular composite graft improves graft efficacy. The current study was designed to test the hypothesis that the biologic milieu of a bone marrow clot will significantly improve the efficacy of such a graft. An established posterior spinal fusion model and cancellous bone matrix was used to compare an enriched cellular composite bone graft alone, bone matrix plus bone marrow clot, and an enriched bone matrix composite graft plus bone marrow clot. Union score, quantitative computed tomography, and mechanical testing were used to define outcome. The union score for the enriched bone matrix plus bone marrow clot composite was superior to the enriched bone matrix alone and the bone matrix plus bone marrow clot. The enriched bone matrix plus bone marrow clot composite also was superior to the enriched bone matrix alone in fusion volume and in fusion area. These data confirm that the addition of a bone marrow clot to an enriched cell-matrix composite graft results in significant improvement in graft performance. Enriched composite grafts prepared using this strategy provide a rapid, simple, safe, and inexpensive method for intraoperative concentration and delivery of bone marrow-derived cells and connective tissue progenitors that may improve the outcome of bone grafting. PMID:12567137

  17. In vitro interactions between rat bone marrow-derived endothelial progenitor cells and hepatic stellate cells: interaction between EPCs and HSCs.

    PubMed

    Liu, Feng; Liu, Zhi-da; Wu, Nan; Wang, Jiang-Hua; Zhang, Heng-Hui; Fei, Ran; Cong, Xu; Chen, Hong-song; Wei, Lai

    2013-08-01

    Transplantation of bone marrow (BM)-derived endothelial progenitor cells (EPCs) has been reported to improve liver fibrosis, but there is no direct evidence for the mechanism of improvement. We investigated the mechanism in vitro by coculturing BM-derived EPCs with activated hepatic stellate cells (HSCs) to mimic the hepatic environment. EPCs and HSCs were cultured alone and indirectly cocultured at a 1:1 ratio in a Transwell system. The characteristics of HSCs and EPCs were examined at different time points. An invasion assay showed the time-dependent effect on degradation of the extracellular matrix (ECM) layer in EPCs cultured alone. Real-time PCR and enzyme-linked immunosorbent assay analysis revealed that EPCs served as a source of matrix metalloproteinase-9 (MMP-9), and MMP-9 expression levels significantly increased during the 2 d of coculture. CFSE labeling showed that EPCs inhibited proliferation of HSCs. Annexin-V/PI staining, erminal deoxynucleotidyl transferase X-dUTP nick end labeling analysis, and (cleaved) caspase-3 activity revealed that EPCs promoted HSC apoptosis. However, the proliferation and apoptosis of EPCs were unaffected by cocultured HSCs. Coculturing increased the expression of inducible nitric oxide synthase, vascular endothelial growth factor, and hepatocyte growth factor (HGF) in EPCs, promoted differentiation of EPCs, and reduced the expression of types I and III collagens and transforming growth factor beta 1. Knockdown of HGF expression attenuated EPC-induced activation of HSC apoptosis and profibrotic ability. These findings demonstrated that BM-derived EPCs could degrade ECM, promoting activated HSC apoptosis, suppressing proliferation and profibrotic ability of activated HSCs. HGF secretion by EPCs plays a key role in inducing activated HSC apoptosis and HSC profibrotic ability. PMID:23722413

  18. Dynamics of bone marrow-derived endothelial progenitor cell/mesenchymal stem cell interaction in co-culture and its implications in angiogenesis

    SciTech Connect

    Aguirre, A.; Planell, J.A.; Engel, E.

    2010-09-17

    Research highlights: {yields} BM-EPCs and MSCs establish complex, self-organizing structures in co-culture. {yields} Co-culture decreases proliferation by cellular self-regulatory mechanisms. {yields} Co-cultured cells present an activated proangiogenic phenotype. {yields} qRT-PCR and cluster analysis identify new target genes playing important roles. -- Abstract: Tissue engineering aims to regenerate tissues and organs by using cell and biomaterial-based approaches. One of the current challenges in the field is to promote proper vascularization in the implant to prevent cell death and promote host integration. Bone marrow endothelial progenitor cells (BM-EPCs) and mesenchymal stem cells (MSCs) are bone marrow resident stem cells widely employed for proangiogenic applications. In vivo, they are likely to interact frequently both in the bone marrow and at sites of injury. In this study, the physical and biochemical interactions between BM-EPCs and MSCs in an in vitro co-culture system were investigated to further clarify their roles in vascularization. BM-EPC/MSC co-cultures established close cell-cell contacts soon after seeding and self-assembled to form elongated structures at 3 days. Besides direct contact, cells also exhibited vesicle transport phenomena. When co-cultured in Matrigel, tube formation was greatly enhanced even in serum-starved, growth factor free medium. Both MSCs and BM-EPCs contributed to these tubes. However, cell proliferation was greatly reduced in co-culture and morphological differences were observed. Gene expression and cluster analysis for wide panel of angiogenesis-related transcripts demonstrated up-regulation of angiogenic markers but down-regulation of many other cytokines. These data suggest that cross-talk occurs in between BM-EPCs and MSCs through paracrine and direct cell contact mechanisms leading to modulation of the angiogenic response.

  19. Fms-like tyrosine kinase 3 ligand is required for thymic dendritic cell generation from bone marrow-derived CD117⁺ hematopoietic progenitor cells.

    PubMed

    Xu, Yunyun; Jiang, Dong; Hu, Yizhou; Li, Yiping; Zhang, Xueguang; Wang, Jian; Wang, Yong

    2015-11-01

    Thymic dendritic cells (TDCs) are a type of dendritic cell (DC) in the thymus, which can enhance the proliferation of thymic T lymphocytes, regulate negative selection and induce central tolerance through autoantigen presentation. However, further investigations using TDCs has been restricted due to insufficient numbers. Therefore, an effective expansion method for TDCs in vitro is urgently required to further examine their biological characteristics. In the present study, a novel system was established using fetal thymus organ culture (FTOC) and a hanging drop culture system in the presence of fms‑like tyrosine kinase 3 ligand (Flt3L), termed the Flt3L/FTOC system. TDCs were successfully generated and expanded from CD117+ bone marrow hematopoietic progenitor cells. Conventional DCs (cDCs; CD11c+B220‑ DCs) and plasmacytoid DCs (pDCs; CD11c+B220+ DCs) were found in the TDCs generated using the Flt3L/FTOC system. These cells exhibited the specific morphological features of DCs, which were confirmed using Giemsa staining. Furthermore, the cytokine and surface marker profiles were also analyzed. Higher expression levels of interferon‑α and interleukin‑12 were observed in the pDCs, compared with the cDCs, and higher expression levels of toll‑like receptor (TLR)7 and TLR9 were found in the pDCs than in the cDCs. In addition, the Flt3L/FTOC‑derived TDCs also exhibited the ability to stimulate the allogenic T cell response. In conclusion, a novel in vitro culture system of thymic cDCs and pDCs using Flt3L was established, and this may provide a methodological basis for understanding the properties of TDCs. PMID:26397863

  20. Assessment of Methods for Rapid Intraoperative Concentration and Selection of Marrow-Derived Connective Tissue Progenitors for Bone Regeneration Using the Canine Femoral Multidefect Model.

    PubMed

    Luangphakdy, Viviane; Boehm, Cynthia; Pan, Hui; Herrick, James; Zaveri, Phil; Muschler, George F

    2016-01-01

    Treatment of large bone defects remains an unsolved clinical challenge, despite a wide array of existing bone graft materials and strategies. Local deficiency in osteogenic connective tissue progenitors (CTP-Os) due to tissue loss is one of the central biological barriers to bone regeneration. Density separation (DS) and selective retention (SR) represent two promising methods that can be used intraoperatively to rapidly concentrate cells and potentially select CTP-Os. This project was designed to compare DS and SR using the canine femoral multidefect (CFMD) model. Mineralized cancellous allograft (MCA) was used as a standardized scaffold for cell transplantation. Two experiments were performed using a cohort of six animals in each comparison. In Cohort I, unprocessed bone marrow aspirate (BMA) clot was compared to DS processing. MCA combined with raw BMA or DS processed cells produced a robust and advanced stage of bone regeneration throughout the defect in 4 weeks with reconstitution of hematopoietic marrow. However, the retention of DS processed cells and CTP-Os in the MCA matrix was low compared to BMA clot. In Cohort II, MCA with DS-T cells (addition of calcium chloride thrombin to induce clotting and enhance cell and CTP-O retention) was compared to MCA with SR cells. A mean of 276 ± 86 million nucleated cells and 29,030 ± 10,510 CTP-Os were implanted per defect in the DS-T group. A mean of 76 ± 42 million nucleated cells and 30,266 ± 15,850 CTP-Os were implanted in the SR group. Bone formation was robust and not different between treatments. Histologically, both groups demonstrated regeneration of hematopoietic marrow tissue. However, SR sites contained more hematopoietic vascular tissues, less fibrosis, and less residual allograft, particularly in the intramedullary cavity, suggesting a more advanced stage of remodeling (p = 0.04). These data demonstrate excellent overall performance of DS and SR processing methods. Both methods achieve a bone

  1. Morphological and proteomic analysis of early stage of osteoblast differentiation in osteoblastic progenitor cells

    SciTech Connect

    Hong, Dun; Chen, Hai-Xiao; Yu, Hai-Qiang; Liang, Yong; Wang, Carrie; Lian, Qing-Quan; Deng, Hai-Teng; Ge, Ren-Shan

    2010-08-15

    Bone remodeling relies on a dynamic balance between bone formation and resorption, mediated by osteoblasts and osteoclasts, respectively. Under certain stimuli, osteoprogenitor cells may differentiate into premature osteoblasts and further into mature osteoblasts. This process is marked by increased alkaline phosphatase (ALP) activity and mineralized nodule formation. In this study, we induced osteoblast differentiation in mouse osteoprogenitor MC3T3-E1 cells and divided the process into three stages. In the first stage (day 3), the MC3T3-E1 cell under osteoblast differentiation did not express ALP or deposit a mineralized nodule. In the second stage, the MC3T3-E1 cell expressed ALP but did not form a mineralized nodule. In the third stage, the MC3T3-E1 cell had ALP activity and formed mineralized nodules. In the present study, we focused on morphological and proteomic changes of MC3T3-E1 cells in the early stage of osteoblast differentiation - a period when premature osteoblasts transform into mature osteoblasts. We found that mean cell area and mean stress fiber density were increased in this stage due to enhanced cell spreading and decreased cell proliferation. We further analyzed the proteins in the signaling pathway of regulation of the cytoskeleton using a proteomic approach and found upregulation of IQGAP1, gelsolin, moesin, radixin, and Cfl1. After analyzing the focal adhesion signaling pathway, we found the upregulation of FLNA, LAMA1, LAMA5, COL1A1, COL3A1, COL4A6, and COL5A2 as well as the downregulation of COL4A1, COL4A2, and COL4A4. In conclusion, the signaling pathway of regulation of the cytoskeleton and focal adhesion play critical roles in regulating cell spreading and actin skeleton formation in the early stage of osteoblast differentiation.

  2. Cathepsin X is secreted by human osteoblasts, digests CXCL-12 and impairs adhesion of hematopoietic stem and progenitor cells to osteoblasts

    PubMed Central

    Staudt, Nicole D.; Aicher, Wilhelm K.; Kalbacher, Hubert; Stevanovic, Stefan; Carmona, Adriana K.; Bogyo, Matthew; Klein, Gerd

    2010-01-01

    Background Hematopoietic stem cells are retained within discrete bone marrow niches through the effects of cell adhesion molecules and chemokine gradients. However, a small proportion of hematopoietic stem cells can also be found trafficking in the peripheral blood. During induced stem cell mobilization a proteolytic microenvironment is generated, but whether proteases are also involved in physiological trafficking of hematopoietic stem cells is not known. In the present study we examined the expression, secretion and function of the cysteine protease cathepsin X by cells of the human bone marrow. Design and Methods Human osteoblasts, bone marrow stromal cells and hematopoietic stem and progenitor cells were analyzed for the secretion of cathepsin X by western blotting, active site labeling, immunofluorescence staining and activity assays. A possible involvement of cathepsin X in cell adhesion and CXCL-12-mediated cell migration was studied in functional assays. Matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) analysis revealed the digestion mechanism of CXCL-12 by cathepsin X. Results Osteoblasts and stromal cells secrete cathepsin X, whereas hematopoietic stem and progenitor cells do not. Using a cathepsin X-selective substrate, we detected the catalytic activity of cathepsin X in cell culture supernatants of osteoblasts. Activated cathepsin X is able to reduce cellular adhesive interactions between CD34+ hematopoietic stem and progenitor cells and adherent osteoblasts. The chemokine CXCL-12, a highly potent chemoattractant for hematopoietic stem cells secreted by osteoblasts, is readily digested by cathepsin X. Conclusions The exo-peptidase cathepsin X has been identified as a new member of the group of CXCL-12-degrading enzymes secreted by non-hematopoietic bone marrow cells. Functional data indicate that cathepsin X can influence hematopoietic stem and progenitor cell trafficking in the bone marrow. PMID:20494937

  3. Impact of parathyroid hormone on bone marrow-derived stem cell mobilization and migration.

    PubMed

    Huber, Bruno C; Grabmaier, Ulrich; Brunner, Stefan

    2014-11-26

    Parathyroid hormone (PTH) is well-known as the principal regulator of calcium homeostasis in the human body and controls bone metabolism via actions on the survival and activation of osteoblasts. The intermittent administration of PTH has been shown to stimulate bone production in mice and men and therefore PTH administration has been recently approved for the treatment of osteoporosis. Besides to its physiological role in bone remodelling PTH has been demonstrated to influence and expand the bone marrow stem cell niche where hematopoietic stem cells, capable of both self-renewal and differentiation, reside. Moreover, intermittent PTH treatment is capable to induce mobilization of progenitor cells from the bone marrow into the bloodstream. This novel function of PTH on modulating the activity of the stem cell niche in the bone marrow as well as on mobilization and regeneration of bone marrow-derived stem cells offers new therapeutic options in bone marrow and stem cell transplantation as well as in the field of ischemic disorders. PMID:25426261

  4. Involvement of marrow-derived endothelial cells in vascularization.

    PubMed

    Larrivée, B; Karsan, A

    2007-01-01

    Until recently, the adult neovasculature was thought to arise only through angiogenesis, the mechanism by which new blood vessels form from preexisting vessels through endothelial cell migration and proliferation. However, recent studies have provided evidence that postnatal neovasculature can also arise though vasculogenesis, a process by which endothelial progenitor cells are recruited and differentiate into mature endothelial cells to form new blood vessels. Evidence for the existence of endothelial progenitors has come from studies demonstrating the ability of bone marrow-derived cells to incorporate into adult vasculature. However, the exact nature of endothelial progenitor cells remains controversial. Because of the lack of definitive markers of endothelial progenitors, the in vivo contribution of progenitor cells to physiological and pathological neovascularization remains unclear. Early studies reported that endothelial progenitor cells actively integrate into the adult vasculature and are critical in the development of many types of vascular-dependent disorders such as neoplastic progression. Moreover, it has been suggested that endothelial progenitor cells can be used as a therapeutic strategy aimed at promoting vascular growth in a variety of ischemic diseases. However, increasing numbers of studies have reported no clear contribution of endothelial progenitors in physiological or pathological angiogenesis. In this chapter, we discuss the origin of the endothelial progenitor cell in the embryo and adult, and we discuss the cell's link to the primitive hematopoietic stem cell. We also review the potential significance of endothelial progenitor cells in the formation of a postnatal vascular network and discuss the factors that may account for the current lack of consensus of the scientific community on this important issue. PMID:17554506

  5. Deletion of BMP receptor type IB decreased bone mass in association with compromised osteoblastic differentiation of bone marrow mesenchymal progenitors.

    PubMed

    Shi, Ce; Iura, Ayaka; Terajima, Masahiko; Liu, Fei; Lyons, Karen; Pan, Haichun; Zhang, Honghao; Yamauchi, Mitsuo; Mishina, Yuji; Sun, Hongchen

    2016-01-01

    We previously found that disruption of two type I BMP receptors, Bmpr1a and Acvr1, respectively, in an osteoblast-specific manner, increased bone mass in mice. BMPR1B, another BMP type I receptor, is also capable of binding to BMP ligands and transduce BMP signaling. However, little is known about the function of BMPR1B in bone. In this study, we investigated the bone phenotype in Bmpr1b null mice and the impacts of loss of Bmpr1b on osteoblasts and osteoclasts. We found that deletion of Bmpr1b resulted in osteopenia in 8-week-old male mice, and the phenotype was transient and gender specific. The decreased bone mass was neither due to the changes in osteoblastic bone formation activity nor osteoclastic bone resorption activity in vivo. In vitro differentiation of Bmpr1b null osteoclasts was increased but resorption activity was decreased. Calvarial pre-osteoblasts from Bmpr1b mutant showed comparable differentiation capability in vitro, while they showed increased BMP-SMAD signaling in culture. Different from calvarial pre-osteoblasts, Bmpr1b mutant bone marrow mesenchymal progenitors showed compromised differentiation in vitro, which may be a reason for the osteopenic phenotype in the mutant mice. In conclusion, our results suggested that BMPR1B plays distinct roles from BMPR1A and ACVR1 in maintaining bone mass and transducing BMP signaling. PMID:27048979

  6. Deletion of BMP receptor type IB decreased bone mass in association with compromised osteoblastic differentiation of bone marrow mesenchymal progenitors

    PubMed Central

    Shi, Ce; Iura, Ayaka; Terajima, Masahiko; Liu, Fei; Lyons, Karen; Pan, Haichun; Zhang, Honghao; Yamauchi, Mitsuo; Mishina, Yuji; Sun, Hongchen

    2016-01-01

    We previously found that disruption of two type I BMP receptors, Bmpr1a and Acvr1, respectively, in an osteoblast-specific manner, increased bone mass in mice. BMPR1B, another BMP type I receptor, is also capable of binding to BMP ligands and transduce BMP signaling. However, little is known about the function of BMPR1B in bone. In this study, we investigated the bone phenotype in Bmpr1b null mice and the impacts of loss of Bmpr1b on osteoblasts and osteoclasts. We found that deletion of Bmpr1b resulted in osteopenia in 8-week-old male mice, and the phenotype was transient and gender specific. The decreased bone mass was neither due to the changes in osteoblastic bone formation activity nor osteoclastic bone resorption activity in vivo. In vitro differentiation of Bmpr1b null osteoclasts was increased but resorption activity was decreased. Calvarial pre-osteoblasts from Bmpr1b mutant showed comparable differentiation capability in vitro, while they showed increased BMP-SMAD signaling in culture. Different from calvarial pre-osteoblasts, Bmpr1b mutant bone marrow mesenchymal progenitors showed compromised differentiation in vitro, which may be a reason for the osteopenic phenotype in the mutant mice. In conclusion, our results suggested that BMPR1B plays distinct roles from BMPR1A and ACVR1 in maintaining bone mass and transducing BMP signaling. PMID:27048979

  7. Lithium-end-capped polylactide thin films influence osteoblast progenitor cell differentiation and mineralization.

    PubMed

    Gomillion, Cheryl T; Lakhman, Rubinder Kaur; Kasi, Rajeswari M; Weiss, R A; Kuhn, Liisa T; Goldberg, A Jon

    2015-02-01

    End-capping by covalently binding functional groups to the ends of polymer chains offers potential advantages for tissue engineering scaffolds, but the ability of such polymers to influence cell behavior has not been studied. As a demonstration, polylactide (PLA) was end-capped with lithium carboxylate ionic groups (hPLA13kLi) and evaluated. Thin films of the hPLA13kLi and PLA homopolymer were prepared with and without surface texturing. Murine osteoblast progenitor cells from collagen 1α1 transgenic reporter mice were used to assess cell attachment, proliferation, differentiation, and mineralization. Measurement of green fluorescent protein expressed by these cells and xylenol orange staining for mineral allowed quantitative analysis. The hPLA13kLi was biologically active, increasing initial cell attachment and enhancing differentiation, while reducing proliferation and strongly suppressing mineralization, relative to PLA. These effects of bound lithium ions (Li(+) ) had not been previously reported, and were generally consistent with the literature on soluble additions of lithium. The surface texturing generated here did not influence cell behavior. These results demonstrate that end-capping could be a useful approach in scaffold design, where a wide range of biologically active groups could be employed, while likely retaining the desirable characteristics associated with the unaltered homopolymer backbone. PMID:24733780

  8. Identification of a novel bone marrow-derived B-cell progenitor population that coexpresses B220 and Thy-1 and is highly enriched for Abelson leukemia virus targets.

    PubMed Central

    Tidmarsh, G F; Heimfeld, S; Whitlock, C A; Weissman, I L; Müller-Sieburg, C E

    1989-01-01

    A novel stage in early B-lymphocyte differentiation has been identified in normal mouse bone marrow cells. Earlier work had demonstrated that bone marrow cells characterized by low levels of Thy-1 and lack of a panel of lineage markers (Thy-1lo Lin- cells) were highly enriched for pluripotent hematopoietic stem cells. In this paper, we present evidence that another bone marrow population, which expressed low levels of Thy-1 and coexpressed B220, a B-lineage-specific form of the leukocyte common antigen, contained early and potent precursors for B lymphocytes upon in vivo transfer to irradiated hosts. These Thy-1lo B220+ cells, comprising 1 to 2% of bone marrow cells, were enriched for large cells in the mitotic cycle; the population lacked significant pluripotent hematopoietic stem cell activity and myeloid-erythroid progenitors. Most strikingly, Thy-1lo B220+ cells represented a highly enriched population of bone marrow cells that could be targets of Abelson murine leukemia virus transformation. We propose that Thy-1lo B220+ bone marrow cells represent the earliest stage of committed lymphocyte progenitors, intermediate in differentiation between Thy-1lo Lin- pluripotent stem cells and, in the B lineage, Thy-1- B220+ pre-B cells. Images PMID:2474759

  9. Bee Venom Accelerates Wound Healing in Diabetic Mice by Suppressing Activating Transcription Factor-3 (ATF-3) and Inducible Nitric Oxide Synthase (iNOS)-Mediated Oxidative Stress and Recruiting Bone Marrow-Derived Endothelial Progenitor Cells.

    PubMed

    Badr, Gamal; Hozzein, Wael N; Badr, Badr M; Al Ghamdi, Ahmad; Saad Eldien, Heba M; Garraud, Olivier

    2016-10-01

    Multiple mechanisms contribute to impaired diabetic wound healing including impaired neovascularization and deficient endothelial progenitor cell (EPC) recruitment. Bee venom (BV) has been used as an anti-inflammatory agent for the treatment of several diseases. Nevertheless, the effect of BV on the healing of diabetic wounds has not been studied. Therefore, in this study, we investigated the impact of BV on diabetic wound closure in a type I diabetic mouse model. Three experimental groups were used: group 1, non-diabetic control mice; group 2, diabetic mice; and group 3, diabetic mice treated with BV. We found that the diabetic mice exhibited delayed wound closure characterized by a significant decrease in collagen production and prolonged elevation of inflammatory cytokines levels in wounded tissue compared to control non-diabetic mice. Additionally, wounded tissue in diabetic mice revealed aberrantly up-regulated expression of ATF-3 and iNOS followed by a marked elevation in free radical levels. Impaired diabetic wound healing was also characterized by a significant elevation in caspase-3, -8, and -9 activity and a marked reduction in the expression of TGF-β and VEGF, which led to decreased neovascularization and angiogenesis of the injured tissue by impairing EPC mobilization. Interestingly, BV treatment significantly enhanced wound closure in diabetic mice by increasing collagen production and restoring the levels of inflammatory cytokines, free radical, TGF-β, and VEGF. Most importantly, BV-treated diabetic mice exhibited mobilized long-lived EPCs by inhibiting caspase activity in the wounded tissue. Our findings reveal the molecular mechanisms underlying improved diabetic wound healing and closure following BV treatment. J. Cell. Physiol. 231: 2159-2171, 2016. © 2016 Wiley Periodicals, Inc. PMID:26825453

  10. Differentiation and regeneration potential of mesenchymal progenitor cells derived from traumatized muscle tissue.

    PubMed

    Jackson, Wesley M; Lozito, Thomas P; Djouad, Farida; Kuhn, Nastaran Z; Nesti, Leon J; Tuan, Rocky S

    2011-11-01

    Mesenchymal stem cell (MSC) therapy is a promising approach to promote tissue regeneration by either differentiating the MSCs into the desired cell type or by using their trophic functions to promote endogenous tissue repair. These strategies of regenerative medicine are limited by the availability of MSCs at the point of clinical care. Our laboratory has recently identified multipotent mesenchymal progenitor cells (MPCs) in traumatically injured muscle tissue, and the objective of this study was to compare these cells to a typical population of bone marrow derived MSCs. Our hypothesis was that the MPCs exhibit multilineage differentiation and expression of trophic properties that make functionally them equivalent to bone marrow derived MSCs for tissue regeneration therapies. Quantitative evaluation of their proliferation, metabolic activity, expression of characteristic cell-surface markers and baseline gene expression profile demonstrate substantial similarity between the two cell types. The MPCs were capable of differentiation into osteoblasts, adipocytes and chondrocytes, but they appeared to demonstrate limited lineage commitment compared to the bone marrow derived MSCs. The MPCs also exhibited trophic (i.e. immunoregulatory and pro-angiogenic) properties that were comparable to those of MSCs. These results suggest that the traumatized muscle derived MPCs may not be a direct substitute for bone marrow derived MSCs. However, because of their availability and abundance, particularly following orthopaedic injuries when traumatized muscle is available to harvest autologous cells, MPCs are a promising cell source for regenerative medicine therapies designed to take advantage of their trophic properties. PMID:21129154

  11. Proliferative activity of vervet monkey bone marrow-derived adherent cells

    SciTech Connect

    Kramvis, A.; Garnett, H.M.

    1987-11-01

    Vervet monkey bone marrow-derived adherent cell population cultured in Fischer's medium supplemented with 12.5% fetal calf serum and 12.5% horse serum consists of two cell shapes: fusiform (type I) and polygonal (type II). Limiting-dilution cloning of the cells suggested that the two morphologically distinct cell types belong to the same cellular system even though they differ in their proliferative capabilities. The labeling index of type II cells, as measured by autoradiography, was found to be consistently lower than that of type I cells. It is probable that these two phenotypes represent different stages of differentiation, where progenitor type I gives rise to type II cells. The bone marrow-derived adherent cells were found to be cytokinetically at rest in vivo, using the thymidine suicide test, and relatively radioresistant with a D0 = 2.1 Gy and n = 2.36 at the time of explantation from the bone. Furthermore, in culture these cells are characterized by a relatively long cell cycle of 60 h, where the length of the S phase is 30 h, G2 is 12 h, M is 6 h, and G1 is 12 h. Thus, the vervet monkey bone marrow-derived adherent cells represent a cell population with a low turnover rate both in vivo and in vitro.

  12. The fate of transplanted xenogeneic bone marrow-derived stem cells in rat intervertebral discs.

    PubMed

    Wei, Aiqun; Tao, Helen; Chung, Sylvia A; Brisby, Helena; Ma, David D; Diwan, Ashish D

    2009-03-01

    Intervertebral disc degeneration is a major cause and a risk factor for chronic low back pain. The potential of using stem cells to treat disc degeneration has been raised. The aims of our study were to assess whether xenogeneic bone-marrow derived stem cells could survive in a rat disc degeneration model and to determine which cell types, if any, survived and differentiated into disc-like cells. Human bone-marrow derived CD34(+) (hematopoietic progenitor cells) and CD34(-) (nonhematopoietic progenitor cells, including mesenchymal stem cells) cells were isolated, fluorescent-labeled, and injected into rat coccygeal discs. The rats were sacrificed at day 1, 10, 21, and 42. Treated discs were examined by histological and immunostaining techniques and compared to control discs. The survival of transplanted cells was further confirmed with a human nuclear specific marker. Fluorescent labeled CD34(-) cells were detected until day 42 in the nucleus pulposus of the injected discs. After 3 weeks these cells had differentiated into cells expressing chondrocytic phenotype (Collagen II and Sox-9). In contrast, the fluorescent labeled CD34(+) cells could not be detected after day 21. No fluorescence-positive cells were detected in the noninjected control discs. Further, no inflammatory cells infiltrated the nucleus pulposus, even though these animals had not received immunosuppressive treatment. Our data provide evidence that transplanted human BM CD34(-) cells survived and differentiated within the relative immune privileged nucleus pulposus of intervertebral disc degeneration. PMID:18853431

  13. Bone Marrow-Derived Cells in the Pathogenesis of Lung Fibrosis

    PubMed Central

    Moore, Bethany B.; Thannickal, Victor J.; Toews, Galen B.

    2016-01-01

    Progressive pulmonary fibrosis is characterized by failed alveolar reepithelialization and fibroblast/myofibroblast accumulation, with deposition of extracellular matrix. This results in loss of lung elasticity, alveolar collapse and fibrosis, impaired gas exchange and progressive decline in pulmonary function. Myofibroblasts represent an activated, contractile cellular phenotype that are potent producers of collagen and other extracellular matrix proteins. It is generally thought that myofibroblasts derive from local tissue fibroblasts. However, recent evidence suggests a portion of the progenitors for these cells may arise from the bone marrow. Fibrocytes, which share both leukocyte and mesenchymal markers, are found in increased numbers in bone marrow and lung of injured mice. Fibrocytes circulate in blood and are recruited to injured sites via chemotactic signals. Studies with bone marrow chimeric and parabiotic mice suggest that fibroblasts (and in some cases myofibroblasts) arise from circulating bone marrow precursors. Chemokine and chemokine receptor interactions are critical for the recruitment of bone marrow-derived progenitors. Once fibrocytes arrive in injured tissues, local factors induce their differentiation into fibroblasts/myofibroblasts. This review will summarize the experimental findings, supporting a role for the participation of bone marrow-derived cells in animal models of lung fibrosis, and potential implications for the pathogenesis of fibrotic lung diseases.

  14. Bone marrow-derived Kruppel-like Factor 10 Controls Re-endothelialization in Response to Arterial Injury

    PubMed Central

    Wara, Akm Khyrul; Manica, Andre; Marchini, Julio F.; Sun, Xinghui; Icli, Basak; Tesmenitsky, Yevgenia; Croce, Kevin; Feinberg, Mark W.

    2013-01-01

    Objective The objective of this study was to investigate the role of Kruppel-like factor (KLF) 10, a zinc-finger transcription factor, in bone marrow-derived cell responses to arterial endothelial injury. Accumulating evidence indicates that bone marrow-derived progenitors are recruited to sites of vascular injury and contribute to endothelial repair. Approach and Results In response to carotid artery endothelial denudation, KLF10 mRNA expression was markedlyincreased in both bone marrow and circulating lin− progenitor cells. To examine the specific role for KLF10 in arterial re-endothelialization, we used two models of endothelial denudation (wire- and thermal-induced injury) of the carotid artery in WT and KLF10−/− mice. WT mice displayed higher areas of re-endothelialization compared to KLF10−/− mice following endothelial injury using either method. Bone marrow (BM) transplant studies revealed that re-constitution of KLF10−/− mice with WT BM fully rescued the defect in re-endothelialization and increased lin−CD34+KDR+ progenitors in the blood and injured carotid arteries. Conversely, reconstitution of WT mice with KLF10−/−BM re-capitulated the defects in re-endothelialization and peripheral cell progenitors. The media from cultured KLF10−/− BM progenitors was markedly inefficient at promoting endothelial cell growth and migration compared to the media from WT progenitors, indicative of defective paracrine trophic effects from KLF10−/− BM progenitors. Finally, BM-derived KLF10−/− lin− progenitors from reconstituted mice had reduced CXCR4 expression and impaired migratory responses. Conclusions Collectively, these observations demonstrate a protective role for BM-derived KLF10 in paracrine and homing responses important to arterial endothelial injury and highlight KLF10 as a possible therapeutic target to promote endothelial repair in vascular disease states. PMID:23685559

  15. Selective Retention of Bone Marrow-Derived Cells to Enhance Spinal Fusion

    PubMed Central

    Matsukura, Yoichi; Nitto, Hironori; Boehm, Cynthia A.; Valdevit, Antonio D.; Kambic, Helen E.; Davros, William J.; Easley, Kirk A.; Powell, Kimerly A.

    2005-01-01

    Connective tissue progenitors can be concentrated rapidly from fresh bone marrow aspirates using some porous matrices as a surface for cell attachment and selective retention, and for creating a cellular graft that is enriched with respect to the number of progenitor cells. We evaluated the potential value of this method using demineralized cortical bone powder as the matrix. Matrix alone, matrix plus marrow, and matrix enriched with marrow cells were compared in an established canine spinal fusion model. Fusions were compared based on union score, fusion mass, fusion volume, and by mechanical testing. Enriched matrix grafts delivered a mean of 2.3 times more cells and approximately 5.6 times more progenitors than matrix mixed with bone marrow. The union score with enriched matrix was superior to matrix alone and matrix plus marrow. Fusion volume and fusion area also were greater with the enriched matrix. These data suggest that the strategy of selective retention provides a rapid, simple, and effective method for concentration and delivery of marrow-derived cells and connective tissue progenitors that may improve the outcome of bone grafting procedures in various clinical settings. PMID:15738828

  16. Repopulation of the Irradiation Damaged Lung with Bone Marrow-derived Cells

    PubMed Central

    Bernard, Mark E.; Kim, Hyun; Rajagopalan, Malolan S.; Stone, Brandon; Salimi, Umar; Rwigema, Jean-Claude; Epperly, Michael W.; Shen, Hongmei; Goff, Julie P.; Franicola, Darcy; Dixon, Tracy; Cao, Shaonan; Zhang, Xichen; Wang, Hong; Stolz, Donna B.; Greenberger, Joel S.

    2012-01-01

    Aim The effect of lung irradiation on reduction of lung stem cells and repopulation with bone marrow-derived cells was measured. Materials and Methods Expression of green fluorescent protein positive cells (GFP+) in the lungs of thoracic irradiated FVB/NHsd mice (Harlan Sprague Dawley, Indianapolis, IN, USA) was determined. This was compared to the repopulation of bone marrow-derived cells found in the lungs from naphthalene treated male FVB/NHsd mice and gangciclovir (GVC) treated FeVBN GFP+ male marrow chimeric HSV-TK-CCSP. The level of mRNA for lung stem cell markers clara cell (CCSP), epithelium 1 (FOXJ1) and surfactant protein C (SP-C), and sorted single cells positive for marrow origin epithelial cells (GFP+ CD45−) was measured. Results The expression of pulmonary stem cells as determined by PCR was reduced most by GCV, then naphthalene, and least by thoracic irradiation. Irradiation, like GCV, reduced mRNA expression of CCSP, CYP2F2, and FOXJ1, while naphthalene reduced that of CCSP and CYP2F2. Ultrastructural analysis showed GFP+ pulmonary cells of bone marrow origin, with the highest frequency being found in GCV-treated groups. Conclusion Bone marrow progenitor cells may not participate in the repopulation of the lung following irradiation. PMID:22210711

  17. Extracellular calcium (Ca2+(o))-sensing receptor in a murine bone marrow-derived stromal cell line (ST2): potential mediator of the actions of Ca2+(o) on the function of ST2 cells

    NASA Technical Reports Server (NTRS)

    Yamaguchi, T.; Chattopadhyay, N.; Kifor, O.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

    1998-01-01

    The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2+(o)) homeostasis by mediating the actions of Ca2+(o) on parathyroid gland and kidney. Bone marrow stromal cells support the formation of osteoclasts from their progenitors as well as the growth of hematopoietic stem cells by secreting humoral factors and through cell to cell contact. Stromal cells also have the capacity to differentiate into bone-forming osteoblasts. Bone resorption by osteoclasts probably produces substantial local increases in Ca2+(o) that could provide a signal for stromal cells in the immediate vicinity, leading us to determine whether such stromal cells express the CaR. In this study, we used the murine bone marrow-derived, stromal cell line, ST2. Both immunocytochemistry and Western blot analysis, using an antiserum specific for the CaR, detected CaR protein in ST2 cells. We also identified CaR transcripts in ST2 cells by Northern analysis using a CaR-specific probe and by RT-PCR with CaR-specific primers, followed by nucleotide sequencing of the amplified products. Exposure of ST2 cells to high Ca2+(o) (4.8 mM) or to the polycationic CaR agonists, neomycin (300 microM) or gadolinium (100 microM), stimulated both chemotaxis and DNA synthesis in ST2 cells. Therefore, taken together, our data strongly suggest that the bone marrow-derived stromal cell line, ST2, possesses both CaR protein and messenger RNA that are very similar if not identical to those in parathyroid and kidney. Furthermore, as ST2 cells have the potential to differentiate into osteoblasts, the CaR in stromal cells could participate in bone turnover by stimulating the proliferation and migration of such cells to sites of bone resorption as a result of local, osteoclast-mediated release of Ca2+(o) and, thereafter, initiating bone formation after their differentiation into osteoblasts.

  18. Therapeutic potential of bone marrow-derived mesenchymal stem cells for cutaneous wound healing.

    PubMed

    Chen, Jerry S; Wong, Victor W; Gurtner, Geoffrey C

    2012-01-01

    Despite advances in wound care, many wounds never heal and become chronic problems that result in significant morbidity and mortality to the patient. Cellular therapy for cutaneous wounds has recently come under investigation as a potential treatment modality for impaired wound healing. Bone marrow-derived mesenchymal stem cells (MSCs) are a promising source of adult progenitor cells for cytotherapy as they are easy to isolate and expand and have been shown to differentiate into various cell lineages. Early studies have demonstrated that MSCs may enhance epithelialization, granulation tissue formation, and neovascularization resulting in accelerated wound closure. It is currently unclear if these effects are mediated through cellular differentiation or by secretion of cytokines and growth factors. This review discusses the proposed biological contributions of MSCs to cutaneous repair and their clinical potential in cell-based therapies. PMID:22787462

  19. Disruption of cell-matrix interactions by heparin enhances mesenchymal progenitor adipocyte differentiation

    SciTech Connect

    Luo Weijun; Shitaye, Hailu; Friedman, Michael; Bennett, Christina N.; Miller, Joshua; MacDougald, Ormond A.; Hankenson, Kurt D.

    2008-11-01

    Differentiation of marrow-derived mesenchymal progenitors to either the osteoblast or adipocyte lineage is reciprocally regulated. Factors that promote osteoblastogenesis inhibit adipogenesis, while adipogenic factors are inhibitory to osteoblast differentiation. Heparin, a soluble glycosaminoglycan, inhibits bone formation in vivo and osteoblast cell differentiation and function in vitro, and has been shown to promote adipocyte differentiation. To elucidate the role that heparin plays in the adipogenic induction of murine mesenchymal progenitors, we studied immortalized marrow stromal cells (IM-MSC), the MSC cell line, ST2, and 3T3L1 pre-adipocytes. Heparin alone was not sufficient to induce adipogenesis, but enhanced the induction under a variety of adipogenic cocktails. This effect was both dose- and time-dependent. Heparin showed a positive effect at concentrations > 0. 1 {mu}g/ml when applied before day 3 during the induction course. Heparin's effect on adipogenesis was independent of cell proliferation, cell density, and extracellular lipid. This effect is likely related to the unique structure of heparin because another polyanionic glycosaminoglycan, dextran sulfate, did not promote adipogenic differentiation. Heparin treatment altered morphology and adhesion characteristics of progenitor cells, resulting in cell rounding and aggregation. As well, heparin counteracted the known inhibitory effect of fibronectin on adipogenesis and decreased basal focal adhesion kinase and paxillin phosphorylation. We conclude that heparin-mediated disruption of cell-matrix adhesion enhances adipogenic potential.

  20. Epigenetic Plasticity Drives Adipogenic and Osteogenic Differentiation of Marrow-derived Mesenchymal Stem Cells.

    PubMed

    Meyer, Mark B; Benkusky, Nancy A; Sen, Buer; Rubin, Janet; Pike, J Wesley

    2016-08-19

    Terminal differentiation of multipotent stem cells is achieved through a coordinated cascade of activated transcription factors and epigenetic modifications that drive gene transcription responsible for unique cell fate. Within the mesenchymal lineage, factors such as RUNX2 and PPARγ are indispensable for osteogenesis and adipogenesis, respectively. We therefore investigated genomic binding of transcription factors and accompanying epigenetic modifications that occur during osteogenic and adipogenic differentiation of mouse bone marrow-derived mesenchymal stem cells (MSCs). As assessed by ChIP-sequencing and RNA-sequencing analyses, we found that genes vital for osteogenic identity were linked to RUNX2, C/EBPβ, retinoid X receptor, and vitamin D receptor binding sites, whereas adipocyte differentiation favored PPARγ, retinoid X receptor, C/EBPα, and C/EBPβ binding sites. Epigenetic marks were clear predictors of active differentiation loci as well as enhancer activities and selective gene expression. These marrow-derived MSCs displayed an epigenetic pattern that suggested a default preference for the osteogenic pathway; however, these patterns were rapidly altered near the Adipoq, Cidec, Fabp4, Lipe, Plin1, Pparg, and Cebpa genes during adipogenic differentiation. Surprisingly, we found that these cells also exhibited an epigenetic plasticity that enabled them to trans-differentiate from adipocytes to osteoblasts (and vice versa) after commitment, as assessed by staining, gene expression, and ChIP-quantitative PCR analysis. The osteogenic default pathway may be subverted during pathological conditions, leading to skeletal fragility and increased marrow adiposity during aging, estrogen deficiency, and skeletal unloading. Taken together, our data provide an increased mechanistic understanding of the epigenetic programs necessary for multipotent differentiation of MSCs that may prove beneficial in the development of therapeutic strategies. PMID:27402842

  1. Intracoronary infusion of a combination of bone marrow-derived stem cells in dogs

    PubMed Central

    Minguell, José J; Florenzano, Fernando M; Ramírez, Manuel R; Martínez, Ramón F; Lasala, Gabriel P

    2010-01-01

    BACKGROUND: Infusion of diverse types of bone marrow cells, as a source of endothelial progenitor cells (EPCs), into the ischemic myocardium is emerging as a promising therapy for coronary ischemia, probably mediated by the formation of new blood vessels. Studies have shown that while the procedure is safe and feasible, efficacy results are contentious. The investigators in the present preclinical translation study hypothesized that the infusion of a combination cell product consisting of EPCs and other cell types, such as mesenchymal stem cells, promotes the formation of more stable and mature blood vessels resulting in improved clinical outcomes. The safety and feasibility of the intracoronary infusion of such a cell combination was assessed in a canine model. METHODS: A mixture of canine autologous mononuclear cells (as the source of EPCs) and ex vivo-expanded bone marrow-derived mesenchymal stem cells or a placebo solution were intracoronarily infused into healthy dogs. Follow-up after cell/placebo infusion included an electrocardiogram, serum cardiac enzyme testing, a transthoracic echocardiography and a histopathological heart examination. RESULTS: On follow-up at all time points after infusion, no significant changes or abnormalities in vital signs, electrocardiogram, transthoracic echocardiography and heart histology were detected. CONCLUSIONS: From a clinical perspective, the safety and feasibility of the protocol used in the present animal study demonstrated clinical relevance and provided direct evidence supporting the intracoronary infusion of combination stem/progenitor cell products. PMID:20631864

  2. Differentiation of skeletal osteogenic progenitor cells to osteoblasts with 3,4-diarylbenzopyran based amide derivatives: Novel osteogenic agents.

    PubMed

    Gupta, Atul; Ahmad, Imran; Kureel, Jyoti; John, Aijaz A; Sultan, Eram; Chanda, Debabrata; Agarwal, Naresh Kumar; Alauddin; Wahajuddin; Prabhaker, S; Verma, Amita; Singh, Divya

    2016-10-01

    A series of 3,4-diarylbenzopyran based amide derivatives was synthesized and evaluated for osteogenic activity in in vitro and in vivo models of osteoporosis. Compounds 17a, 21b-c and 22a-b showed significant osteogenic activity in osteoblast differentiation assay. Among the synthesized compounds, 22b was identified as lead molecule which showed significant osteogenic activity at 1 pM concentration in osteoblast differentiation assay and at 1 mg kg(-1) body weight dose in estrogen deficient balb/c mice model. In vitro bone mineralization and expression of osteogenic marker genes viz BMP-2, RUNX-2, OCN, and collagen type 1 further confirmed the osteogenic potential of 22b. Gene expression study for estrogen receptor α and β (ER-α and ER-β) in mouse calvarial osteoblasts (MCOs) unveiled that possibly 22b exerted osteogenic efficacy via activation of Estrogen receptor-β preferentially. In vivo pharmacokinetic, estrogenicity and acute toxicity studies of 22b showed that it had good bioavailability and was devoid of uterine estrogenicity at 1 mg kg(-1) and inherent toxicity up to 1000 mg kg(-1) body weight dose respectively. PMID:27236065

  3. Bone marrow-derived stem cells and respiratory disease.

    PubMed

    Jones, Carla P; Rankin, Sara M

    2011-07-01

    Adult bone marrow contains a number of discrete populations of progenitor cells, including endothelial, mesenchymal, and epithelial progenitor cells and fibrocytes. In the context of a range of diseases, endothelial progenitor cells have been reported to promote angiogenesis, mesenchymal stem cells are potent immunosuppressors but can also contribute directly to tissue regeneration, and fibrocytes have been shown to induce tissue fibrosis. This article provides an overview of the basic biology of these different subsets of progenitor cells, reporting their distinct phenotypes and functional activities. The differences in their secretomes are highlighted, and the relative role of cellular differentiation vs paracrine effects of progenitor cells is considered. The article reviews the literature examining the contribution of progenitor cells to the pathogenesis of respiratory disease, and discusses recent studies using bone marrow progenitor cells as stem cell therapies in the context of pulmonary hypertension, COPD, and asthma. PMID:21729891

  4. Fluid shear stress stimulates prostaglandin and nitric oxide release in bone marrow-derived preosteoclast-like cells

    NASA Technical Reports Server (NTRS)

    McAllister, T. N.; Du, T.; Frangos, J. A.

    2000-01-01

    Bone is a porous tissue that is continuously perfused by interstitial fluid. Fluid flow, driven by both vascular pressure and mechanical loading, may generate significant shear stresses through the canaliculi as well as along the bone lining at the endosteal surface. Both osteoblasts and osteocytes produce signaling factors such as prostaglandins and nitric in response to fluid shear stress (FSS); however, these humoral agents appear to have more profound affects on osteoclast activity at the endosteal surface. We hypothesized that osteoclasts and preosteoclasts may also be mechanosensitive and that osteoclast-mediated autocrine signaling may be important in bone remodeling. In this study, we investigated the effect of FSS on nitric oxide (NO), prostaglandin E(2) (PGE(2)), and prostacyclin (PGI(2)) release by neonatal rat bone marrow-derived preosteoclast-like cells. These cells were tartrate-resistant acid phosphatase (TRAP) positive, weakly nonspecific esterase (NSE) positive, and capable of fusing into calcitonin-responsive, bone-resorbing, multinucleated cells. Bone marrow-derived preosteoclast-like cells exposed for 6 h to a well-defined FSS of 16 dynes/cm(2) produced NO at a rate of 7.5 nmol/mg protein/h, which was 10-fold that of static controls. This response was completely abolished by 100 microM N(G)-amino-L-arginine (L-NAA). Flow also stimulated PGE(2) production (3.9 microg/mg protein/h) and PGI(2) production (220 pg/mg protein/h). L-NAA attenuated flow-induced PGE(2) production by 30%, suggesting that NO may partially modulate PGE(2) production. This is the first report demonstrating that marrow derived cells are sensitive to FSS and that autocrine signaling in these cells may play an important role in load-induced remodeling and signal transduction in bone. Copyright 2000 Academic Press.

  5. Sirtuin1 (Sirt1) promotes cortical bone formation by preventing β-catenin sequestration by FoxO transcription factors in osteoblast progenitors.

    PubMed

    Iyer, Srividhya; Han, Li; Bartell, Shoshana M; Kim, Ha-Neui; Gubrij, Igor; de Cabo, Rafael; O'Brien, Charles A; Manolagas, Stavros C; Almeida, Maria

    2014-08-29

    A decline of the levels and activity of Sirtuin1 (Sirt1), a NAD(+) class III histone deacetylase, with age contributes to the development of several diseases including type 2 diabetes, neurodegeneration, inflammation, and cancer. The anti-aging effects of Sirt1 evidently result from the deacetylation of many transcription factors and co-factors including members of the Forkhead box O (FoxO) family and β-catenin. Wnt/β-catenin is indispensable for osteoblast generation. FoxOs, on the other hand, sequester β-catenin and inhibit osteoprogenitor proliferation. Here, we have deleted Sirt1 in osteoprogenitors expressing Osterix1 (Osx1)-Cre and their descendants. Sirt1(ΔOsx1) mice had lower cortical thickness in femora and vertebrae because of reduced bone formation at the endocortical surface. In line with this, osteoprogenitor cell cultures from the Sirt1(ΔOsx1) mice exhibited lower alkaline phosphatase activity and mineralization, as well as decreased proliferation and increased apoptosis. These changes were associated with decreased Wnt/β-catenin signaling and expression of cyclin D1 and resulted from increased binding of FoxOs to β-catenin. These findings demonstrate that Sirt1-induced deacetylation of FoxOs unleashes Wnt signaling. A decline in Sirt1 activity in osteoblast progenitors with aging may, therefore, contribute to the age-related loss of bone mass. Together with evidence that Sirt1 activators increase bone mass in aged mice, our results also suggest that Sirt1 could be a therapeutic target for osteoporosis. PMID:25002589

  6. Osteoanagenesis after transplantation of bone marrow-derived mesenchymal stem cells using polyvinylidene chloride film as a scaffold.

    PubMed

    Hamajima, Soichiro; Hayashi, Tatsuhide; Sato, Yamato; Sasaki, Keisuke; Kawai, Tatsushi

    2011-01-01

    The aim of this study was to develop a new cell transplantation technique for osteoanagenesis at bone defect sites. Polyvinylidene chloride (PVDC) film was evaluated because of its good biocompatibility and flexibility. We used this film as both a cell scaffold and a barrier membrane. Initially, the cell compatibility of the PVDC film for fibroblast-like cells and osteoblast-like cells was confirmed. Subsequently, bone marrow cells were obtained from rats and cultured on PVDC films in two kinds of medium. The PVDC films with bone marrow-derived mesenchymal stem cells (MSCs) were then applied to critical-sized bone defects in the calvarial bone of rats. After the transplantation, the surgical sites were dissected out and evaluated by soft X-ray radiography, micro-CT analysis and histological examinations. The bone marrow-derived MSC-transplanted rats showed greater bone regeneration than the control rats. Therefore, PVDC film is considered to be useful as a scaffold for bone regeneration. PMID:21946492

  7. Bone marrow-derived stem cell therapy for metastatic brain cancers.

    PubMed

    Kaneko, Yuji; Tajiri, Naoki; Staples, Meaghan; Reyes, Stephanny; Lozano, Diego; Sanberg, Paul R; Freeman, Thomas B; van Loveren, Harry; Kim, Seung U; Borlongan, Cesar V

    2015-01-01

    We propose that stem cell therapy may be a potent treatment for metastatic melanoma in the brain. Here we discuss the key role of a leaky blood-brain barrier (BBB) that accompanies the development of brain metastases. We review the need to characterize the immunological and inflammatory responses associated with tumor-derived BBB damage in order to reveal the contribution of this brain pathological alteration to the formation and growth of brain metastatic cancers. Next, we discuss the potential repair of the BBB and attenuation of brain metastasis through transplantation of bone marrow-derived mesenchymal stem cells with the endothelial progenitor cell phenotype. In particular, we review the need for evaluation of the efficacy of stem cell therapy in repairing a disrupted BBB in an effort to reduce neuroinflammation, eventually attenuating brain metastatic cancers. The demonstration of BBB repair through augmented angiogenesis and vasculogenesis will be critical to establishing the potential of stem cell therapy for the treatment/prevention of metastatic brain tumors. The overarching hypothesis we advanced here is that BBB breakdown is closely associated with brain metastatic cancers of melanoma, exacerbating the inflammatory response of the brain during metastasis, and ultimately worsening the outcome of metastatic brain cancers. Abrogating this leaky BBB-mediated inflammation via stem cell therapy represents a paradigm-shifting approach to treating brain cancer. This review article discusses the pros and cons of cell therapy for melanoma brain metastases. PMID:25310691

  8. Novel animal models for tracking the fate and contributions of bone marrow derived cells in diabetic healing.

    PubMed

    Caskey, Robert C; Liechty, Kenneth W

    2013-01-01

    There is a vast wealth of information to be gained by tracking both the fate and contribution of individual cell types to the wound healing response. This is particularly important in research focused on impaired healing, such as diabetic wound healing, where the number or function of one or more specific cell types may be abnormal and contribute to the observed healing derangements. Specifically, diabetic wounds have been shown to have an overactive inflammatory response and decreased angiogenesis. The ability to track specific cell types participating in these responses would dramatically improve our understanding of the cellular derangements in diabetic healing. In this chapter, we review two novel chimeric models based on the leptin deficient Db/Db mouse. The use of these models allows for the tracking of bone marrow derived inflammatory and progenitor cell populations as well as the determination of the molecular contributions of these cell populations to the wound healing response. PMID:24029932

  9. Altered MicroRNA Expression Profile in Exosomes during Osteogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells

    PubMed Central

    Zhang, Shui-Jun; Zhao, Chen; Qiu, Bin-Song; Gu, Hai-Feng; Hong, Jian-Fei; Cao, Li; Chen, Yu; Xia, Bing; Bi, Qin; Wang, Ya-Ping

    2014-01-01

    The physiological role of microRNAs (miRNAs) in osteoblast differentiation remains elusive. Exosomal miRNAs isolated from human bone marrow-derived mesenchymal stem cells (BMSCs) culture were profiled using miRNA arrays containing probes for 894 human matured miRNAs. Seventy-nine miRNAs (∼8.84%) could be detected in exosomes isolated from BMSC culture supernatants when normalized to endogenous control genes RNU44. Among them, nine exosomal miRNAs were up regulated and 4 miRNAs were under regulated significantly (Relative fold>2, p<0.05) when compared with the values at 0 day with maximum changes at 1 to 7 days. Five miRNAs (miR-199b, miR-218, miR-148a, miR-135b, and miR-221) were further validated and differentially expressed in the individual exosomal samples from hBMSCs cultured at different time points. Bioinformatic analysis by DIANA-mirPath demonstrated that RNA degradation, mRNA surveillance pathway, Wnt signaling pathway, RNA transport were the most prominent pathways enriched in quantiles with differential exosomal miRNA patterns related to osteogenic differentiation. These data demonstrated exosomal miRNA is a regulator of osteoblast differentiation. PMID:25503309

  10. p62 is required to retain short-term repopulating and myeloid progenitor cells through inhibition of IKK/NF-κB/Ccl4 signaling at the bone marrow macrophage-osteoblast niche

    PubMed Central

    Chang, Kyung Hee; Sengupta, Amitava; Nayak, Ramesh C.; Duran, Angeles; Lee, Sang Jun; Pratt, Ronald G.; Wellendorf, Ashley M.; Hill, Sarah E.; Watkins, Marcus; Gonzalez-Nieto, Daniel; Aronow, Bruce J.; Starczynowski, Daniel T.; Civitelli, Roberto; Diaz-Meco, Maria T.; Moscat, Jorge; Cancelas, Jose A.

    2014-01-01

    In the bone marrow (BM), hematopoietic progenitors (HP) reside in specific anatomical niches near osteoblasts (Ob), macrophages (MΦ) and other cells forming the BM microenvironment. A connection between immunosurveillance and traffic of HP has been demonstrated but the regulatory signals that instruct immune regulation on HP circulation are unknown. We discovered that the BM microenvironment deficiency of p62, an autophagy regulator and signal organizer, results in loss of autophagic repression of macrophage contact-dependent activation of Ob NF-κB signaling. Consequently, Ob p62-deficient mice lose bone, Ob Ccl4 expression and HP chemotaxis towards Cxcl12 resulting in egress of short-term hematopoietic stem cells and myeloid progenitors. Finally, Ccl4 expression and myeloid progenitor egress are reversed by the deficiency of the p62 PB1 binding partner Nbr1. A functional ‘MΦ-Ob niche’ is required for myeloid progenitor/short-term stem cell retention, in which Ob p62 is required to maintain NF-κB signaling repression, osteogenesis and BM progenitor retention. PMID:25533346

  11. Adipose lineage specification of bone marrow-derived myeloid cells

    PubMed Central

    Majka, Susan M.; Miller, Heidi L.; Sullivan, Timothy; Erickson, Paul F.; Kong, Raymond; Weiser-Evans, Mary; Nemenoff, Raphael; Moldovan, Radu; Morandi, Shelley A.; Davis, James A.; Klemm, Dwight J.

    2012-01-01

    We have reported the production of white adipocytes in adipose tissue from hematopoietic progenitors arising from bone marrow. However, technical challenges have hindered detection of this adipocyte population by certain other laboratories. These disparate results highlight the need for sensitive and definitive techniques to identify bone marrow progenitor (BMP)-derived adipocytes. In these studies we exploited new models and methods to enhance detection of this adipocyte population. Here we showed that confocal microscopy with spectrum acquisition could effectively identify green fluorescent protein (GFP) positive BMP-derived adipocytes by matching their fluorescence spectrum to that of native GFP. Likewise, imaging flow cytometry made it possible to visualize intact unilocular and multilocular GFP-positive BMP-derived adipocytes and distinguished them from non-fluorescent adipocytes and cell debris in the cytometer flow stream. We also devised a strategy to detect marker genes in flow-enriched adipocytes from which stromal cells were excluded. This technique also proved to be an efficient means for detecting genetically labeled adipocytes and should be applicable to models in which marker gene expression is low or absent. Finally, in vivo imaging of mice transplanted with BM from adipocyte-targeted luciferase donors showed a time-dependent increase in luciferase activity, with the bulk of luciferase activity confined to adipocytes rather than stromal cells. These results confirmed and extended our previous reports and provided proof-of-principle for sensitive techniques and models for detection and study of these unique cells. PMID:23700536

  12. Transgelin is a TGFβ-inducible gene that regulates osteoblastic and adipogenic differentiation of human skeletal stem cells through actin cytoskeleston organization.

    PubMed

    Elsafadi, M; Manikandan, M; Dawud, R A; Alajez, N M; Hamam, R; Alfayez, M; Kassem, M; Aldahmash, A; Mahmood, A

    2016-01-01

    Regenerative medicine is a novel approach for treating conditions in which enhanced bone regeneration is required. We identified transgelin (TAGLN), a transforming growth factor beta (TGFβ)-inducible gene, as an upregulated gene during in vitro osteoblastic and adipocytic differentiation of human bone marrow-derived stromal (skeletal) stem cells (hMSC). siRNA-mediated gene silencing of TAGLN impaired lineage differentiation into osteoblasts and adipocytes but enhanced cell proliferation. Additional functional studies revealed that TAGLN deficiency impaired hMSC cell motility and in vitro transwell cell migration. On the other hand, TAGLN overexpression reduced hMSC cell proliferation, but enhanced cell migration, osteoblastic and adipocytic differentiation, and in vivo bone formation. In addition, deficiency or overexpression of TAGLN in hMSC was associated with significant changes in cellular and nuclear morphology and cytoplasmic organelle composition as demonstrated by high content imaging and transmission electron microscopy that revealed pronounced alterations in the distribution of the actin filament and changes in cytoskeletal organization. Molecular signature of TAGLN-deficient hMSC showed that several genes and genetic pathways associated with cell differentiation, including regulation of actin cytoskeleton and focal adhesion pathways, were downregulated. Our data demonstrate that TAGLN has a role in generating committed progenitor cells from undifferentiated hMSC by regulating cytoskeleton organization. Targeting TAGLN is a plausible approach to enrich for committed hMSC cells needed for regenerative medicine application. PMID:27490926

  13. The Effect of Quercetin on the Osteogenesic Differentiation and Angiogenic Factor Expression of Bone Marrow-Derived Mesenchymal Stem Cells.

    PubMed

    Zhou, Yuning; Wu, Yuqiong; Jiang, Xinquan; Zhang, Xiuli; Xia, Lunguo; Lin, Kaili; Xu, Yuanjin

    2015-01-01

    Bone marrow-derived mesenchymal stem cells (BMSCs) are widely used in regenerative medicine in light of their ability to differentiate along the chondrogenic and osteogenic lineages. As a type of traditional Chinese medicine, quercetin has been preliminarily reported to promote osteogenic differentiation in osteoblasts. In the present study, the effects of quercetin on the proliferation, viability, cellular morphology, osteogenic differentiation and angiogenic factor secretion of rat BMSCs (rBMSCs) were examined by MTT assay, fluorescence activated cell sorter (FACS) analysis, real-time quantitative PCR (RT-PCR) analysis, alkaline phosphatase (ALP) activity and calcium deposition assays, and Enzyme-linked immunosorbent assay (ELISA). Moreover, whether mitogen-activated protein kinase (MAPK) signaling pathways were involved in these processes was also explored. The results showed that quercetin significantly enhanced the cell proliferation, osteogenic differentiation and angiogenic factor secretion of rBMSCs in a dose-dependent manner, with a concentration of 2 μM achieving the greatest stimulatory effect. Moreover, the activation of the extracellular signal-regulated protein kinases (ERK) and p38 pathways was observed in quercetin-treated rBMSCs. Furthermore, these induction effects could be repressed by either the ERK inhibitor PD98059 or the p38 inhibitor SB202190, respectively. These data indicated that quercetin could promote the proliferation, osteogenic differentiation and angiogenic factor secretion of rBMSCs in vitro, partially through the ERK and p38 signaling pathways. PMID:26053266

  14. The Effect of Quercetin on the Osteogenesic Differentiation and Angiogenic Factor Expression of Bone Marrow-Derived Mesenchymal Stem Cells

    PubMed Central

    Zhou, Yuning; Wu, Yuqiong; Jiang, Xinquan; Zhang, Xiuli; Xia, Lunguo; Lin, Kaili; Xu, Yuanjin

    2015-01-01

    Bone marrow-derived mesenchymal stem cells (BMSCs) are widely used in regenerative medicine in light of their ability to differentiate along the chondrogenic and osteogenic lineages. As a type of traditional Chinese medicine, quercetin has been preliminarily reported to promote osteogenic differentiation in osteoblasts. In the present study, the effects of quercetin on the proliferation, viability, cellular morphology, osteogenic differentiation and angiogenic factor secretion of rat BMSCs (rBMSCs) were examined by MTT assay, fluorescence activated cell sorter (FACS) analysis, real-time quantitative PCR (RT-PCR) analysis, alkaline phosphatase (ALP) activity and calcium deposition assays, and Enzyme-linked immunosorbent assay (ELISA). Moreover, whether mitogen-activated protein kinase (MAPK) signaling pathways were involved in these processes was also explored. The results showed that quercetin significantly enhanced the cell proliferation, osteogenic differentiation and angiogenic factor secretion of rBMSCs in a dose-dependent manner, with a concentration of 2 μM achieving the greatest stimulatory effect. Moreover, the activation of the extracellular signal-regulated protein kinases (ERK) and p38 pathways was observed in quercetin-treated rBMSCs. Furthermore, these induction effects could be repressed by either the ERK inhibitor PD98059 or the p38 inhibitor SB202190, respectively. These data indicated that quercetin could promote the proliferation, osteogenic differentiation and angiogenic factor secretion of rBMSCs in vitro, partially through the ERK and p38 signaling pathways. PMID:26053266

  15. Bone marrow-derived pancreatic stellate cells in rats.

    PubMed

    Sparmann, Gisela; Kruse, Marie-Luise; Hofmeister-Mielke, Nicole; Koczan, Dirk; Jaster, Robert; Liebe, Stefan; Wolff, Daniel; Emmrich, Jörg

    2010-03-01

    Origin and fate of pancreatic stellate cells (PSCs) before, during and after pancreatic injury are a matter of debate. The crucial role of PSCs in the pathogenesis of pancreatic fibrosis is generally accepted. However, the turnover of the cells remains obscure. The present study addressed the issue of a potential bone marrow (BM) origin of PSCs. We used a model of stable hematopoietic chimerism by grafting enhanced green fluorescence protein (eGFP)-expressing BM cells after irradiation of acceptor rats. Chimerism was detected by FACS analysis of eGFP-positive cells in the peripheral blood. Dibutyltin dichloride (DBTC) was used to induce acute pancreatic inflammation with subsequent recovery over 4 weeks. Investigations have been focused on isolated cells to detect the resting PSC population. The incidence of eGFP-positive PSC obtained from the pancreas of chimeric rats was approximately 7% in healthy pancreatic tissue and increased significantly to a mean of 18% in the restored pancreas 4 weeks after DBTC-induced acute inflammation. Our results suggest that BM-derived progenitor cells represent a source of renewable stellate cells in the pancreas. Increased numbers of resting PSCs after regeneration point toward enhanced recruitment of BM-derived cells to the pancreas and/or re-acquisition of a quiescent state after inflammation-induced activation. PMID:20101265

  16. Bone marrow-derived cell regulation of skeletal muscle regeneration.

    PubMed

    Sun, Dongxu; Martinez, Carlo O; Ochoa, Oscar; Ruiz-Willhite, Lourdes; Bonilla, Jose R; Centonze, Victoria E; Waite, Lindsay L; Michalek, Joel E; McManus, Linda M; Shireman, Paula K

    2009-02-01

    Limb regeneration requires the coordination of multiple stem cell populations to recapitulate the process of tissue formation. Therefore, bone marrow (BM) -derived cell regulation of skeletal muscle regeneration was examined in mice lacking the CC chemokine receptor 2 (CCR2). Myofiber size, numbers of myogenic progenitor cells (MPCs), and recruitment of BM-derived cells and macrophages were assessed after cardiotoxin-induced injury of chimeric mice produced by transplanting BM from wild-type (WT) or CCR2(-/-) mice into irradiated WT or CCR2(-/-) host mice. Regardless of the host genotype, muscle regeneration and recruitment of BM-derived cells and macrophages were similar in mice replenished with WT BM, whereas BM-derived cells and macrophage accumulation were decreased and muscle regeneration was impaired in all animals receiving CCR2(-/-) BM. Furthermore, numbers of MPCs (CD34(+)/Sca-1(-)/CD45(-) cells) were significantly increased in mice receiving CCR2(-/-) BM despite the decreased size of regenerated myofibers. Thus, the expression of CCR2 on BM-derived cells regulated macrophage recruitment into injured muscle, numbers of MPC, and the extent of regenerated myofiber size, all of which were independent of CCR2 expression on host-derived cells. Future studies in regenerative medicine must include consideration of the role of BM-derived cells, possibly macrophages, in CCR2-dependent events that regulate effective skeletal muscle regeneration. PMID:18827026

  17. Viscoelastic properties of human mesenchymally-derived stem cells and primary osteoblasts, chondrocytes, and adipocytes

    PubMed Central

    Darling, Eric M.; Topel, Matthew; Zauscher, Stefan; Vail, Thomas P.; Guilak, Farshid

    2010-01-01

    The mechanical properties of single cells play important roles in regulating cell-matrix interactions, potentially influencing the process of mechanotransduction. Recent studies also suggest that cellular mechanical properties may provide novel biological markers, or “biomarkers,” of cell phenotype, reflecting specific changes that occur with disease, differentiation, or cellular transformation. Of particular interest in recent years has been the identification of such biomarkers that can be used to determine specific phenotypic characteristics of stem cells that separate them from primary, differentiated cells. The goal of this study was to determine the elastic and viscoelastic properties of three primary cell types of mesenchymal lineage (chondrocytes, osteoblasts, and adipocytes) and to test the hypothesis that primary differentiated cells exhibit distinct mechanical properties compared to adult stem cells (adipose-derived or bone marrow-derived mesenchymal stem cells). In an adherent, spread configuration, chondrocytes, osteoblasts, and adipocytes all exhibited significantly different mechanical properties, with osteoblasts being stiffer than chondrocytes and both being stiffer than adipocytes. Adipose-derived and mesenchymal stem cells exhibited similar properties to each other, but were mechanically distinct from primary cells, particularly when comparing a ratio of elastic to relaxed moduli. These findings will help more accurately model the cellular mechanical environment in mesenchymal tissues, which could assist in describing injury thresholds and disease progression or even determining the influence of mechanical loading for tissue engineering efforts. Furthermore, the identification of mechanical properties distinct to stem cells could result in more successful sorting procedures to enrich multipotent progenitor cell populations. PMID:17825308

  18. Connective tissue progenitor cell growth characteristics on textured substrates.

    PubMed

    Mata, Alvaro; Boehm, Cynthia; Fleischman, Aaron J; Muschler, George F; Roy, Shuvo

    2007-01-01

    Growth characteristics of human connective tissue progenitor (CTP) cells were investigated on smooth and textured substrates, which were produced using MEMS (microelectromechanical systems) fabrication technology. Human bone marrow derived cells were cultured for 9 days under conditions promoting osteoblastic differentiation on polydimethylsiloxane (PDMS) substrates comprising smooth (non-patterned) surfaces (SMOOTH), 4 different cylindrical post micro-textures (POSTS) that were 7-10 microm high and 5, 10, 20, and 40 microm diameter, respectively, and channel micro-textures (CHANNELS) with curved cross-sections that were 11 microm high, 45 microm wide, and separated by 5 microm wide ridges. Standard glass-tissue culture surfaces were used as controls. Micro-textures resulted in the modification of CTP morphology, attachment, migration, and proliferation characteristics. Specifically, cells on POSTS exhibited more contoured morphology with closely packed cytoskeletal actin microfilaments compared to the more random orientation in cells grown on SMOOTH. CTP colonies on 10 gm-diameter POSTS exhibited higher cell number than any other POSTS, and a significant increase in cell number (442%) compared to colonies on SMOOTH (71%). On CHANNELS, colonies tended to be denser (229%) than on POSTS (up to 140% on 10 microm POSTS), and significantly more so compared to those on SMOOTH (104%). PMID:18019838

  19. Bone Marrow-Derived Cells Contribute to Fibrosis in the Chronically Failing Heart

    PubMed Central

    Chu, Po-Yin; Mariani, Justin; Finch, Samara; McMullen, Julie R.; Sadoshima, Junichi; Marshall, Tanneale; Kaye, David M.

    2010-01-01

    Cardiac fibrosis contributes significantly to the phenotype of the chronically failing heart. It is not clear whether in this setting the fibrosis is contributed by native cardiac fibroblasts or alternatively by recruitment of cells arising from the bone marrow. We aimed to determine the contribution of bone marrow-derived cells to cardiac fibrosis in the failing heart and to investigate potentially contributing cytokines. Bone marrow-derived fibrocyte recruitment to the failing heart was studied in a transgenic (Mst1 mice) model of dilated cardiomyopathy. In conjunction, we examined the role of stromal-derived factor-1 (SDF-1), a key chemoattractant, by assessing myocardial expression and secretion by cardiomyocytes and in clinical samples. Bone marrow-derived cells were recruited in significantly greater numbers in Mst1 versus control mice (P < 0.001), contributing 17 ± 4% of the total fibroblast load in heart failure. Patients with heart failure had higher plasma levels of SDF-1 than healthy control subjects (P < 0.01). We found that cardiomyocytes constitutively secrete SDF-1, which is significantly up-regulated by angiotensin II. SDF-1 was shown to increases cardiac fibroblast migration by 59% (P < 0.05). Taken together, our data suggest that recruitment of bone marrow-derived cells under the influence of factors, including SDF-1, may play an important role in the pathogenesis of cardiac fibrosis in heart failure. PMID:20150435

  20. Enhanced adipogenic differentiation of bovine bone marrow-derived mesenchymal stem cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Until now, the isolation and characterization of bovine bone marrow-derived mesenchymal stem cells (bBM-MSCs) have not been established, which prompted us to optimize the differentiation protocol for bBM-MSCs. In this study, bBM-MSCs were freshly isolated from three 6-month-old cattle and used for p...

  1. Bone marrow-derived cells are differentially involved in pathological and physiological retinal angiogenesis in mice

    SciTech Connect

    Zou, He; Otani, Atsushi; Oishi, Akio; Yodoi, Yuko; Kameda, Takanori; Kojima, Hiroshi; Yoshimura, Nagahisa

    2010-01-08

    Purpose: Bone marrow-derived cells have been shown to play roles in angiogenesis. Although these cells have been shown to promote angiogenesis, it is not yet clear whether these cells affect all types of angiogenesis. This study investigated the involvement of bone marrow-derived cells in pathological and physiological angiogenesis in the murine retina. Materials and methods: The oxygen-induced retinopathy (OIR) model was used as a retinal angiogenesis model in newborn mice. To block the influence of bone marrow-derived cells, the mice were irradiated with a 4-Gy dose of radiation from a {sup 137}Cs source. Irradiation was performed in four different conditions with radio dense 2-cm thick lead disks; (1) H group, the head were covered with these discs to protect the eyes from radiation; (2) A group, all of the body was covered with these discs; (3) N group, mice were completely unshielded; (4) C group, mice were put in the irradiator but were not irradiated. On P17, the retinal areas showing pathological and physiological retinal angiogenesis were measured and compared to the retinas of nonirradiated mice. Results: Although irradiation induced leukocyte depletion, it did not affect the number of other cell types or body weight. Retinal nonperfusion areas were significantly larger in irradiated mice than in control mice (P < 0.05), indicating that physiological angiogenesis was impaired. However, the formation of tuft-like angiogenesis processes was more prominent in the irradiated mice (P < 0.05), indicating that pathological angiogenesis was intact. Conclusions: Bone marrow-derived cells seem to be differentially involved in the formation of physiological and pathological retinal vessels. Pathological angiogenesis in the murine retina does not require functional bone marrow-derived cells, but these cells are important for the formation of physiological vessels. Our results add a new insight into the pathology of retinal angiogenesis and bolster the hypothesis that

  2. Cigarette smoke inhibits recruitment of bone-marrow-derived stem cells to the uterus.

    PubMed

    Zhou, Yuping; Gan, Ye; Taylor, Hugh S

    2011-02-01

    Cigarette smoking leads to female infertility and a decreased incidence of endometriosis. Bone marrow derived stem cells are recruited to uterine endometrium and endometriosis. The effect of cigarette smoking on stem cell recruitment to any organ is uncharacterized. We hypothesized that bone marrow-derived mesenchymal stem cell recruitment to the uterus and differentiation would be diminished by cigarette smoke. We used human mesenchymal stem cells (hMSC) in vitro and a mouse model of cigarette smoke exposure. After myeloablation female C57BL/6J received bone marrow cells from males. Mice were exposed to room air or smoke from unfiltered cigarettes. Immunofluorescence and Y-FISH was performed on uterine sections. In vitro hMSCs were treated with 8-Br-cAMP to induce endometrial cell differentiation with or without cigarette smoke extract (CSE) and decidualization assessed morphologically and by prolactin expression. After 4 weeks the total number of Y-chromosome cells in the uterus was reduced by 68% in the smoke exposed mice. Both leukocytes and bone marrow derived endometrial cells were reduced by 60% and 73%, respectively. Differentiation of bone marrow derived cell to endometrial epithelial cells was reduced by 84%. hMSC treated with CSE failed to show cytological characteristics of decidualization. mRNA levels of the decidualization marker prolactin were decreased by 90% in CSE treated cells. Smoking inhibits both recruitment of bone marrow derived stem cells to uterus and stem cell differentiation. Inhibition of stem cells recruitment may be a general mechanism by which smoking leads to long term organ damage through inability to repair or regenerate multiple tissues. PMID:20955787

  3. Bioactive lipid coating of bone allografts directs engraftment and fate determination of bone marrow-derived cells in rat GFP chimeras.

    PubMed

    Das, Anusuya; Segar, Claire E; Chu, Yihsuan; Wang, Tiffany W; Lin, Yong; Yang, Chunxi; Du, Xeujun; Ogle, Roy C; Cui, Quanjun; Botchwey, Edward A

    2015-09-01

    Bone grafting procedures are performed to treat wounds incurred during wartime trauma, accidents, and tumor resections. Endogenous mechanisms of repair are often insufficient to ensure integration between host and donor bone and subsequent restoration of function. We investigated the role that bone marrow-derived cells play in bone regeneration and sought to increase their contributions by functionalizing bone allografts with bioactive lipid coatings. Polymer-coated allografts were used to locally deliver the immunomodulatory small molecule FTY720 in tibial defects created in rat bone marrow chimeras containing genetically-labeled bone marrow for monitoring cell origin and fate. Donor bone marrow contributed significantly to both myeloid and osteogenic cells in remodeling tissue surrounding allografts. FTY720 coatings altered the phenotype of immune cells two weeks post-injury, which was associated with increased vascularization and bone formation surrounding allografts. Consequently, degradable polymer coating strategies that deliver small molecule growth factors such as FTY720 represent a novel therapeutic strategy for harnessing endogenous bone marrow-derived progenitors and enhancing healing in load-bearing bone defects. PMID:26125501

  4. The synergistic effect of VEGF and biomorphic silicon carbides topography on in vivo angiogenesis and human bone marrow derived mesenchymal stem cell differentiation.

    PubMed

    Díaz-Rodríguez, P; Gómez-Amoza, J L; Landin, M

    2015-08-01

    Topographical features of biomaterials are able to modulate cell attachment, spreading and differentiation. The addition of growth factors to implantable biomaterials can modify these cellular responses, enhancing their therapeutic potential. The aim of this research is to establish the influence of biomorphic silicon carbide ceramics (bioSiCs) surface topography on the proliferation and osteoblastic differentiation of mesenchymal stem cells and the potential synergistic effect of the ceramic porous structure together with vascular endothelial growth factor loading (VEGF) on the surface mediated osteoblastic differentiation. Three porous bioSiCs with important differences in their microstructure were obtained from different natural precursors. Samples loaded with or without VEGF through ionic interactions were cultured with human umbilical vein endothelial cells (HUVEC) or bone marrow derived mesenchymal stem cells (hMSCs). Cell behaviour and protein activity with regard to bioSiC porous structure and surface properties were analysed. An in vivo model (Chick Chorioallantoic Membrane; CAM) was used to assess the capability of the VEGF loaded systems to promote angiogenesis. Experimental data show that loaded systems were able to control the release of VEGF for up to 15 d ensuring the activity of the protein, increasing the proliferation of HUVECs and the formation of new blood vessels in the CAM. It was found that the selection of bioSiCs with a higher pore size promoted a higher concentration of osteoblastic differentiation markers of MSCs cultured on the surface of bioSiCs. Furthermore, the addition of VEGF to the systems was able to promote a faster osteoblastic differentiation according to the qPCR results, suggesting a synergy between both the surface properties and the controlled release of the growth factor. The VEGF loaded sapelli bioSiC was found to be the most promising material for bone tissue engineering applications. PMID:26238485

  5. Effects of Line and Pillar Array Microengineered SiO2 Thin Films on the Osteogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells.

    PubMed

    Carvalho, Angela; Pelaez-Vargas, Alejandro; Hansford, Derek J; Fernandes, Maria H; Monteiro, Fernando J

    2016-02-01

    A primary goal in bone tissue engineering is the design of implants that induce controlled, guided, and rapid healing. The events that normally lead to the integration of an implant into bone and determine the performance of the device occur mainly at the tissue-implant interface. Topographical surface modification of a biomaterial might be an efficient tool for inducing stem cell osteogenic differentiation and replace the use of biochemical stimuli. The main goal of this work was to develop micropatterned bioactive silica thin films to induce the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) only through topographical stimuli. Line and pillar micropatterns were developed by a combination of sol-gel/soft lithography and characterized by scanning electron microscopy, atomic force microscopy, and contact angle measurements. hMSCs were cultured onto the microfabricated thin films and flat control for up to 21 days under basal conditions. The micropatterned groups induced levels of osteogenic differentiation and expression of osteoblast-associated markers higher than those of the flat controls. Via comparison of the micropatterns, the pillars caused a stronger response of the osteogenic differentiation of hMSCs with a higher level of expression of osteoblast-associated markers, ALP activity, and extracellular matrix mineralization after the cells had been cultured for 21 days. These findings suggest that specific microtopographic cues can direct hMSCs toward osteogenic differentiation. PMID:26771563

  6. Bone Marrow-Derived Stem Cells: a Mixed Blessing in the Multifaceted World of Diabetic Complications.

    PubMed

    Mangialardi, Giuseppe; Madeddu, Paolo

    2016-05-01

    Diabetes is one of the main economic burdens in health care, which threatens to worsen dramatically if prevalence forecasts are correct. What makes diabetes harmful is the multi-organ distribution of its microvascular and macrovascular complications. Regenerative medicine with cellular therapy could be the dam against life-threatening or life-altering complications. Bone marrow-derived stem cells are putative candidates to achieve this goal. Unfortunately, the bone marrow itself is affected by diabetes, as it can develop a microangiopathy and neuropathy similar to other body tissues. Neuropathy leads to impaired stem cell mobilization from marrow, the so-called mobilopathy. Here, we review the role of bone marrow-derived stem cells in diabetes: how they are affected by compromised bone marrow integrity, how they contribute to other diabetic complications, and how they can be used as a treatment for these. Eventually, we suggest new tactics to optimize stem cell therapy. PMID:27025211

  7. Adeno associated viral-mediated intraosseous labeling of bone marrow derived cells for CNS tracking.

    PubMed

    Selenica, Maj-Linda B; Reid, Patrick; Pena, Gabriela; Alvarez, Jennifer; Hunt, Jerry B; Nash, Kevin R; Morgan, Dave; Gordon, Marcia N; Lee, Daniel C

    2016-05-01

    Inflammation, including microglial activation in the CNS, is an important hallmark in many neurodegenerative diseases. Microglial stimuli not only impact the brain microenvironment by production and release of cytokines and chemokines, but also influence the activity of bone marrow derived cells and blood born macrophage populations. In many diseases including brain disorders and spinal cord injury, researchers have tried to harbor the neuroprotective and repair properties of these subpopulations. Hematopoietic bone marrow derived cells (BMDCs) are of great interest, especially during gene therapy because certain hematopoietic cell subpopulations traffic to the sites of injury and inflammation. The aim of this study was to develop a method of labeling endogenous bone marrow derived cells through intraosseous impregnation of recombinant adeno-associated virus (rAAV) or lentivirus. We utilized rAAV serotype 9 (rAAV-9) or lentivirus for gene delivery of green florescence protein (GFP) to the mouse bone marrow cells. Flow cytometry showed that both viruses were able to efficiently transduce mouse bone marrow cells in vivo. However, the rAAV9-GFP viral construct transduced BMDCs more efficiently than the lentivirus (11.2% vs. 6.8%), as indicated by cellular GFP expression. We also demonstrate that GFP labeled cells correspond to bone marrow cells of myeloid origin using CD11b as a marker. Additionally, we characterized the ability of bone marrow derived, GFP labeled cells to extravasate into the brain parenchyma upon acute and subchronic neuroinflammatory stimuli in the mouse CNS. Viral mediated over expression of chemokine (C-C motif) ligand 2 (CCL2) or intracranial injection of lipopolysaccharide (LPS) recruited GFP labeled BMDCs from the periphery into the brain parenchyma compared to vehicle treated mice. Altogether our findings demonstrate a useful method of labeling endogenous BMDCs via viral transduction and the ability to track subpopulations throughout the body

  8. Chronic foot-shock stress potentiates the influx of bone marrow-derived microglia into hippocampus.

    PubMed

    Brevet, Marie; Kojima, Hideto; Asakawa, Akihiro; Atsuchi, Kaori; Ushikai, Miharu; Ataka, Koji; Inui, Akio; Kimura, Hiroshi; Sevestre, Henri; Fujimiya, Mineko

    2010-07-01

    For several years, a new population of microglia derived from bone marrow has been described in multiple settings such as infection, trauma, and neurodegenerative disease. The aim of this study was to investigate the migration of bone marrow-derived cells to the brain parenchyma after stress exposure. Stress exposure was performed in mice that had received bone marrow transplantation from GFP mice, allowing identification of blood-derived elements within the brain. Electric foot-shock exposure was chosen because of its ability to serve as fundamental and physical stress in mice. Bone marrow-derived GFP(+) cells migrated to the ventral part of the hippocampus and acquired a ramified microglia-like morphology. Microglia marker Iba1 was expressed by 100% of the ramified cells, whereas ramified cells were negative for the astrocyte marker GFAP. Compared with the case in the control group, ramified cells significantly increased after chronic exposure to stress (5 days). One month after 5 days of stress exposure, ramified cells significantly decreased in ventral hippocampus compared with the group examined immediately after the last stress exposure. We report for the first time the migration of bone marrow-derived cells to the ventral hippocampus after stress exposure. These cells have the characteristics of microglia. Mechanisms responsible for this migration and their roles in the brain remain to be determined. PMID:20155811

  9. In Vivo Transplantation of Autogenous Marrow-Derived Cells Following Rapid Intraoperative Magnetic Separation Based on Hyaluronan to Augment Bone Regeneration

    PubMed Central

    Joshi, Powrnima; Fleury, Sean; Luangphakdy, Viviane; Shinohara, Kentaro; Pan, Hui; Boehm, Cynthia; Vasanji, Amit; Hefferan, Theresa E.; Walker, Esteban; Yaszemski, Michael; Hascall, Vincent; Zborowski, Maciej

    2013-01-01

    Introduction This project was designed to test the hypothesis that rapid intraoperative processing of bone marrow based on hyaluronan (HA) could be used to improve the outcome of local bone regeneration if the concentration and prevalence of marrow-derived connective tissue progenitors (CTPs) could be increased and nonprogenitors depleted before implantation. Methods HA was used as a marker for positive selection of marrow-derived CTPs using magnetic separation (MS) to obtain a population of HA-positive cells with an increased CTP prevalence. Mineralized cancellous allograft (MCA) was used as an osteoconductive carrier scaffold for loading of HA-positive cells. The canine femoral multidefect model was used and four cylindrical defects measuring 10 mm in diameter and 15 mm in length were grafted with MCA combined with unprocessed marrow or with MS processed marrow that was enriched in HA+ CTPs and depleted in red blood cells and nonprogenitors. Outcome was assessed at 4 weeks using quantitative 3D microcomputed tomography (micro-CT) analysis of bone formation and histomorphological assessment. Results Histomorphological assessment showed a significant increase in new bone formation and in the vascular sinus area in the MS-processed defects. Robust bone formation was found throughout the defect area in both groups (defects grafted with unprocessed marrow or with MS processed marrow.) Percent bone volume in the defects, as assessed by micro-CT, was greater in defects engrafted with MS processed cells, but the difference was not statistically significant. Conclusion Rapid intraoperative MS processing to enrich CTPs based on HA as a surface marker can be used to increase the concentration and prevalence of CTPs. MCA grafts supplemented with heparinized bone marrow or MS processed cells resulted in a robust and advanced stage of bone regeneration at 4 weeks. A greater new bone formation and vascular sinus area was found in defects grafted with MS processed cells

  10. Bone marrow-derived cells migrate to the liver and contribute to the generation of different cell types in chronic Schistosoma mansoni infection.

    PubMed

    Azevedo, Carine Machado; Solano de Freitas Souza, Bruno; Andrade de Oliveira, Sheilla; Paredes, Bruno Diaz; Barreto, Elton Sá; Neto, Hélio Almeida; Ribeiro dos Santos, Ricardo; Pereira Soares, Milena Botelho

    2015-12-01

    The main pathogenic event caused by Schistosoma mansoni infection is characterized by a granulomatous inflammatory reaction around parasite eggs and fibrosis in the liver. We have previously shown that transplantation of bone marrow cells (BMC) promotes a reduction in liver fibrosis in chronically S. mansoni-infected mice. Here we investigated the presence and phenotype of bone marrow-derived cells in livers of S. mansoni-infected mice. During the chronic phase of infection, C57BL/6 mice had an increased number of circulating mesenchymal stem cells and endothelial progenitor cells in the peripheral blood when compared to uninfected controls. In order to investigate the fate of BMC in the liver, we generated bone marrow chimeric mice by transplanting BMC from transgenic green fluorescent protein (GFP) mice into lethally irradiated wild-type C57BL/6 mice. S. mansoni-infected chimeric mice did not demonstrate increased mortality and developed similar liver histopathological features, when compared to wild-type S. mansoni-infected mice. GFP(+) bone marrow-derived cells were found in the liver parenchyma, particularly in periportal regions. CD45(+)GFP(+) cells were found in the granulomas. Flow cytometry analysis of digested liver tissue characterized GFP(+) cells as lymphocytes, myeloid cells and stem cells. GFP(+) cells were also found in areas of collagen deposition, although rare GFP(+) cells expressed the myofibroblast cell marker α-SMA. Additionally GFP(+) endothelial cells (co-stained with von Willebrand factor) were frequently observed, while BMC-derived hepatocytes (GFP(+) albumin(+) cells) were sparsely found in the liver of chimeric mice chronically infected with S. mansoni. In conclusion, BMC are recruited to the liver during chronic experimental infection with S. mansoni and contribute to the generation of different cell types involved, not only in disease pathogenesis, but possibly in liver regeneration and repair. PMID:26297681

  11. A Method for Generation of Bone Marrow-Derived Macrophages from Cryopreserved Mouse Bone Marrow Cells

    PubMed Central

    Lima, Djalma S.; Zamboni, Dario S.

    2010-01-01

    The broad use of transgenic and gene-targeted mice has established bone marrow-derived macrophages (BMDM) as important mammalian host cells for investigation of the macrophages biology. Over the last decade, extensive research has been done to determine how to freeze and store viable hematopoietic human cells; however, there is no information regarding generation of BMDM from frozen murine bone marrow (BM) cells. Here, we establish a highly efficient protocol to freeze murine BM cells and further generate BMDM. Cryopreserved murine BM cells maintain their potential for BMDM differentiation for more than 6 years. We compared BMDM obtained from fresh and frozen BM cells and found that both are similarly able to trigger the expression of CD80 and CD86 in response to LPS or infection with the intracellular bacteria Legionella pneumophila. Additionally, BMDM obtained from fresh or frozen BM cells equally restrict or support the intracellular multiplication of pathogens such as L. pneumophila and the protozoan parasite Leishmania (L.) amazonensis. Although further investigation are required to support the use of the method for generation of dendritic cells, preliminary experiments indicate that bone marrow-derived dendritic cells can also be generated from cryopreserved BM cells. Overall, the method described and validated herein represents a technical advance as it allows ready and easy generation of BMDM from a stock of frozen BM cells. PMID:21179419

  12. Intravitreal Implantation of Genetically Modified Autologous Bone Marrow-Derived Stem Cells for Treating Retinal Disorders.

    PubMed

    Tracy, Christopher J; Sanders, Douglas N; Bryan, Jeffrey N; Jensen, Cheryl A; Castaner, Leilani J; Kirk, Mark D; Katz, Martin L

    2016-01-01

    A number of retinal degenerative diseases may be amenable to treatment with continuous intraocular delivery of therapeutic agents that cannot be delivered effectively to the retina via systemic or topical administration. Among these disorders are lysosomal storage diseases resulting from deficiencies in soluble lysosomal enzymes. Most cells, including those of the retina, are able to take up these enzymes and incorporate them in active form into their lysosomes. In theory, therefore, continuous intraocular administration of a normal form of a soluble lysosomal enzyme should be able to cure the molecular defect in the retinas of subjects lacking this enzyme. Experiments were conducted to determine whether genetically modified bone marrow-derived stem cells implanted into the vitreous could be used as -vehicles for continuous delivery of such enzymes to the retina. Bone marrow-derived mesenchymal stem cells (MSCs) from normal mice were implanted into the vitreous of mice undergoing retinal degeneration as a result of a mutation in the PPT1 gene. The implanted cells appeared to survive indefinitely in the vitreous without proliferating or invading the retina. This indicates that intravitreal implantation of MSCs is likely a safe means of long-term delivery of proteins synthesized by the implanted cells. Experiments have been initiated to test the efficacy of using genetically modified autologous MSCs to inhibit retinal degeneration in a canine model of neuronal ceroid lipofuscinosis. PMID:26427461

  13. Intravitreal Implantation of Genetically Modified Autologous Bone Marrow-Derived Stem Cells for Treating Retinal Disorders

    PubMed Central

    Tracy, Christopher J.; Sanders, Douglas N.; Bryan, Jeffrey N.; Jensen, Cheryl A.; Castaner, Leilani J.; Kirk, Mark D.; Katz, Martin L.

    2016-01-01

    A number of retinal degenerative diseases may be amenable to treatment with continuous intraocular delivery of therapeutic agents that cannot be delivered effectively to the retina via systemic or topical administration. Among these disorders are lysosomal storage diseases resulting from deficiencies in soluble lysosomal enzymes. Most cells, including those of the retina, are able to take up these enzymes and incorporate them in active form into their lysosomes. In theory, therefore, continuous intraocular administration of a normal form of a soluble lysosomal enzyme should be able to cure the molecular defect in the retinas of subjects lacking this enzyme. Experiments were conducted to determine whether genetically modified bone marrow-derived stem cells implanted into the vitreous could be used as vehicles for continuous delivery of such enzymes to the retina. Bone marrow-derived mesenchymal stem cells (MSCs) from normal mice were implanted into the vitreous of mice undergoing retinal degeneration as a result of a mutation in the PPT1 gene. The implanted cells appeared to survive indefinitely in the vitreous without proliferating or invading the retina. This indicates that intravitreal implantation of MSCs is likely a safe means of long-term delivery of proteins synthesized by the implanted cells. Experiments have been initiated to test the efficacy of using genetically modified autologous MSCs to inhibit retinal degeneration in a canine model of neuronal ceroid lipofuscinosis. PMID:26427461

  14. Onset of apoprotein E secretion during differentiation of mouse bone marrow-derived mononuclear phagocytes

    SciTech Connect

    Werb, Z.; Chin, J.R.

    1983-10-01

    A number of macrophage functions were sequentially expressed when the bone marrow precursors of mononuclear phagocytes differentiated in culture in the presence of a specific growth factor, colony-stimulating factor-1. The authors defined the expression of apoprotein E (ApoE), a major secreted protein of resident peritoneal macrophages, during maturation of adherent bone marrow-derived mononuclear phagocytes into macrophages. By 5 d the bone marrow macrophages were active secretory cells, but few cells contained intracellular immunoreactive ApoE, and little, if any, ApoE was secreted. ApoE secretion was initiated at 9 d, and this correlated with an increase in the percentage of macrophages containing intracellular ApoE. The onset of ApoE secretion was selective, and little change occurred in the other major secreted proteins detected by (/sup 35/S)methionine incorporation. In parallel, the high rate of plasminogen activator secretion, which peaked at 7 d, decreased markedly. ApoE secretion was not associated with altered expression of the macrophage surface antigen, la, or with secretion of fibronectin. Virtually all cells in independent colonies of bone marrow-derived macrophages eventually expressed ApoE. The proliferating monocyte/macrophage-like cell lines P388D1, J774.2, WHEI-3, RAW 264.1, and MGI.D/sup +/ secreted little or no ApoE. These data establish that ApoE secretion is developmentally regulated.

  15. Bone regenerates via dedifferentiation of osteoblasts in the zebrafish fin.

    PubMed

    Knopf, Franziska; Hammond, Christina; Chekuru, Avinash; Kurth, Thomas; Hans, Stefan; Weber, Christopher W; Mahatma, Gina; Fisher, Shannon; Brand, Michael; Schulte-Merker, Stefan; Weidinger, Gilbert

    2011-05-17

    While mammals have a limited capacity to repair bone defects, zebrafish can completely regenerate amputated bony structures of their fins. Fin regeneration is dependent on formation of a blastema, a progenitor cell pool accumulating at the amputation plane. It is unclear which cells the blastema is derived from, whether it forms by dedifferentiation of mature cells, and whether blastema cells are multipotent. We show that mature osteoblasts dedifferentiate and form part of the blastema. Osteoblasts downregulate expression of intermediate and late bone differentiation markers and induce genes expressed by bone progenitors. Dedifferentiated osteoblasts proliferate in a FGF-dependent manner and migrate to form part of the blastema. Genetic fate mapping shows that osteoblasts only give rise to osteoblasts in the regenerate, indicating that dedifferentiation is not associated with the attainment of multipotency. Thus, bone can regenerate from mature osteoblasts via dedifferentiation, a finding with potential implications for human bone repair. PMID:21571227

  16. Compensatory cellular reactions to nonsteroidal anti-inflammatory drugs on osteogenic differentiation in canine bone marrow-derived mesenchymal stem cells.

    PubMed

    Oh, Namgil; Kim, Sangho; Hosoya, Kenji; Okumura, Masahiro

    2014-05-01

    The suppressive effects of nonsteroidal anti-inflammatory drugs (NSAIDs) on the bone healing process have remained controversial, since no clinical data have clearly shown the relationship between NSAIDs and bone healing. The aim of this study was to assess the compensatory response of canine bone marrow-derived mesenchymal stem cells (BMSCs) to several classes of NSAIDs, including carprofen, meloxicam, indomethacin and robenacoxib, on osteogenic differentiation. Each of the NSAIDs (10 µM) was administered during 20 days of the osteogenic process with human recombinant IL-1β (1 ng/ml) as an inflammatory stimulator. Gene expression of osteoblast differentiation markers (alkaline phosphatase and osteocalcin), receptors of PGE2 (EP2 and EP4) and enzymes for prostaglandin (PG) E2 synthesis (COX-1, COX-2, cPGES and mPGES-1) was measured by using quantitative reverse transcription-polymerase chain reaction. Protein production levels of alkaline phosphatase, osteocalcin and PGE2 were quantified using an alkaline phosphatase activity assay, osteocalcin immunoassay and PGE2 immunoassay, respectively. Histologic analysis was performed using alkaline phosphatase staining, von Kossa staining and alizarin red staining. Alkaline phosphatase and calcium deposition were suppressed by all NSAIDs. However, osteocalcin production showed no significant suppression by NSAIDs. Gene expression levels of PGE2-related receptors and enzymes were upregulated during continuous treatment with NSAIDs, while certain channels for PGE2 synthesis were utilized differently depending on the kind of NSAIDs. These data suggest that canine BMSCs have a compensatory mechanism to restore PGE2 synthesis, which would be an intrinsic regulator to maintain differentiation of osteoblasts under NSAID treatment. PMID:24419976

  17. Evaluation of rhBMP-2 and bone marrow derived stromal cell mediated bone regeneration using transgenic fluorescent protein reporter mice

    PubMed Central

    Gohil, Shalini V.; Adams, Douglas J.; Maye, Peter; Rowe, David W.; Nair, Lakshmi S.

    2016-01-01

    The aim of the study is use of transgenic fluorescent protein reporter mouse models to understand the cellular processes in recombinant human bone morphogenetic protein-2 (rhBMP-2) mediated bone formation. Bilateral parietal calvarial bone defects in Col3.6Topaz transgenic fluorescent osteoblast reporter mouse were used to understand the bone formation in the presence and absence of rhBMP2 and/or Col3.6Cyan bone marrow derived stromal cells (BMSCs), using collagen-hydroxyapatite matrix (Healos) as a biomaterial. The bone regeneration was not confined to the site of BMP-2 implantation and significant bone formation was observed in the neighboring defect site. Osteogenic cellular activity with overlying alizarin complexone staining was observed in both the defects indicating host cell induced mineralization. However, implantation of BMSCs along with rhBMP-2 demonstrated a donor cell derived bone formation. The presence of rhBMP-2 did not support host cell recruitment in the presence of donor cells. This study demonstrates the potential of multiple fluorescent reporters to understand the cellular processes involved in the bone regeneration process using biomaterials, growth factors, and/or stem cells. PMID:24677665

  18. Bone-marrow-derived mesenchymal stem cells inhibit gastric aspiration lung injury and inflammation in rats.

    PubMed

    Zhou, Jing; Jiang, Liyan; Long, Xuan; Fu, Cuiping; Wang, Xiangdong; Wu, Xiaodan; Liu, Zilong; Zhu, Fen; Shi, Jindong; Li, Shanqun

    2016-09-01

    Gastric aspiration lung injury is one of the most common clinical events. This study investigated the effects of bone-marrow-derived mesenchymal stem cells (BMSCs) on combined acid plus small non-acidified particle (CASP)-induced aspiration lung injury. Enhanced green fluorescent protein (EGFP(+) ) or EGFP(-) BMSCs or 15d-PGJ2 were injected via the tail vein into rats immediately after CASP-induced aspiration lung injury. Pathological changes in lung tissues, blood gas analysis, the wet/dry weight ratio (W/D) of the lung, levels of total proteins and number of total cells and neutrophils in bronchoalveolar lavage fluid (BALF) were determined. The cytokine levels were measured using ELISA. Protein expression was determined by Western blot. Bone-marrow-derived mesenchymal stem cells treatment significantly reduced alveolar oedema, exudation and lung inflammation; increased the arterial partial pressure of oxygen; and decreased the W/D of the lung, the levels of total proteins and the number of total cells and neutrophils in BALF in the rats with CASP-induced lung injury. Bone-marrow-derived mesenchymal stem cells treatment decreased the levels of tumour necrosis factor-α and Cytokine-induced neutrophil chemoattractant (CINC)-1 and the expression of p-p65 and increased the levels of interleukin-10 and 15d-PGJ2 and the expression of peroxisome proliferator-activated receptor (PPAR)-γ in the lung tissue in CASP-induced rats. Tumour necrosis factor-α stimulated BMSCs to secrete 15d-PGJ2 . A tracking experiment showed that EGFP(+) BMSCs were able to migrate to local lung tissues. Treatment with 15d-PGJ2 also significantly inhibited CASP-induced lung inflammation and the production of pro-inflammatory cytokines. Our results show that BMSCs can protect lung tissues from gastric aspiration injury and inhibit lung inflammation in rats. A beneficial effect might be achieved through BMSC-derived 15d-PGJ2 activation of the PPAR-γ receptor, reducing the production of

  19. Comparison of human adipose-derived stem cells and bone marrow-derived stem cells in a myocardial infarction model.

    PubMed

    Rasmussen, Jeppe Grøndahl; Frøbert, Ole; Holst-Hansen, Claus; Kastrup, Jens; Baandrup, Ulrik; Zachar, Vladimir; Fink, Trine; Simonsen, Ulf

    2014-02-01

    Treatment of myocardial infarction (MI) with bone marrow-derived mesenchymal stem cells and recently also adipose-derived stem cells has shown promising results. In contrast to clinical trials and their use of autologous bone marrow-derived cells from the ischemic patient, the animal MI models are often using young donors and young, often immune-compromised, recipient animals. Our objective was to compare bone marrow-derived mesenchymal stem cells with adipose-derived stem cells from an elderly ischemic patient in the treatment of MI using a fully grown non-immune-compromised rat model. Mesenchymal stem cells were isolated from adipose tissue and bone marrow and compared with respect to surface markers and proliferative capability. To compare the regenerative potential of the two stem cell populations, male Sprague-Dawley rats were randomized to receive intramyocardial injections of adipose-derived stem cells, bone marrow-derived mesenchymal stem cells, or phosphate-buffered saline 1 week following induction of MI. After 4 weeks, left ventricular ejection fraction (LVEF) was improved in the adipose-derived stem cell group, and scar wall thickness was greater compared with the saline group. Adipose-derived as well as bone marrow-derived mesenchymal stem cells prevented left ventricular end diastolic dilation. Neither of the cell groups displayed increased angiogenesis in the myocardium compared with the saline group. Adipose-derived stem cells from a human ischemic patient preserved cardiac function following MI, whereas this could not be demonstrated for bone marrow-derived mesenchymal stem cells, with only adipose-derived stem cells leading to an improvement in LVEF. Neither of the stem cell types induced myocardial angiogenesis, raising the question whether donor age and health have an effect on the efficacy of stem cells used in the treatment of MI. PMID:23211469

  20. Suppression of ornithine decarboxylase promotes osteogenic differentiation of human bone marrow-derived mesenchymal stem cells.

    PubMed

    Tsai, Yo-Hsian; Lin, Kuan-Lian; Huang, Yuan-Pin; Hsu, Yi-Chiang; Chen, Chung-Hwan; Chen, Yuhsin; Sie, Min-Hua; Wang, Gwo-Jaw; Lee, Mon-Juan

    2015-07-22

    Ornithine decarboxylase (ODC) is the rate-limiting enzyme for polyamine biosynthesis. Suppression of ODC by its irreversible inhibitor, α-difluoromethylornithine (DFMO), or by RNA interference through siRNA, enhanced osteogenic gene expression and alkaline phosphatase activity, and accelerated matrix mineralization of human bone marrow-derived mesenchymal stem cells (hBMSCs). Besides, adipogenic gene expression and lipid accumulation was attenuated, indicating that the enhanced osteogenesis was accompanied by down-regulation of adipogenesis when ODC was suppressed. A decrease in the intracellular polyamine content of hBMSCs during osteogenic induction was observed, suggesting that the level of endogenous polyamines is regulated during differentiation of hBMSCs. This study elucidates the role of polyamine metabolism in the lineage commitment of stem cells and provides a potential new indication for DFMO as bone-stimulating drug. PMID:26140984

  1. Bone Marrow-Derived Cells as a Therapeutic Approach to Optic Nerve Diseases.

    PubMed

    Mesentier-Louro, Louise A; Zaverucha-do-Valle, Camila; Rosado-de-Castro, Paulo H; Silva-Junior, Almir J; Pimentel-Coelho, Pedro M; Mendez-Otero, Rosalia; Santiago, Marcelo F

    2016-01-01

    Following optic nerve injury associated with acute or progressive diseases, retinal ganglion cells (RGCs) of adult mammals degenerate and undergo apoptosis. These diseases have limited therapeutic options, due to the low inherent capacity of RGCs to regenerate and due to the inhibitory milieu of the central nervous system. Among the numerous treatment approaches investigated to stimulate neuronal survival and axonal extension, cell transplantation emerges as a promising option. This review focuses on cell therapies with bone marrow mononuclear cells and bone marrow-derived mesenchymal stem cells, which have shown positive therapeutic effects in animal models of optic neuropathies. Different aspects of available preclinical studies are analyzed, including cell distribution, potential doses, routes of administration, and mechanisms of action. Finally, published and ongoing clinical trials are summarized. PMID:26649049

  2. Bone Marrow-Derived Cells as a Therapeutic Approach to Optic Nerve Diseases

    PubMed Central

    Mesentier-Louro, Louise A.; Zaverucha-do-Valle, Camila; Rosado-de-Castro, Paulo H.; Silva-Junior, Almir J.; Pimentel-Coelho, Pedro M.; Mendez-Otero, Rosalia; Santiago, Marcelo F.

    2016-01-01

    Following optic nerve injury associated with acute or progressive diseases, retinal ganglion cells (RGCs) of adult mammals degenerate and undergo apoptosis. These diseases have limited therapeutic options, due to the low inherent capacity of RGCs to regenerate and due to the inhibitory milieu of the central nervous system. Among the numerous treatment approaches investigated to stimulate neuronal survival and axonal extension, cell transplantation emerges as a promising option. This review focuses on cell therapies with bone marrow mononuclear cells and bone marrow-derived mesenchymal stem cells, which have shown positive therapeutic effects in animal models of optic neuropathies. Different aspects of available preclinical studies are analyzed, including cell distribution, potential doses, routes of administration, and mechanisms of action. Finally, published and ongoing clinical trials are summarized. PMID:26649049

  3. Bone marrow derived stem cells in regenerative medicine as Advanced Therapy Medicinal Products

    PubMed Central

    Astori, Giuseppe; Soncin, Sabrina; Lo Cicero, Viviana; Siclari, Francesco; Sürder, Daniel; Turchetto, Lucia; Soldati, Gianni; Moccetti, Tiziano

    2010-01-01

    Bone marrow derived stem cells administered after minimal manipulation represent an important cell source for cellbased therapies. Clinical trial results, have revealed both safety and efficacy of the cell reinfusion procedure in many cardiovascular diseases. Many of these early clinical trials were performed in a period before the entry into force of the US and European regulation on cellbased therapies. As a result, conflicting data have been generated on the effectiveness of those therapies in certain conditions as acute myocardial infarction. As more academic medical centers and private companies move toward exploiting the full potential of cellbased medicinal products, needs arise for the development of the infrastructure necessary to support these investigations. This review describes the regulatory environment surrounding the production of cell based medicinal products and give practical aspects for cell isolation, characterization, production following Good Manufacturing Practice, focusing on the activities associated with the investigational new drug development. PMID:20589167

  4. Use of bone marrow derived stem cells in trauma and orthopaedics: A review of current concepts

    PubMed Central

    Pastides, Philip S; Welck, Matthew J; Khan, Wasim S

    2015-01-01

    There is a considerable amount of interest in the future role of bone marrow-derived stem cells (BMDSCs) and tissue engineering techniques to manage conditions within the musculoskeletal system. Repair of soft tissue and bone defects, in the early stages of injury, may lead to a reduction in progression of symptoms. Furthermore, troublesome soft tissue injuries that are notoriously fraught with problems either in healing or function, could be augmented with such techniques. The aim of this review paper is to look at the advances in such strategies to tackle these problems and assess how BMDSCs, with the aid of growth factors and scaffolds, are being used in vitro, animal and even human models to treat problems within the field of trauma and orthopaedics. There is plenty of evidence that the results are encouraging and thus gaining momentum toward their use in human studies. PMID:26191493

  5. Generation and characterization of bovine bone marrow-derived macrophage cell line.

    PubMed

    Xiao, Jiajia; Xie, Rongxia; Li, Qiaoqiao; Chen, Wuju; Zhang, Yong

    2016-05-01

    Macrophages, as the forefront of innate immune defense, have an important role in the host responses to mycobacterial infection. Therefore, a stable macrophage cell line is needed for future bovine immune system research on the bacterial infection. In this study, we established a bovine macrophage cell line by introducing the human telomerase reverse transcriptase (hTERT) gene into bovine bone marrow-derived macrophages (bBMMs). The TERT-bBMMs cells expressed macrophage surface antigen (CD11b, CD282) and upregulated expression of the cytokines IL-1β, IL-6, IL-10, IL-12, TNF-α in response to bacterial invasion. These results demonstrate that this cell line provide reliable cell model system for future studies on interactions between the bovine macrophages and Mycobacterium tuberculosis. PMID:26936441

  6. Immunoregulatory effects of bone marrow-derived mesenchymal stem cells in the nasal polyp microenvironment.

    PubMed

    Pezato, Rogério; de Almeida, Danilo Cândido; Bezerra, Thiago Freire; Silva, Fernando de Sá; Perez-Novo, Claudina; Gregório, Luís Carlos; Voegels, Richard Louis; Câmara, Niels Olsen; Bachert, Claus

    2014-01-01

    Nasal polyposis is a severe, chronic inflammatory condition of the paranasal sinuses and is frequently associated with asthma and aspirin sensitivity. Mesenchymal stem cells exhibit a potent immunosuppressive effect in several inflammatory conditions, and their role in nasal polyposis remains little explored. Hence, we investigated whether bone marrow-derived mesenchymal stem cells could modulate cell phenotype in the nasal polyp milieu. After coculture with mesenchymal stem cells, the frequency of these inflammatory cells was found to decrease. Furthermore, mesenchymal stem cells promoted strong inhibition of CD4+ and CD8+ T cell proliferation, increased the frequency of CD4+CD25+Foxp3 T cells, and changed the global cytokine profile from an inflammatory to an anti-inflammatory response. We believe that mesenchymal stem cells may be a very useful adjunct for investigation of the inflammatory process in nasal polyposis, contributing to better understanding of the inflammatory course of this condition. PMID:24707116

  7. Different Balance of Wnt Signaling in Adult and Fetal Bone Marrow-Derived Mesenchymal Stromal Cells.

    PubMed

    Paciejewska, Maja M; Maijenburg, Marijke W; Gilissen, Christian; Kleijer, Marion; Vermeul, Kim; Weijer, Kees; Veltman, Joris A; von Lindern, Marieke; van der Schoot, C Ellen; Voermans, Carlijn

    2016-06-15

    Mesenchymal stromal cells (MSCs) are applied as novel therapeutics for their regenerative and immune-suppressive capacities. Clinical applications, however, require extensive expansion of MSCs. Fetal bone marrow-derived MSCs (FBMSCs) proliferate faster than adult bone marrow-derived MSC (ABMSCs). To optimize expansion and function of MSC in general, we explored the differences between ABMSC and FBMSC. Gene expression profiling implicated differential expression of genes encoding proteins in the Wnt signaling pathway, including excreted inhibitors of Wnt signaling, particularly by ABMSC. Both MSC types had a similar basal level of canonical Wnt signaling. Abrogation of autocrine Wnt production by inhibitor of Wnt production-2 (IWP2) reduced canonical Wnt signaling and cell proliferation of FBMSCs, but hardly affected ABMSC. Addition of exogenous Wnt3a, however, induced expression of the target genes lymphocyte enhancer-binding factor (LEF) and T-cell factor (TCF) faster and at lower Wnt3a levels in ABMSC compared to FBMSC. Medium replacement experiments indicated that ABMSC produce an inhibitor of Wnt signaling that is effective on ABMSC itself but not on FBMSC, whereas FBMSC excrete (Wnt) factors that stimulate proliferation of ABMSC. In contrast, FBMSC were not able to support hematopoiesis, whereas ABMSC displayed hematopoietic support sensitive to IWP2, the inhibitor of Wnt factor excretion. In conclusion, ABMSC and FBMSC differ in their Wnt signature. While FBMSC produced factors, including Wnt signals, that enhanced MSC proliferation, ABMSC produced Wnt factors in a setting that enhanced hematopoietic support. Thus, further unraveling the molecular basis of this phenomenon may lead to improvement of clinical expansion protocols of ABMSCs. PMID:27154244

  8. Spatial and Temporal Coordination of Bone Marrow-Derived Cell Activity During Arteriogenesis: Regulation of the Endogenous Response and Therapeutic Implications

    PubMed Central

    Meisner, Joshua K.; Price, Richard J.

    2010-01-01

    Arterial occlusive disease (AOD) is the leading cause of morbidity and mortality through the developed world, which creates a significant need for effective therapies to halt disease progression. Despite success of animal and small-scale human therapeutic arteriogenesis studies, this promising concept for treating AOD has yielded largely disappointing results in large-scale clinical trials. One reason for this lack of successful translation is that endogenous arteriogenesis is highly dependent on a poorly understood sequence of events and interactions between bone marrow derived cells (BMCs) and vascular cells, which makes designing effective therapies difficult. We contend that the process follows a complex, ordered sequence of events with multiple, specific BMC populations recruited at specific times and locations. Here we present the evidence suggesting roles for multiple BMC populations from neutrophils and mast cells to progenitor cells and propose how and where these cell populations fit within the sequence of events during arteriogenesis. Disruptions in these various BMC populations can impair the arteriogenesis process in patterns that characterize specific patient populations. We propose that an improved understanding of how arteriogenesis functions as a system can reveal individual BMC populations and functions that can be targeted for overcoming particular impairments in collateral vessel development. PMID:21044213

  9. Donor-matched comparison of dental pulp stem cells and bone marrow-derived mesenchymal stem cells in a rat model

    PubMed Central

    Alge, Daniel L.; Zhou, Dan; Adams, Lyndsey L.; Wyss, Brandon K.; Shadday, Matthew D.; Woods, Erik J.; Chu, T.M. Gabriel; Goebel, W. Scott

    2010-01-01

    Dental pulp stem cells (DPSC) have drawn much interest for the regeneration of mineralized tissues, and several studies have compared DPSC to bone marrow-derived mesenchymal stem cells (BMMSC). However, conflicting results, possibly due to donor-associated variability, have been published and the regenerative potential of DPSC is currently unclear. In the present study we have sought to address this problem using a donor-matched experimental design to robustly compare the biological properties of DPSC and BMMSC. All experiments were performed using cells isolated from a single adult Sprague-Dawley rat. Our results show that DPSC and BMMSC had similar morphologies and flow cytometry profiles, were capable of forming colonies in vitro, and were capable of osteogenic, chondrogenic, and adipogenic differentiation. However, quantitative comparisons revealed that DPSC had a faster population doubling time and a higher percentage of stem/progenitor cells in the population as determined by clonogenic assays. Furthermore, while both cell populations formed mineral in vitro, DPSC had significantly higher alkaline phosphatase activity than BMMSC after three weeks in osteogenic medium. These data show several key differences between DPSC and BMMSC and support the possibility of using DPSC for mineralized tissue regeneration. PMID:19842108

  10. miR-210 promotes osteoblastic differentiation through inhibition of AcvR1b.

    PubMed

    Mizuno, Yosuke; Tokuzawa, Yoshimi; Ninomiya, Yuichi; Yagi, Ken; Yatsuka-Kanesaki, Yukiko; Suda, Tatsuo; Fukuda, Toru; Katagiri, Takenobu; Kondoh, Yasumitsu; Amemiya, Tomoyuki; Tashiro, Hideo; Okazaki, Yasushi

    2009-07-01

    Although microRNAs (miRNAs) are involved in many biological processes, the mechanisms whereby miRNAs regulate osteoblastic differentiation are poorly understood. Here, we found that BMP-4-induced osteoblastic differentiation of bone marrow-derived ST2 stromal cells was promoted and repressed after transfection of sense and antisense miR-210, respectively. A reporter assay demonstrated that the activin A receptor type 1B (AcvR1b) gene was a target for miR-210. Furthermore, inhibition of transforming growth factor-beta (TGF-beta)/activin signaling in ST2 cells with SB431542 promoted osteoblastic differentiation. We conclude that miR-210 acts as a positive regulator of osteoblastic differentiation by inhibiting the TGF-beta/activin signaling pathway through inhibition of AcvR1b. PMID:19520079

  11. Restoration of the GM2 ganglioside metabolism in bone marrow-derived stromal cells from Tay-Sachs disease animal model.

    PubMed

    Martino, S; Cavalieri, C; Emiliani, C; Dolcetta, D; Cusella De Angelis, M G; Chigorno, V; Severini, G M; Sandhoff, K; Bordignon, C; Sonnino, S; Orlacchio, A

    2002-08-01

    The therapeutic potential of bone marrow-derived stromal cells for the therapy of Tay-Sachs disease is primarily related to the restoration of their own GM2 ganglioside storage. With this aim, we produced bone marrow-derived stromal cells from the adult Tay-Sachs animal model and transduced them with a retroviral vector encoding for the alpha-subunit of the lysosomal enzyme beta-hexosaminidase A (E.C. 3.2.1.52). Our results demonstrate that transduced Tay-Sachs bone marrow-derived stromal cells have beta-hexosaminidase A comparable to that of bone marrow-derived stromal cells from wild-type mice. Moreover, beta-hexosaminidase A in transduced Tay-Sachs bone marrow-derived stromal cells was able to hydrolyze the GM2 ganglioside in a feeding experiment, thus demonstrating the correction of the altered phenotype. PMID:12374215

  12. Sepsis-Induced Osteoblast Ablation Causes Immunodeficiency.

    PubMed

    Terashima, Asuka; Okamoto, Kazuo; Nakashima, Tomoki; Akira, Shizuo; Ikuta, Koichi; Takayanagi, Hiroshi

    2016-06-21

    Sepsis is a host inflammatory response to severe infection associated with high mortality that is caused by lymphopenia-associated immunodeficiency. However, it is unknown how lymphopenia persists after the accelerated lymphocyte apoptosis subsides. Here we show that sepsis rapidly ablated osteoblasts, which reduced the number of common lymphoid progenitors (CLPs). Osteoblast ablation or inducible deletion of interleukin-7 (IL-7) in osteoblasts recapitulated the lymphopenic phenotype together with a lower CLP number without affecting hematopoietic stem cells (HSCs). Pharmacological activation of osteoblasts improved sepsis-induced lymphopenia. This study demonstrates a reciprocal interaction between the immune and bone systems, in which acute inflammation induces a defect in bone cells resulting in lymphopenia-associated immunodeficiency, indicating that bone cells comprise a therapeutic target in certain life-threatening immune reactions. PMID:27317262

  13. Different Forms of Tenascin-C with Tenascin-R Regulate Neural Differentiation in Bone Marrow-Derived Human Mesenchymal Stem Cells

    PubMed Central

    Tsai, Hung-Li; Chiu, Wen-Ta; Fang, Chia-Lang; Hwang, Shiaw-Min; Renshaw, Perry F.

    2014-01-01

    Mesenchymal stem cells (MSCs) are currently thought to transdifferentiate into neural lineages under specific microenvironments. Studies have reported that the tenascin family members, tenascin-C (TnC) and tenascin-R (TnR), regulate differentiation and migration, in addition to neurite outgrowth and survival in numerous types of neurons and mesenchymal progenitor cells. However, the mechanisms by which TnC and TnR affect neuronal differentiation are not well understood. In this study, we hypothesized that different forms of tenascin might regulate the neural transdifferentiation of human bone marrow-derived mesenchymal stem cells. Human MSCs were cultured in media incorporated with soluble tenascins, or on precoated tenascins. In a qualitative polymerase chain reaction analysis, adding a soluble TnC and TnR mixture to the medium significantly enhanced the expression of neuronal and glial markers, whereas no synaptic markers were expressed. Conversely, in groups of cells treated with coated TnC, hMSCs showed neurite outgrowth and synaptic marker expression. After being treated with coated TnR, hMSCs exhibited neuronal differentiation; however, it inhibited neurite outgrowth and synaptic marker expression. A combination of TnC and TnR significantly promoted hMSC differentiation in neurons or oligodendrocytes, induced neurite formation, and inhibited differentiation into astrocytes. Furthermore, the effect of the tenascin mixture showed dose-dependent effects, and a mixture ratio of 1:1 to 1:2 (TnC:TnR) provided the most obvious differentiation of neurons and oligodendrocytes. In a functional blocking study, integrin α7 and α9β1-blocking antibodies inhibited, respectively, 80% and 20% of mRNA expression by hMSCs in the coated tenascin mixture. In summary, the coated combination of TnC and TnR appeared to regulate neural differentiation signaling through integrin α7 and α9β1 in bone marrow-derived hMSCs. Our findings demonstrate novel mechanisms by which tenascin

  14. Bone marrow and bone marrow derived mononuclear stem cells therapy for the chronically ischemic myocardium

    SciTech Connect

    Waksman, Ron; Baffour, Richard

    2003-09-01

    Bone marrow stem cells have been shown to differentiate into various phenotypes including cardiomyocytes, vascular endothelial cells and smooth muscle. Bone marrow stem cells are mobilized and home in to areas of injured myocardium where they are involved in tissue repair. In addition, bone marrow secretes multiple growth factors, which are essential for angiogenesis and arteriogenesis. In some patients, these processes are not enough to avert clinical symptoms of ischemic disease. Therefore, in vivo administration of an adequate number of stem cells would be a significant therapeutic advance. Unfractionated bone marrow derived mononuclear stem cells, which contain both hematopoietic and nonhematopoietic cells may be more appropriate for cell therapy. Studies in animal models suggest that implantation of different types of stem cells improve angiogenesis and arteriogenesis, tissue perfusion as well as left ventricular function. Several unanswered questions remain. For example, the optimal delivery approach, dosage and timing of the administration of cell therapy as well as durability of improvements need to be studied. Early clinical studies have demonstrated safety and feasibility of various cell therapies in ischemic disease. Randomized, double blind and placebo-controlled clinical trials need to be completed to determine the effectiveness of stem cell.

  15. Data on nitric oxide production by human bone marrow-derived mesenchymal stromal cells.

    PubMed

    Najar, Mehdi; Fayyad-Kazan, Mohammad; Fayyad-Kazan, Hussein; Meuleman, Nathalie; Bron, Dominique; Lagneaux, Laurence

    2016-09-01

    Due to its anti-inflammatory and immunosuppressive potential, Nitric oxide (NO), a gaseous radical, is of special importance during graft-versus-host diseases (GVHD) and feoto-maternal tolerance. NO is a major mediator of murine mesenchymal stromal cells (MSCs)-immunosuppressive capacity. In this data article, we characterized NO production by human bone marrow-derived MSCs (hBMSCs). MSCs, isolated from healthy donors (n=5), were defined according to the International Society for cellular Therapy (ISCT) guidelines. Based on a fluorometric detection system, and upon using Nitrite ([Formula: see text])/Nitrate ( [Formula: see text]) Assay Kit, the amounts of NO metabolites ( [Formula: see text] and [Formula: see text]) produced by hBMSCs, being grown in a culture medium either lacking (constitutive condition) or containing IL-4, IL-10 or a pro-inflammatory cytokine cocktail made of IL-1β, TNF-α, IFN-α and IFN-γ, were assessed. All assays were carried out in triplicates and the mean values are reported. The data from this study supports and corroborates the discussion associated with our previously published work entitled "The Immunomodulatory Potential of Mesenchymal Stromal Cells: A Story of a Regulatory Network" (Najar et al., 2016) [1]. PMID:27536712

  16. Bone marrow-derived macrophages exclusively expressed caveolin-2: The role of inflammatory activators and hypoxia.

    PubMed

    Maceckova, Michaela; Martiskova, Hana; Koudelka, Adolf; Kubala, Lukas; Lojek, Antonin; Pekarova, Michaela

    2015-11-01

    Caveolins are specific proteins involved in regulation of signal transduction to intracellular space. Still, their contribution to immune functions has not been completely clarified. Thus, we decided to characterize the expression of caveolins in bone marrow-derived macrophages (BMDMs) under resting and inflammatory conditions. The effect of classical activators (lipopolysaccharide, LPS; interferon-gamma, IFN-γ) was further potentiated with hypoxic (5% O2) conditions. The activation of p44/42-extracellular signal-regulated kinases 1 and 2 (ERK1/2) and expression of caveolin-1, -2, and -3, hypoxia inducible factor-1 alpha (HIF-1α), as well as inducible nitric oxide synthase (iNOS) was monitored using the Western blot technique. The production of nitric oxide (NO) and tumor necrosis factor-alpha (TNFα) was analyzed by Griess method or ELISA, respectively. BMDMs were also transfected with siRNA against caveolin-2. Importantly, our study showed for the first time that BMDMs expressed only caveolin-2, and its level decreased after activation of macrophages with LPS, IFN-γ, and/or hypoxia. The expression of caveolin-2 negatively correlates with the iNOS and HIF-1α protein levels, as well as with the LPS/IFN-γ- and hypoxia-induced activation of ERK1/2. We concluded that caveolin-2 is most probably involved in regulation of pro-inflammatory responses of BMDMs, triggered via activation of ERK1/2. PMID:26215374

  17. Multiple Tumor Types May Originate from Bone Marrow-Derived Cells1*

    PubMed Central

    Liu, Chunfang; Chen, Zhongwei; Chen, Zhihong; Zhang, Tao; Lu, Yuan

    2006-01-01

    Abstract It was believed that tumors originated from the transformation of their tissue-specific stem cells. However, bone marrow-derived cells (BMDCs), which possess an unexpected degree of plasticity and often reside in other tissues, might also represent a potential source of malignancy. To study whether BMDCs play a role in the source of other tumors, BMDCs from mice were treated with 3-methycholanthrene until malignant transformation was achieved. Here we show that transformed BMDCs could form many tumor types, including epithelial tumors, neural tumors, muscular tumors, tumors of fibroblasts, blood vessel endothelial tumors, and tumors of poor differentiation in vivo. Moreover, a single transformed BMDC has the ability to self-renew, differentiate spontaneously into various types of tumor cells in vitro, express markers associated with multipotency, and form teratoma in vivo. These data suggest that multipotent cancer stem cells seemed to originate from transformed BMDCs. Conclusively, these findings reveal that BMDCs might be a source of many tumor types, even teratoma. In addition, multipotent cancer stem cells might originate from malignant transformed BMDCs. PMID:16984729

  18. Retinal Electrophysiological Effects of Intravitreal Bone Marrow Derived Mesenchymal Stem Cells in Streptozotocin Induced Diabetic Rats

    PubMed Central

    Akkoç, Tolga; Eraslan, Muhsin; Şahin, Özlem; Özkara, Selvinaz; Vardar Aker, Fugen; Subaşı, Cansu; Karaöz, Erdal; Akkoç, Tunç

    2016-01-01

    Diabetic retinopathy is the most common cause of legal blindness in developed countries at middle age adults. In this study diabetes was induced by streptozotocin (STZ) in male Wistar albino rats. After 3 months of diabetes, rights eye were injected intravitreally with green fluorescein protein (GFP) labelled bone marrow derived stem cells (BMSC) and left eyes with balanced salt solution (Sham). Animals were grouped as Baseline (n = 51), Diabetic (n = 45), Diabetic+BMSC (n = 45 eyes), Diabetic+Sham (n = 45 eyes), Healthy+BMSC (n = 6 eyes), Healthy+Sham (n = 6 eyes). Immunohistology analysis showed an increased retinal gliosis in the Diabetic group, compared to Baseline group, which was assessed with GFAP and vimentin expression. In the immunofluorescence analysis BMSC were observed to integrate mostly into the inner retina and expressing GFP. Diabetic group had prominently lower oscillatory potential wave amplitudes than the Baseline group. Three weeks after intravitreal injection Diabetic+BMSC group had significantly better amplitudes than the Diabetic+Sham group. Taken together intravitreal BMSC were thought to improve visual function. PMID:27300133

  19. Chemically Functionalized Silk for Human Bone Marrow-Derived Mesenchymal Stem Cells Proliferation and Differentiation.

    PubMed

    Zheng, Ke; Chen, Ying; Huang, Wenwen; Lin, Yinan; Kaplan, David L; Fan, Yimin

    2016-06-15

    To produce biocompatible, mechanically robust, and conductive materials for bone tissue engineering, chemical oxidation using sodium hyprochlorite (NaClO) was utilized to introduce carboxyl groups onto silk fibroin (SF). A final carboxyl content of 1.09 mM/g SF was obtained, corresponding to ∼47% of the primary hydroxymethyl groups on the silk. Interestingly, both infrared (IR) spectroscopy and circular dichroism (CD) spectra demonstrated that the resulting oxidized silk (OxSF) self-assembled into β-sheet structures under aqueous conditions and this contributed to the mechanical properties of the as-prepared silk-based scaffolds and the mineralized OxSF scaffolds (M-OxSF). The OxSF scaffolds had a compressive modulus of 211 ± 75 KPa in the hydrated state, 10 times higher than that of the SF scaffolds, and the modulus of the M-OxSF scaffolds was increased to 758 ± 189 KPa. Human bone marrow-derived mesenchymal stem cells (hMSCs) grown on the scaffolds during osteogenesis showed that the OxSF scaffolds supported the proliferation and differentiation of hMSCs in vitro. PMID:27177120

  20. On the origin of human adipocytes and the contribution of bone marrow-derived cells.

    PubMed

    Rydén, Mikael

    2016-01-01

    In the last decade, results in both animal models and humans have demonstrated that white adipocytes are generated over the entire life-span. This adds to the plasticity of adipose tissue and alterations in adipocyte turnover are linked to metabolic dysfunction. Adipocytes are derived from precursors present primarily in the perivascular areas of adipose tissue but their precise origin remains unclear. The multipotent differentiation capacity of bone marrow-derived cells (BMDC) has prompted the suggestion that BMDC may contribute to different cell tissue pools, including adipocytes. However, data in murine transplantation models have been conflicting and it has been a matter of debate whether BMDC actually differentiate into adipocytes or just fuse with resident fat cells. To resolve this controversy in humans, we recently performed a study in 65 subjects that had undergone bone marrow transplantation. Using a set of newly developed assays including single cell genome-wide analyses of mature adipocytes, we demonstrated that bone marrow contributes with approximately 10 % to the adipocyte pool. This proportion was more than doubled in obesity, suggesting that BMDC may constitute a reserve pool for adipogenesis, particularly upon weight gain. This commentary discusses the possible relevance of these and other recent findings for human pathophysiology. PMID:27617752

  1. Distinct functional responses to stressors of bone marrow derived dendritic cells from diverse inbred chicken lines.

    PubMed

    Van Goor, Angelica; Slawinska, Anna; Schmidt, Carl J; Lamont, Susan J

    2016-10-01

    Differences in responses of chicken bone marrow derived dendritic cells (BMDC) to in vitro treatment with lipopolysaccharide (LPS), heat, and LPS + heat were identified. The Fayoumi is more disease resistant and heat tolerant than the Leghorn line. Nitric Oxide (NO) production, phagocytic ability, MHC II surface expression and mRNA expression were measured. NO was induced in BMDC from both lines in response to LPS and LPS + heat stimulation; Fayoumi produced more NO with LPS treatment. Fayoumi had higher phagocytic ability and MHC II surface expression. Gene expression for the heat-related genes BAG3, HSP25, HSPA2, and HSPH1 was strongly induced with heat and few differences existed between lines. Expression for the immune-related genes CCL4, CCL5, CD40, GM-CSF, IFN-γ, IL-10, IL-12β, IL-1β, IL-6, IL-8, and iNOS was highly induced in response to LPS and different between lines. This research contributes to the sparse knowledge of genetic differences in chicken BMDC biology and function. PMID:27238770

  2. Bone marrow-derived cells serve as proangiogenic macrophages but not endothelial cells in wound healing

    PubMed Central

    Okuno, Yuji; Nakamura-Ishizu, Ayako; Kishi, Kazuo; Suda, Toshio

    2011-01-01

    Bone marrow-derived cells (BMDCs) contribute to postnatal vascular growth by differentiating into endothelial cells or secreting angiogenic factors. However, the extent of their endothelial differentiation highly varies according to the angiogenic models used. Wound healing is an intricate process in which the skin repairs itself after injury. As a process also observed in cancer progression, neoangiogenesis into wound tissues is profoundly involved in this healing process, suggesting the contribution of BMDCs. However, the extent of the differentiation of BMDCs to endothelial cells in wound healing is unclear. In this study, using the green fluorescent protein-bone marrow chim-eric experiment and high resolution confocal microscopy at a single cell level, we observed no endothelial differentiation of BMDCs in 2 acute wound healing models (dorsal excisional wound and ear punch) and a chronic wound healing model (decubitus ulcer). Instead, a major proportion of BMDCs were macrophages. Indeed, colony-stimulating factor 1 (CSF-1) inhibition depleted approximately 80% of the BMDCs at the wound healing site. CSF-1–mutant (CSF-1op/op) mice showed significantly reduced neoangiogenesis into the wound site, supporting the substantial role of BMDCs as macrophages. Our data show that the proangiogenic effects of macrophages, but not the endothelial differentiation, are the major contribution of BMDCs in wound healing. PMID:21411758

  3. Sertoli cells promote proliferation of bone marrow-derived mesenchymal stem cells in co-culture.

    PubMed

    Zhang, Fenxi; Lu, Ming; Liu, Hengxing; Ren, Tongming; Miao, Yingying; Wang, Jingjing

    2016-05-01

    Bone marrow-derived mesenchymal stem cells (BMSCs) are a major source for cell transplantation. The proliferative ability of BMSCs is an important determinant of the efficiency of transplant therapy. Sertoli cells are "nurse" cells for development of sperm cells. Our recent study showed that Sertoli cells promoted proliferation of human umbilical cord mesenchymal stem cells (hUCMSCs) in co-culture. Studies by other groups also showed that Sertoli cells promoted growth of endothelial cells and neural stem cells. In this study, we investigated the effect of Sertoli cells on proliferation of BMSCs. Our results showed that Sertoli cells in co-culture significantly enhanced proliferation of BMSCs (P < 0.01). Moreover, co-culture with Sertoli cells also markedly increased mRNA and/or protein expressions of Mdm2, p-Akt and Cyclin D1, and decreased p53 expression in BMSCs (P < 0.01 or < 0.05). These findings indicate that Sertoli cells have the potential to enhance proliferation of BMSCs. PMID:27319049

  4. Age-associated metabolic dysregulation in bone marrow-derived macrophages stimulated with lipopolysaccharide.

    PubMed

    Fei, Fan; Lee, Keith M; McCarry, Brian E; Bowdish, Dawn M E

    2016-01-01

    Macrophages are major contributors to age-associated inflammation. Metabolic processes such as oxidative phosphorylation, glycolysis and the urea cycle regulate inflammatory responses by macrophages. Metabolic profiles changes with age; therefore, we hypothesized that dysregulation of metabolic processes could contribute to macrophage hyporesponsiveness to LPS. We examined the intracellular metabolome of bone marrow-derived macrophages from young (6-8 wk) and old (18-22 mo) mice following lipopolysaccharide (LPS) stimulation and tolerance. We discovered known and novel metabolites that were associated with the LPS response of macrophages from young mice, which were not inducible in macrophages from old mice. Macrophages from old mice were largely non-responsive towards LPS stimulation, and we did not observe a shift from oxidative phosphorylation to glycolysis. The critical regulatory metabolites succinate, γ-aminobutyric acid, arginine, ornithine and adenosine were increased in LPS-stimulated macrophages from young mice, but not macrophages from old mice. A shift between glycolysis and oxidative phosphorylation was not observed during LPS tolerance in macrophages from either young or old mice. Metabolic bottlenecks may be one of the mechanisms that contribute to the dysregulation of LPS responses with age. PMID:26940652

  5. Multiple loci govern the bone marrow-derived immunoregulatory mechanism controlling dominant resistance to autoimmune orchitis.

    PubMed Central

    Meeker, N D; Hickey, W F; Korngold, R; Hansen, W K; Sudweeks, J D; Wardell, B B; Griffith, J S; Teuscher, C

    1995-01-01

    The existence of immunoregulatory genes conferring dominant resistance to autoimmunity is well documented. In an effort to better understand the nature and mechanisms of action of these genes, we utilized the murine model of autoimmune orchitis as a prototype. When the orchitis-resistant strain DBA/2J is crossed with the orchitis-susceptible strain BALB/cByJ, the F1 hybrid is completely resistant to the disease. By using reciprocal radiation bone marrow chimeras, the functional component mediating this resistance was mapped to the bone marrow-derived compartment. Resistance is not a function of either low-dose irradiation- or cyclophosphamide (20 mg/kg)-sensitive immunoregulatory cells, but can be adoptively transferred by primed splenocytes. Genome exclusion mapping identified three loci controlling the resistant phenotype. Orch3 maps to chromosome 11, whereas Orch4 and Orch5 map to the telomeric and centromeric regions of chromosome 1, respectively. All three genes are linked to a number of immunologically relevant candidate loci. Most significant, however, is the linkage of Orch3 to Idd4 and Orch5 to Idd5, two susceptibility genes which play a role in autoimmune insulin-dependent type 1 diabetes mellitus in the nonobese diabetic mouse. PMID:7777570

  6. Vanadate inhibits dexamethasone-induced apoptosis of rat bone marrow-derived mesenchymal stem cells.

    PubMed

    Fan, Qie; Zhan, Xinli; Li, Xiaofeng; Zhao, Jinmin; Chen, Yueping

    2015-01-01

    Apoptosis of bone marrow-derived mesenchymal stem cells (BM-MSCs) has been shown to contribute to the development of osteoporosis, which is often the result of long-term use of glucocorticoid drugs such as dexamethasone (Dex). However, it remains unknown whether Dex induces apoptosis of BM-MSCs, and whether a chemical agent like vanadate can block such effects. To investigate these two issues, we isolated BM-MSCs from SD rats and treated the cells with different doses of Dex. We found that Dex induced apoptosis in dose- and time-dependent manners. Pretreating BM-MSCs with vanadate prevented Dex-induced apoptosis. Furthermore, we found that expression of caspases (3, 8, and 9) increased in Dex-treated BM-MSC and was attenuated by vanadate pretreatment. These results not only demonstrate the role of vanadate in the inhibition of Dex-induced apoptosis of BM-MSCs, but also reveal the therapeutic potential of vanadate in glucocorticoid-mediated osteoporosis. PMID:25887871

  7. A Modified Method of Insulin Producing Cells' Generation from Bone Marrow-Derived Mesenchymal Stem Cells

    PubMed Central

    Czubak, Paweł; Putowski, Lechosław

    2014-01-01

    Type 1 diabetes mellitus is a result of autoimmune destruction of pancreatic insulin producing β-cells and so far it can be cured only by insulin injection, by pancreas transplantation, or by pancreatic islet cells' transplantation. The methods are, however, imperfect and have a lot of disadvantages. Therefore new solutions are needed. The best one would be the use of differentiated mesenchymal stem cells (MSCs). In the present study, we investigated the potential of the bone marrow-derived MSCs line for in vitro differentiation into insulin producing cells (IPSs). We applied an 18-day protocol to differentiate MSCs. Differentiating cells formed cell clusters some of which resembled pancreatic islet-like cells. Using dithizone we confirmed the presence of insulin in the cells. What is more, the expression of proinsulin C-peptide in differentiated IPCs was analyzed by flow cytometry. For the first time, we investigated the influence of growth factors' concentration on IPCs differentiation efficiency. We have found that an increase in the concentration of growth factors up to 60 ng/mL of β-FGF/EGF and 30 ng/mL of activin A/β-cellulin increases the percentage of IPCs. Further increase of growth factors does not show any increase of the percentage of differentiated cells. Our findings suggest that the presented protocol can be adapted for differentiation of insulin producing cells from stem cells. PMID:25405207

  8. A modified method of insulin producing cells' generation from bone marrow-derived mesenchymal stem cells.

    PubMed

    Czubak, Paweł; Bojarska-Junak, Agnieszka; Tabarkiewicz, Jacek; Putowski, Lechosław

    2014-01-01

    Type 1 diabetes mellitus is a result of autoimmune destruction of pancreatic insulin producing β-cells and so far it can be cured only by insulin injection, by pancreas transplantation, or by pancreatic islet cells' transplantation. The methods are, however, imperfect and have a lot of disadvantages. Therefore new solutions are needed. The best one would be the use of differentiated mesenchymal stem cells (MSCs). In the present study, we investigated the potential of the bone marrow-derived MSCs line for in vitro differentiation into insulin producing cells (IPSs). We applied an 18-day protocol to differentiate MSCs. Differentiating cells formed cell clusters some of which resembled pancreatic islet-like cells. Using dithizone we confirmed the presence of insulin in the cells. What is more, the expression of proinsulin C-peptide in differentiated IPCs was analyzed by flow cytometry. For the first time, we investigated the influence of growth factors' concentration on IPCs differentiation efficiency. We have found that an increase in the concentration of growth factors up to 60 ng/mL of β-FGF/EGF and 30 ng/mL of activin A/β-cellulin increases the percentage of IPCs. Further increase of growth factors does not show any increase of the percentage of differentiated cells. Our findings suggest that the presented protocol can be adapted for differentiation of insulin producing cells from stem cells. PMID:25405207

  9. Immune Dysfunction Associated with Abnormal Bone Marrow-Derived Mesenchymal Stroma Cells in Senescence Accelerated Mice

    PubMed Central

    Li, Ming; Guo, Kequan; Adachi, Yasushi; Ikehara, Susumu

    2016-01-01

    Senescence accelerated mice (SAM) are a group of mice that show aging-related diseases, and SAM prone 10 (SAMP10) show spontaneous brain atrophy and defects in learning and memory. Our previous report showed that the thymus and the percentage of T lymphocytes are abnormal in the SAMP10, but it was unclear whether the bone marrow-derived mesenchymal stroma cells (BMMSCs) were abnormal, and whether they played an important role in regenerative medicine. We thus compared BMMSCs from SAMP10 and their control, SAM-resistant (SAMR1), in terms of cell cycle, oxidative stress, and the expression of PI3K and mitogen-activated protein kinase (MAPK). Our cell cycle analysis showed that cell cycle arrest occurred in the G0/G1 phase in the SAMP10. We also found increased reactive oxygen stress and decreased PI3K and MAPK on the BMMSCs. These results suggested the BMMSCs were abnormal in SAMP10, and that this might be related to the immune system dysfunction in these mice. PMID:26840301

  10. The Healing Effect of Bone Marrow-Derived Stem Cells in Knee Osteoarthritis: A Case Report.

    PubMed

    Mehrabani, Davood; Mojtahed Jaberi, Fereidoon; Zakerinia, Maryam; Hadianfard, Mohammad Javad; Jalli, Reza; Tanideh, Nader; Zare, Shahrokh

    2016-05-01

    Osteoarthritis (OA) is a prevalent chronic disease impacting on quality of life and has societal and economical burden increasing with age. Yet, no confirmed pharmacological, biological or surgical therapy could prevent the progressive destruction of OA joint. Mesenchymal stem cells (MSCs) with immunosuppressive activities emerged a potential therapy. We describe a magnetic resonance images (MRI) approved 47 years old nomad female suffering from a severe right knee OA. After intra-articular injection of 36×10(6) passage 2 of bone marrow-derived stem cells (BMSCs), the patient's functional status of the knee, the number of stairs she could climb, the pain on visual analog scale (VAS) and walking distance improved after two months post-transplantation. MRI revealed an extension of the repaired tissue over subchondral bone. So as MSC transplantation is a simple technique, resulted into pain relief, minimized donor-site morbidity, provided a better quality of life, significantly improved cartilage quality with no need to hospitalization or surgery, cell transplantation can be considered as a reliable alternative treatment for chronic knee OA. Therefore these findings can be added to the literature on using BMSCs for treatment of OA. PMID:27579273

  11. Imperatorin Suppresses Degranulation and Eicosanoid Generation in Activated Bone Marrow-Derived Mast Cells

    PubMed Central

    Jeong, Kyu-Tae; Lee, Eujin; Park, Na-Young; Kim, Sun-Gun; Park, Hyo-Hyun; Lee, Jiean; Lee, Youn Ju; Lee, Eunkyung

    2015-01-01

    Imperatorin has been known to exert many biological functions including anti-inflammatory activity. In this study, we investigated the inhibitory effects of imperatorin on the production of inflammatory mediators in mouse bone marrow-derived mast cells (BMMC). Imperatorin inhibited degranulation and the generation of eicosanoids (leukotriene C4 (LTC4) and prostaglandin D2 (PGD2)) in IgE/antigen (Ag)-stimulated BMMC. To elucidate the molecular mechanism involved in this process, we investigated the effect of imperatorin on intracellular signaling in BMMC. Biochemical analyses of the IgE/Ag-mediated signaling pathway demonstrated that imperatorin dramatically attenuated degranulation and the production of 5-lipoxygenase-dependent LTC4 and cyclooxygenase-2-dependent PGD2 through the inhibition of intracellular calcium influx/phospholipase Cγ1, cytosolic phospholipase A2/mitogen-activated protein kinases and/or nuclear factor-κB pathways in BMMC. These results suggest that the effects of imperatorin on inhibition of degranulation and eicosanoid generation through the suppression of multiple steps of IgE/Ag-mediated signaling pathways would be beneficial for the prevention of allergic inflammation. PMID:26336581

  12. Recognization of receptors on bone marrow-derived dendritic cells bound with Pholiota nameko polysaccharides.

    PubMed

    Li, Haiping; Tao, Yongqing; Zhao, Pei; Ban, Xiaopan; Zhi, Dexian; Li, Guoliang; Wang, Fenlin; Yang, Xiaoli; Huai, Lihua

    2015-01-01

    Three major active polysaccharides isolated from Pholiota nameko (PNPS), including PNPS-1, PNPS-2 and PNPS-3, had been proved to inhibit the maturation of the murine bone marrow-derived dendritic cells (BMDCs). This paper recognized the affinity bind between PNPS and the five receptors (TLR2, TLR4, CD14, Dectin-1 and Mannose receptor) on BMDCs, using the bio-layer interferometry (BLI)-based biosensor technology developed by ForteBio on Octet RED system (Fortebio, Inc.). From the primary binding experiment, the gradient binding experiment and the inhibition binding experiment between the receptor proteins and PNPS, combined with the binding experiment between PNPS and the BMDCs membranes, we found that PNPS-1, PNPS-2 and PNPS-3 presented strong affinity bind with both TLR2 and Dectin-1 on BMDCs, only PNPS-3 with Mannose receptor. These data confirmed that PNPS could interact with TLR2, Dectin-1 and Mannose receptor that were very important for the affinity bind of these receptors and PNPS, which triggered the further stimulation on BMDCs. PMID:25158288

  13. Resistance of LPS-activated bone marrow derived macrophages to apoptosis mediated by dexamethasone

    PubMed Central

    Haim, Yasmin Ohana; Unger, Naamit Deshet; Souroujon, Miriam C.; Mittelman, Moshe; Neumann, Drorit

    2014-01-01

    Glucocorticoids (GC) display pleiotropic effects on the immune system. Macrophages are a major target for GC action. Here we show that dexamethasone (DEX), a synthetic GC, decreased viability of naïve bone marrow-derived macrophages (BMDM), involving an apoptotic mechanism. Administration of DEX together with lipopolysaccharide (LPS) protected BMDM against DEX-mediated cell death, suggesting that activated BMDM respond to DEX differently than naïve BMDM. An insight to the molecular basis of LPS actions was provided by a 7 fold increase in mRNA levels of glucocorticoid receptor beta (GRβ), a GR dominant-negative splice variant which inhibits GRα's transcriptional activity. LPS did not inhibit all DEX-mediated effects on BMDM; DEX significantly reduced the percentage of BMDM expressing high levels of the cell surface markers F4/80 and CD11b and led to a decrease in macrophage inflammatory protein 1 alpha (MIP1-α) mRNA and protein levels. These two DEX-mediated effects were not prevented by LPS. Our finding that LPS did not reduce the DEX-induced elevation of glucocorticoid-induced leucine zipper (GILZ), a mediator of GCs anti-inflammatory actions, may provide an underlying mechanism. These findings enable a better understanding of clinical states, such as sepsis, in which macrophages are activated by endotoxins and treatment by GCs is considered. PMID:24608810

  14. Engineering interaction between bone marrow derived endothelial cells and electrospun surfaces for artificial vascular graft applications.

    PubMed

    Ahmed, Furqan; Dutta, Naba K; Zannettino, Andrew; Vandyke, Kate; Choudhury, Namita Roy

    2014-04-14

    The aim of this investigation was to understand and engineer the interactions between endothelial cells and the electrospun (ES) polyvinylidene fluoride-co-hexafluoropropylene (PVDF-HFP) nanofiber surfaces and evaluate their potential for endothelialization. Elastomeric PVDF-HFP samples were electrospun to evaluate their potential use as small diameter artificial vascular graft scaffold (SDAVG) and compared with solvent cast (SC) PVDF-HFP films. We examined the consequences of fibrinogen adsorption onto the ES and SC samples for endothelialisation. Bone marrow derived endothelial cells (BMEC) of human origin were incubated with the test and control samples and their attachment, proliferation, and viability were examined. The nature of interaction of fibrinogen with SC and ES samples was investigated in detail using ELISA, XPS, and FTIR techniques. The pristine SC and ES PVDF-HFP samples displayed hydrophobic and ultrahydrophobic behavior and accordingly, exhibited minimal BMEC growth. Fibrinogen adsorbed SC samples did not significantly enhance endothelial cell binding or proliferation. In contrast, the fibrinogen adsorbed electrospun surfaces showed a clear ability to modulate endothelial cell behavior. This system also represents an ideal model system that enables us to understand the natural interaction between cells and their extracellular environment. The research reported shows potential of ES surfaces for artificial vascular graft applications. PMID:24564790

  15. The healing effect of bone marrow-derived stem cells in acute radiation syndrome

    PubMed Central

    Mortazavi, Seyed Mohammad Javad; Shekoohi-Shooli, Fatemeh; Aghamir, Seyed Mahmood Reza; Mehrabani, Davood; Dehghanian, Amirreza; Zare, Shahrokh; Mosleh-Shirazi, Mohammad Amin

    2016-01-01

    Objectives: To determine the effect of bone marrow-derived mesenchymal stem cells (BMSCs) on regeneration of bone marrow and intestinal tissue and survival rate in experimental mice with acute radiation syndrome (ARS). Methods: Forty mice were randomly divided into two equal groups of A receiving no BMSC transplantation and B receiving BMSCs. BMSCs were isolated from the bone marrow and cultured in DMEM media. Both groups were irradiated with 10 Gy (dose rate 0.28 Gy/ min) 60CO during 35 minutes with a field size of 35×35 for all the body area. Twenty-four hours after γ irradiation, 150×103 cells of passage 5 in 150 µl medium were injected intravenously into the tail. Animals were euthanized one and two weeks after cell transplantation. They were evaluated histologically for any changes in bone marrow and intestinal tissues. The survival rate in mice were also determined. Results: A significant increase for bone marrow cell count and survival rate were observed in group B in comparison to group A. Histological findings denoted to a healing in sample tissues. Conclusion: BMSCs could significantly reduce the side effects of ARS and increase the survival rate and healing in injured tissue. As such their transplantation may open a window in treatment of patients with ARS. PMID:27375707

  16. Caffeine inhibits the viability and osteogenic differentiation of rat bone marrow-derived mesenchymal stromal cells

    PubMed Central

    Zhou, Y; Guan, XX; Zhu, ZL; Guo, J; Huang, YC; Hou, WW; Yu, HY

    2010-01-01

    BACKGROUND AND PURPOSE Caffeine is consumed extensively in Europe and North America. As a risk factor for osteoporosis, epidemiological studies have observed that caffeine can decrease bone mineral density, adversely affect calcium absorption and increase the risk of bone fracture. However, the exact mechanisms have not been fully investigated. Here, we examined the effects of caffeine on the viability and osteogenesis of rat bone marrow-derived mesenchymal stromal cells (rBMSCs). EXPERIMENTAL APPROACH Cell viability, apoptosis and necrosis were quantified using thymidine incorporation and flow cytometry. Sequential gene expressions in osteogenic process were measured by real-time PCR. cAMP, alkaline phosphatase and osteocalcin were assessed by immunoassay, spectrophotometry and radioimmunoassay, respectively. Mineralization was determined by calcium deposition. KEY RESULTS After treating BMSCs with high caffeine concentrations (0.1–1 mM), their viability decreased in a concentration-dependent manner. This cell death was primarily due to necrosis and, to a small extent, apoptosis. Genes and protein sequentially expressed in osteogenesis, including Cbfa1/Runx2, collagen I, alkaline phosphatase and its protein, were significantly downregulated except for osteocalcin and its protein. Moreover, caffeine inhibited calcium deposition in a concentration- and time-dependent manner, but increased intracellular cAMP in a concentration-dependent manner. CONCLUSIONS AND IMPLICATIONS By suppressing the commitment of BMSCs to the osteogenic lineage and selectively inhibiting gene expression, caffeine downregulated some important events in osteogenesis and ultimately affected bone mass. PMID:20726981

  17. The Healing Effect of Bone Marrow-Derived Stem Cells in Knee Osteoarthritis: A Case Report

    PubMed Central

    Mehrabani, Davood; Mojtahed Jaberi, Fereidoon; Zakerinia, Maryam; Hadianfard, Mohammad Javad; Jalli, Reza; Tanideh, Nader; Zare, Shahrokh

    2016-01-01

    Osteoarthritis (OA) is a prevalent chronic disease impacting on quality of life and has societal and economical burden increasing with age. Yet, no confirmed pharmacological, biological or surgical therapy could prevent the progressive destruction of OA joint. Mesenchymal stem cells (MSCs) with immunosuppressive activities emerged a potential therapy. We describe a magnetic resonance images (MRI) approved 47 years old nomad female suffering from a severe right knee OA. After intra-articular injection of 36×106 passage 2 of bone marrow-derived stem cells (BMSCs), the patient’s functional status of the knee, the number of stairs she could climb, the pain on visual analog scale (VAS) and walking distance improved after two months post-transplantation. MRI revealed an extension of the repaired tissue over subchondral bone. So as MSC transplantation is a simple technique, resulted into pain relief, minimized donor-site morbidity, provided a better quality of life, significantly improved cartilage quality with no need to hospitalization or surgery, cell transplantation can be considered as a reliable alternative treatment for chronic knee OA. Therefore these findings can be added to the literature on using BMSCs for treatment of OA. PMID:27579273

  18. Age-associated metabolic dysregulation in bone marrow-derived macrophages stimulated with lipopolysaccharide

    PubMed Central

    Fei, Fan; Lee, Keith M.; McCarry, Brian E.; Bowdish, Dawn M. E.

    2016-01-01

    Macrophages are major contributors to age-associated inflammation. Metabolic processes such as oxidative phosphorylation, glycolysis and the urea cycle regulate inflammatory responses by macrophages. Metabolic profiles changes with age; therefore, we hypothesized that dysregulation of metabolic processes could contribute to macrophage hyporesponsiveness to LPS. We examined the intracellular metabolome of bone marrow-derived macrophages from young (6–8 wk) and old (18–22 mo) mice following lipopolysaccharide (LPS) stimulation and tolerance. We discovered known and novel metabolites that were associated with the LPS response of macrophages from young mice, which were not inducible in macrophages from old mice. Macrophages from old mice were largely non-responsive towards LPS stimulation, and we did not observe a shift from oxidative phosphorylation to glycolysis. The critical regulatory metabolites succinate, γ-aminobutyric acid, arginine, ornithine and adenosine were increased in LPS-stimulated macrophages from young mice, but not macrophages from old mice. A shift between glycolysis and oxidative phosphorylation was not observed during LPS tolerance in macrophages from either young or old mice. Metabolic bottlenecks may be one of the mechanisms that contribute to the dysregulation of LPS responses with age. PMID:26940652

  19. Donor age and cell passage affects differentiation potential of murine bone marrow-derived stem cells

    PubMed Central

    Kretlow, James D; Jin, Yu-Qing; Liu, Wei; Zhang, Wen Jie; Hong, Tan-Hui; Zhou, Guangdong; Baggett, L Scott; Mikos, Antonios G; Cao, Yilin

    2008-01-01

    Background Bone marrow-derived mesenchymal stem cells (BMSCs) are a widely researched adult stem cell population capable of differentiation into various lineages. Because many promising applications of tissue engineering require cell expansion following harvest and involve the treatment of diseases and conditions found in an aging population, the effect of donor age and ex vivo handling must be understood in order to develop clinical techniques and therapeutics based on these cells. Furthermore, there currently exists little understanding as to how these two factors may be influenced by one another. Results Differences in the adipogenic, chondrogenic, and osteogenic differentiation capacity of murine MSCs harvested from donor animals of different age and number of passages of these cells were observed. Cells from younger donors adhered to tissue culture polystyrene better and proliferated in greater number than those from older animals. Chondrogenic and osteogenic potential decreased with age for each group, and adipogenic differentiation decreased only in cells from the oldest donors. Significant decreases in differentiation potentials due to passage were observed as well for osteogenesis of BMSCs from the youngest donors and chondrogenesis of the cells from the oldest donors. Conclusion Both increasing age and the number of passages have lineage dependent effects on BMSC differentiation potential. Furthermore, there is an obvious interplay between donor age and cell passage that in the future must be accounted for when developing cell-based therapies for clinical use. PMID:18957087

  20. Age-associated metabolic dysregulation in bone marrow-derived macrophages stimulated with lipopolysaccharide

    NASA Astrophysics Data System (ADS)

    Fei, Fan; Lee, Keith M.; McCarry, Brian E.; Bowdish, Dawn M. E.

    2016-03-01

    Macrophages are major contributors to age-associated inflammation. Metabolic processes such as oxidative phosphorylation, glycolysis and the urea cycle regulate inflammatory responses by macrophages. Metabolic profiles changes with age; therefore, we hypothesized that dysregulation of metabolic processes could contribute to macrophage hyporesponsiveness to LPS. We examined the intracellular metabolome of bone marrow-derived macrophages from young (6–8 wk) and old (18–22 mo) mice following lipopolysaccharide (LPS) stimulation and tolerance. We discovered known and novel metabolites that were associated with the LPS response of macrophages from young mice, which were not inducible in macrophages from old mice. Macrophages from old mice were largely non-responsive towards LPS stimulation, and we did not observe a shift from oxidative phosphorylation to glycolysis. The critical regulatory metabolites succinate, γ-aminobutyric acid, arginine, ornithine and adenosine were increased in LPS-stimulated macrophages from young mice, but not macrophages from old mice. A shift between glycolysis and oxidative phosphorylation was not observed during LPS tolerance in macrophages from either young or old mice. Metabolic bottlenecks may be one of the mechanisms that contribute to the dysregulation of LPS responses with age.

  1. Human term placenta-derived mesenchymal stromal cells are less prone to osteogenic differentiation than bone marrow-derived mesenchymal stromal cells.

    PubMed

    Pilz, Gregor A; Ulrich, Christine; Ruh, Manuel; Abele, Harald; Schäfer, Richard; Kluba, Torsten; Bühring, Hans-Jörg; Rolauffs, Bernd; Aicher, Wilhelm K

    2011-04-01

    Mesenchymal stromal cells (MSC) can be isolated from different tissues. They are capable of differentiating in vitro, for example, to osteoblasts, chondrocytes, or adipocytes. In contrast to CD34 for hematopoietic stem cells, a distinct MSC-defining antibody is not available. Further, for hematopoietic cells lineage-defining antigens such as CD3 or CD20 are known. In contrast, for MSC-derived cells lineage-associated cell surface markers are far from being established. We therefore investigated expression of cell surface antigens on human term placenta-derived MSC (pMSC) in more detail and correlated expression pattern to the osteogenic differentiation capacity of the MSC. We report that pMSC expressed the typical cell surface antigens at levels comparable to bone marrow-derived MSC (bmMSC), including CD73, CD90, and CD105, but did not express CD11b, CD34, and CD45. Further, CD164, TNAP, and the W5C5 antigens were detected on pMSC, whereas CD349 was not observed. Some pMSC expressed CD146 at low or moderate levels, and their osteogenic differentiation potential was weak. In contrast, bmMSC expressed CD146 at high levels, expression of alkaline phosphatase was significantly higher, and they presented a pronounced osteogenic differentiation potential. We conclude that MSC from different sources differ in their expression of distinct markers, and that this may correlate in part with their lineage determination. Thus, a higher percentage of bmMSC expressed CD146 at prominent levels and such cells may be better suited for bone repair. In contrast, many pMSC expressed CD146 at low or moderate levels. They, therefore, may be suitable for applications in which osteogenic differentiation is undesirable. PMID:21047215

  2. The effect of rat bone marrow derived mesenchymal stem cells transplantation for restoration of olfactory disorder.

    PubMed

    Jo, Hyogyeong; Jung, Minyoung; Seo, Dong Jin; Park, Dong Joon

    2015-11-13

    The purpose of the study was to investigate the effect of bone marrow-derived mesenchymal stem cells (BMSCs) transplantation on olfactory epithelium (OE) of morphologic and functional restoration following neural Sensorineural Disorder in rats. Except the Normal group, twenty-one rats underwent Triton X-100 (TX-100) irrigation to induce degeneration of OE, and then BMSCs and PBS were treated from the both medial canthus to the rear part of the both nasal cavity into the experimental group and then were observed for restoration according to time point. At two and four weeks after transplantation with BMSCs, restoration of OE was observed with olfactory marker protein (OMP) and behavioral test. And we observed the expression of OMP, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). After TX-100 irrigation, the OE almost disappeared in 3 days. At four weeks after transplantation with BMSCs, the thickness and cellular composition of OE was considerably restored to normal group and expression of OMP was markedly increased when compared with PBS group and reduced the searching time in the behavioral test. Furthermore at two weeks after treatment with BMSCs, expression of NGF and BDNF was greatly increased when compared with PBS group. However at four weeks after treatment with BMSCs, expression of NGF and BDNF was slightly decreased. Our results suggest the BMSCs transplantation affect restoration of OE and olfaction, most likely via regulation of the neurotrophic factor expression, especially the expression of NGF and BDNF and has a possibility of a new therapeutic strategy for the treatment of olfactory disorder caused by the degeneration of OE. PMID:26427869

  3. RAT CYTOMEGALOVIRUS INFECTION DEPLETES MHC II IN BONE MARROW DERIVED DENDRITIC CELLS

    PubMed Central

    Baca Jones, Carmen C.; Kreklywich, Craig N.; Messaoudi, Ilhem; Vomaske, Jennifer; McCartney, Erin; Orloff, Susan L.; Nelson, Jay A.; Streblow, Daniel N.

    2009-01-01

    While cytomegalovirus (CMV) infects and replicates in a multitude of cell types, the ability of the virus to replicate in antigen presenting cells (APCs) is believed to play a critical role in the viral dissemination and latency. CMV infection of APCs and manipulation of their function is an important area of investigation. CMV down regulation of MHC II is reportedly mediated by the HCMV proteins US2, US3, UL83, UL111a (vIL10) or through the induction of cellular IL10. In this study, we demonstrate that rat CMV (RCMV) significantly reduces MHC II expression by mechanisms that do not involve orthologues of the known HCMV genes nor by an increase in cellular IL10. Rat bone marrow derived dendritic cells (BMDC) were highly susceptible to infection with RCMV and a recombinant RCMV expressing eGFP. RCMV infection of BMDCs depleted both surface and intracellular MHC II to nearly undetectable levels as well as reduced surface expression of MHC I. The effect on MHC II only occurred in the infected GFP positive cells and is mediated by an immediate early or early viral gene product. Furthermore, treatment of uninfected immature DCs with virus-free conditioned supernatants from infected cells failed to down regulate MHC II. RCMV depletion of MHC II was sensitve to treatment with lysosomal inhibitors but not proteasomal inhibitors suggesting that the mechanism of RCMV mediated down-regulation of MHC II occurs through endocytic degradation. Since RCMV does not encode homologues of US2, US3, UL83 or UL111a, these data indicate a novel mechanism for RCMV depletion of MHC II. PMID:19349057

  4. Bone marrow-derived cells rescue salivary gland function in mice with head and neck irradiation

    PubMed Central

    Sumita, Yoshinori; Liu, Younan; Khalili, Saeed; Maria, Ola M.; Xia, Dengsheng; Key, Sharon; Cotrim, Ana P.; Mezey, Eva; Tran, Simon D.

    2012-01-01

    Treatment for most patients with head and neck cancers includes ionizing radiation. A consequence of this treatment is irreversible damage to salivary glands (SGs), which is accompanied by a loss of fluid-secreting acinar-cells and a considerable decrease of saliva output. While there are currently no adequate conventional treatments for this condition, cell-based therapies are receiving increasing attention to regenerate SGs. In this study, we investigated whether bone marrow-derived cells (BMDCs) can differentiate into salivary epithelial cells and restore SG function in head and neck irradiated mice. BMDCs from male mice were transplanted into the tail-vein of 18 Gy-irradiated female mice. Salivary output was increased in mice that received BMDCs transplantation at week 8 and 24 post-irradiation. At 24 weeks after irradiation (IR), harvested SGs (submandibular and parotid glands) of BMDC-treated mice had greater weights than those of non-treated mice. Histological analysis shows that SGs of treated mice demonstrated an increased level of tissue regenerative activity such as blood vessel formation and cell proliferation, while apoptotic activity was increased in non-transplanted mice. The expression of stem cell markers (Sca-1 or c-kit) was detected in BMDC-treated SGs. Finally, we detected an increased ratio of acinar-cell area and approximately 9% of Y-chromosome-positive (donor-derived) salivary epithelial cells in BMDC-treated mice. We propose here that cell therapy using BMDCs can rescue the functional damage of irradiated SGs by direct differentiation of donor BMDCs into salivary epithelial cells. PMID:20933096

  5. Susceptibility of bone marrow-derived macrophages to influenza virus infection is dependent on macrophage phenotype

    PubMed Central

    Campbell, Gillian M.; Nicol, Marlynne Q.; Dransfield, Ian; Shaw, Darren J.; Nash, Anthony A.

    2015-01-01

    The role of the macrophage in influenza virus infection is complex. Macrophages are critical for resolution of influenza virus infections but implicated in morbidity and mortality in severe infections. They can be infected with influenza virus and consequently macrophage infection is likely to have an impact on the host immune response. Macrophages display a range of functional phenotypes, from the prototypical pro-inflammatory classically activated cell to alternatively activated anti-inflammatory macrophages involved in immune regulation and wound healing. We were interested in how macrophages of different phenotype respond to influenza virus infection and therefore studied the infection of bone marrow-derived macrophages (BMDMs) of classical and alternative phenotype in vitro. Our results show that alternatively activated macrophages are more readily infected and killed by the virus than classically activated. Classically activated BMDMs express the pro-inflammatory markers inducible nitric oxide synthase (iNOS) and TNF-α, and TNF-α expression was further upregulated following infection. Alternatively activated macrophages express Arginase-1 and CD206; however, following infection, expression of these markers was downregulated whilst expression of iNOS and TNF-α was upregulated. Thus, infection can override the anti-inflammatory state of alternatively activated macrophages. Importantly, however, this results in lower levels of pro-inflammatory markers than those produced by classically activated cells. Our results showed that macrophage phenotype affects the inflammatory macrophage response following infection, and indicated that modulating the macrophage phenotype may provide a route to develop novel strategies to prevent and treat influenza virus infection. PMID:26297234

  6. Susceptibility of bone marrow-derived macrophages to influenza virus infection is dependent on macrophage phenotype.

    PubMed

    Campbell, Gillian M; Nicol, Marlynne Q; Dransfield, Ian; Shaw, Darren J; Nash, Anthony A; Dutia, Bernadette M

    2015-10-01

    The role of the macrophage in influenza virus infection is complex. Macrophages are critical for resolution of influenza virus infections but implicated in morbidity and mortality in severe infections. They can be infected with influenza virus and consequently macrophage infection is likely to have an impact on the host immune response. Macrophages display a range of functional phenotypes, from the prototypical pro-inflammatory classically activated cell to alternatively activated anti-inflammatory macrophages involved in immune regulation and wound healing. We were interested in how macrophages of different phenotype respond to influenza virus infection and therefore studied the infection of bone marrow-derived macrophages (BMDMs) of classical and alternative phenotype in vitro. Our results show that alternatively activated macrophages are more readily infected and killed by the virus than classically activated. Classically activated BMDMs express the pro-inflammatory markers inducible nitric oxide synthase (iNOS) and TNF-α, and TNF-α expression was further upregulated following infection. Alternatively activated macrophages express Arginase-1 and CD206; however, following infection, expression of these markers was downregulated whilst expression of iNOS and TNF-α was upregulated. Thus, infection can override the anti-inflammatory state of alternatively activated macrophages. Importantly, however, this results in lower levels of pro-inflammatory markers than those produced by classically activated cells. Our results showed that macrophage phenotype affects the inflammatory macrophage response following infection, and indicated that modulating the macrophage phenotype may provide a route to develop novel strategies to prevent and treat influenza virus infection. PMID:26297234

  7. CD44 promotes the migration of bone marrow-derived mesenchymal stem cells toward glioma

    PubMed Central

    YIN, QIANG; ZHOU, YANG-YANG; WANG, PENG; MA, LI; LI, PENG; WANG, XIAO-GUANG; SHE, CHUN-HUA; LI, WEN-LIANG

    2016-01-01

    Previous in vivo and in vitro studies have shown that human mesenchymal stem cells (MSCs) exhibit tropism for gliomas. However, the mechanism underlying this directed migration remains unclear. The aim of the present study was to investigate the possible mechanism underlying platelet-derived growth factor-BB (PDGF-BB)-induced chemotactic migration of bone marrow-derived MSCs (BMSCs) toward glioma. Rat glioma C6 cell-conditioned medium was utilized to evaluate the chemotactic response of BMSCs toward glioma using an in vitro migration assay. Recombinant rat PDGF-BB was added to C6 cell-conditioned medium to assess its effect on the tropism of BMSCs. The effect of PDGF-BB on the expression levels of cluster of differentiation (CD)44 in BMSCs was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence assays. The results revealed that chemotactic migration was induced in BMSCs by rat glioma C6 cell-conditioned medium, which was enhanced by PDGF-BB treatment in a dose-dependent manner. Furthermore, RT-PCR and immunofluorescence assays showed that CD44 expression was upregulated in BMSCs following treatment with 40 ng/ml PDGF-BB for 12 h. Additionally, 3-h pretreatment with the anti-CD44 neutralizing antibody OX-50 was observed to attenuate the tropism of BMSCs toward glioma in the presence or absence of PDGF-BB. The results of the present study indicate that CD44 mediates the tropism of BMSCs toward glioma, and PDGF-BB promotes the migration of BMSCs toward glioma via the upregulation of CD44 expression in BMSCs. These findings suggest CD44 inhibition may be a potential therapeutic target for the treatment of glioma. PMID:27073479

  8. Bone marrow-derived cells contribute to NDEA-induced lung squamous cell carcinoma.

    PubMed

    Luo, Dan; Liu, Dengqun; Zhou, Xiangdong; Yang, Shiming; Tang, Chunlan; Liu, Guoxiang

    2013-02-01

    Bone marrow-derived stem cells (BMDCs) have the ability to differentiate into lung epithelial cells in response to damage; however, their role in squamous cell carcinoma (SCC) formation is unknown. This study aimed to determine whether BMDC-derived lung epithelial cells could contribute to SCC formation. A model of lung SCC induced with N-nitrosodiethylamine (NDEA) in recipient female mice transplanted with green fluorescent protein (GFP)-positive BMDCs from male donors was established. Incorporation of BMDCs in lung tissue was determined using immunohistochemistry and immunofluorescence to detect GFP expression and fluorescence in situ hybridization to Y chromosomes. BMDC appeared at three stages of lung SCC progression: metaplasia, dysplasia, and carcinoma. There was a significantly higher proportion of GFP-positive (GFP(+)) cells within SCC than was found in metaplasia and dysplasia 16 weeks post-transplantation (both P < 0.017); GFP(+) BMDCs were also observed in clusters within several SCC nests. Furthermore, most GFP(+) cells in SCC were pancytokeratin-positive (PCK(+)) epithelial cells, and some exhibited proliferative activity as determined by Ki67 staining (9.7 ± 3.92 %). The presence of GFP(+)Ki67(+)PCK(+) cells within SCC nests suggested that some donor BMDCs differentiated into proliferating epithelial cells. Finally, analysis of p63 expression, a marker of SCC cells, indicated that the presence of GFP(+)p63(+) cells (green) in inner parts of the SCC. These findings strongly suggest that BMDC-derived lung epithelial cells could participate in lung SCC formation and partially contribute to tumor growth, which might have significant potential implications for both clinical cancer therapy using BMDCs. PMID:23055190

  9. Therapeutic effects of bone marrow-derived mesenchymal stem cells on radiation-induced lung injury.

    PubMed

    Xia, Chengcheng; Chang, Pengyu; Zhang, Yuyu; Shi, Weiyan; Liu, Bin; Ding, Lijuan; Liu, Min; Gao, Ling; Dong, Lihua

    2016-02-01

    Radiation-induced lung injury (RILI) is a fatal condition featured by interstitial pneumonitis and fibrosis. Mesenchymal stem cells (MSCs) have been widely used for treating RILI in rodent models. In the present study, we aimed to investigate whether the therapeutic effects of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) on RILI were in a dose-dependent manner. A total of 100 mice were randomly divided into: a control group (n=25), subject to lung irradiation and injection of phosphate-buffered solution (PBS) via the tail vein; and the hBM-MSC group, subject to lung irradiation followed by injection of a low dose (1x103 hBM-MSCs/g), medium dose (5x103 hBM-MSCs/g) and high dose (1x104 hBM-MSCs/g) of hBM-MSCs in PBS through the tail vein, respectively. After sacrifice, the pulmonary tissues were subject to hematoxylin and eosin (H&E) staining, Masson's trichrome staining and immunohistochemical staining to investigate the pathological changes. Immunofluorescent staining was performed to evaluate the differentiation capacity of hBM-MSCs in vivo by analyzing the expression of SPC and PECAM. hBM-MSCs improved the survival rate and histopathological features in the irradiated mice, especially in the low-dose group. Marked decrease in collagen deposition was noted in the irradiated mice treated using a low dose of hBM-MSCs. In addition, hBM-MSCs attenuated secretion and expression of IL-10 and increased the expression of TNF-α. Furthermore, hBM-MSCs had the potential to differentiate into functional cells upon lung injury. Low-dose hBM-MSCs contributed to functional recovery in mice with RILI. PMID:26717975

  10. Low level light promotes the proliferation and differentiation of bone marrow derived mesenchymal stem cells

    NASA Astrophysics Data System (ADS)

    Ahn, Jin-Chul; Rhee, Yun-Hee; Choi, Sun-Hyang; Kim, Dae Yu; Chung, Phil-Sang

    2015-03-01

    Low-level light irradiation (LLLI) reported to stimulate the proliferation or differentiation of a variety of cell types. However, very little is known about the effect of light therapy on stem cells. The aim of the present study was to evaluate the effect of LLLI on the molecular physiological change of human bone marrow derived stem cells (hBMSC) by wavelength (470, 630, 660, 740 and 850, 50mW). The laser diode was performed with different time interval (0, 7.5, 15, 30J/cm2, 50mW) on hBMSC. To determine the molecular physiological changes of cellular level of hBMSC, the clonogenic assay, ATP assay, reactive oxygen species (ROS) detection, mitochondria membrane potential (MMPΦ) staining and calcium efflux assay were assessed after irradiation. There was a difference between with and without irradiation on hBMSCs. An energy density up to 30 J/cm² improved the cell proliferation in comparison to the control group. Among these irradiated group, 630 and 660nm were significantly increased the cell proliferation. The cellular level of ATP and calcium influx was increased with energy dose-dependent in all LLLI groups. Meanwhile, ROS and MMPΦ were also increased after irradiation except 470nm. It can be concluded that LLLI using infrared light and an energy density up to 30 J/cm² has a positive stimulatory effect on the proliferation or differentiation of hBMSCs. Our results suggest that LLLI may influence to the mitochondrial membrane potential activity through ATP synthesis and increased cell metabolism which leads to cell proliferation and differentiation.

  11. Establishment and characterization of mouse bone marrow-derived mast cell hybridomas

    SciTech Connect

    Kawahara, Takeshi

    2012-11-01

    Interleukin (IL)-3-dependent mouse bone marrow-derived mast cells (BMMCs) are an important model for studying the function of mucosal-type mast cells. In the present study, BMMCs were successfully immortalized by cell fusion using a hypoxanthine-aminopterin-thymidine medium-sensitive variant of P815 mouse mastocytoma (P815-6TgR) as a partner cell line. The established mouse mast cell hybridomas (MMCHs) expressed {alpha}, {beta}, and {gamma} subunits of high-affinity immunoglobulin E (IgE) receptor (Fc{epsilon}RI) and possessed cytoplasmic granules devoid of or partially filled with electron-dense material. Four independent MMCH clones continuously proliferated without supplemental exogenous IL-3 and showed a degranulation response on stimulation with IgE+antigen. Furthermore, histamine synthesis and release by degranulation were confirmed in MMCH-D5, a MMCH clone that showed the strongest degranulation response. MMCH-D5 exhibited elevated levels of IL-3, IL-4, IL-13, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor (TNF)-{alpha}, and cyclooxygenase 2, and production of prostaglandin D{sub 2} and leukotriene C{sub 4} in response to IgE-induced stimulation. MMCH clones also expressed Toll-like receptors (TLRs) 1, 2, 4, and 6 and showed elevated levels of TNF-{alpha} expression in response to stimulation with TLR2 and TLR4 ligands. The MMCHs established using this method should be suitable for studies on Fc{epsilon}RI- and TLR-mediated effector functions of mast cells.

  12. Bone marrow derived myeloid cells orchestrate antiangiogenic resistance in glioblastoma through coordinated molecular networks.

    PubMed

    Achyut, B R; Shankar, Adarsh; Iskander, A S M; Ara, Roxan; Angara, Kartik; Zeng, Peng; Knight, Robert A; Scicli, Alfonso G; Arbab, Ali S

    2015-12-28

    Glioblastoma (GBM) is a hypervascular and malignant form of brain tumors. Anti-angiogenic therapies (AAT) were used as an adjuvant against VEGF-VEGFR pathway to normalize blood vessels in clinical and preclinical studies, which resulted into marked hypoxia and recruited bone marrow derived cells (BMDCs) to the tumor microenvironment (TME). In vivo animal models to track BMDCs and investigate molecular mechanisms in AAT resistance are rare. We exploited recently established chimeric mouse to develop orthotopic U251 tumor, which uses as low as 5 × 10(6) GFP+ BM cells in athymic nude mice and engrafted >70% GFP+ cells within 14 days. Our unpublished data and published studies have indicated the involvement of immunosuppressive myeloid cells in therapeutic resistance in glioma. Similarly, in the present study, vatalanib significantly increased CD68+ myeloid cells, and CD133+, CD34+ and Tie2+ endothelial cell signatures. Therefore, we tested inhibition of CSF1R+ myeloid cells using GW2580 that reduced tumor growth by decreasing myeloid (Gr1+ CD11b+ and F4/80+) and angiogenic (CD202b+ and VEGFR2+) cell signatures in TME. CSF1R blockade significantly decreased inflammatory, proangiogenic and immunosuppressive molecular signatures compared to vehicle, vatalanib or combination. TCK1 or CXCL7, a potent chemoattractant and activator of neutrophils, was observed as most significantly decreased cytokine in CSF1R blockade. ERK MAPK pathway was involved in cytokine network regulation. In conclusion, present study confirmed the contribution of myeloid cells in GBM development and therapeutic resistance using chimeric mouse model. We identified novel molecular networks including CXCL7 chemokine as a promising target for future studies. Nonetheless, survival studies are required to assess the beneficial effect of CSF1R blockade. PMID:26404753

  13. Increased Differentiation Capacity of Bone Marrow-Derived Mesenchymal Stem Cells in Aquaporin-5 Deficiency

    PubMed Central

    Yi, Fei; Khan, Muhammad; Gao, Hongwen; Hao, Feng; Sun, Meiyan; Zhong, Lili; Lu, Changzheng; Feng, Xuechao

    2012-01-01

    Mesenchymal stem cells (MSCs) are adult stem cells with a self-renewal and multipotent capability and express extensively in multitudinous tissues. We found that water channel aquaporin-5 (AQP5) is expressed in bone marrow-derived MSCs (BMMSCs) in the plasma membrane pattern. BMMSCs from AQP5−/− mice showed significantly lower plasma membrane water permeability than those from AQP5+/+ mice. In characterizing the cultured BMMSCs from AQP5−/− and AQP5+/+ mice, we found no obvious differences in morphology and proliferation between the 2 genotypes. However, the multiple differentiation capacity was significantly higher in AQP5−/− than AQP5+/+ BMMSCs as revealed by representative staining by Oil Red O (adipogenesis); Alizarin Red S and alkaline phosphatase (ALP; osteogenesis); and type II collagen and Safranin O (chondrogenesis) after directional induction. Relative mRNA expression levels of 3 lineage differentiation markers, including PPARγ2, C/EBPα, adipsin, collagen 1a, osteopontin, ALP, collagen 11a, collagen 2a, and aggrecan, were significantly higher in AQP5−/− -differentiating BMMSCs, supporting an increased differentiation capacity of AQP5−/− BMMSCs. Furthermore, a bone-healing process was accelerated in AQP5−/− mice in a drill-hole injury model. Mechanistic studies indicated a significantly lower apoptosis rate in AQP5−/− than AQP5+/+ BMMSCs. Apoptosis inhibitor Z-VAD-FMK increased the differentiation capacity to a greater extent in AQP5+/+ than AQP5−/− BMMSCs. We conclude that AQP5-mediated high plasma membrane water permeability enhances the apoptosis rate of differentiating BMMSCs, thus decreasing their differentiation capacity. These data implicate AQP5 as a novel determinant of differentiation of BMMSCs and therefore a new molecular target for regulating differentiation of BMMSCs during tissue repair and regeneration. PMID:22420587

  14. Euphorbia supina inhibits inflammatory mediators in mouse bone marrow-derived mast cells and macrophages.

    PubMed

    Chae, Hee-Sung; Song, Hyuk-Hwan; Kim, Young-Mi; Lee, Hyeong-Kyu; Oh, Sei-Ryang; Chin, Young-Won

    2015-12-01

    Euphorbia supina has been traditionally used for the treatment of furuncle and bloody diarrhea relevant to the inflammatory process. It has been proven to have a variety of pharmacological efficacies including antiarthritic, detoxification, hemostatic, and diuretic activities. RAW 264.7 macrophages and bone marrow-derived mast cells (BMMCs) were used to determine the anti-inflammatory and anti-allergic effects of E. supina (ES). NO production was assayed by measuring the nitrite content of the supernatants of cultured RAW 264.7 cells. β-hexosaminidase, a marker of mast cell degranulation, was quantitated by spectrophotometric analysis. ELISA was used for the analysis of interleukin-6 expression, and Western blotting was used to analyze 5-LOX, iNOS, and MAPK activation. The relevant gene expression upon ES treatment was measured by RT-PCR. ES inhibited inducible nitric oxide synthase (iNOS) in RAW 264.7 cells, and IL-6 and LTC4 production in PMA- and A23187-induced BMMCs along with the downregulation of 5-LOX gene expression. Furthermore, in the present study, a decrease in p-ERK, p-JNK, and p-P38 expression, as well as the suppression of degranulation, were observed by treatment with ES. Further in vivo study revealed that ES treatment also remarkably inhibited xylene-induced mouse ear edema and MPO levels in mice ears. This study demonstrates that ES has a potential regulatory effect on the expression of inflammatory mediators through the inhibition of both the phosphorylation of MAPK signaling and the activation of degranulation. PMID:26386544

  15. Genetic stability of bone marrow-derived human mesenchymal stromal cells in the Quantum System

    PubMed Central

    Jones, Mark; Varella-Garcia, Marileila; Skokan, Margaret; Bryce, Steven; Schowinsky, Jeffrey; Peters, Rebecca; Vang, Boah; Brecheisen, Michelle; Startz, Thomas; Frank, Nathan; Nankervis, Brian

    2014-01-01

    Background aims The Quantum® Cell Expansion System (Quantum; Terumo BCT, Inc, Lakewood, CO, USA) is a novel hollow fiber-based device that automates and closes the cell culture process, reducing labor intensive tasks such as manual cell culture feeding and harvesting. The manual cell selection and expansion processes for the production of clinical-scale quantities of bone marrow-derived human mesenchymal stromal cells (BM-hMSCs) have been successfully translated onto the Quantum platform previously. The formerly static, manual, in vitro process performed primarily on tissue culture polystyrene substrates may raise the question of whether BM-hMSCs cultured on a hollow fiber platform yields comparable cell quality. Methods A rigorous battery of assays was used to determine the genetic stability of BM-hMSCs selected and produced with the Quantum. In this study, genetic stability was determined by assessing spectral karyotype, micronucleus formation and tumorigenicity to resolve chromosomal aberrations in the stem cell population. Cell phenotype, adherent growth kinetics and tri-lineage differentiation were also evaluated. HMSC bone marrow aspirates, obtained from three approved donors, were expanded in parallel using T225 culture flasks and the Quantum. Results BM-hMSCs harvested from the Quantum demonstrated immunophenotype, morphology and tri-lineage differentiation capacity characteristics consistent with the International Society of Cell Therapy standard for hMSCs. Cell populations showed no malignant neoplastic formation in athymic mice 60 days post-transplant, no clonal chromosomal aberrations were observed and no DNA damage was found as measured by micronucleus formation. Conclusions Quantum-produced BM-hMSCs are of comparable quality and demonstrate analogous genetic stability to BM-hMSCs cultured on tissue culture polystyrene substrates. PMID:23992670

  16. Bone marrow-derived mesenchymal stromal cells inhibit Th2-mediated allergic airways inflammation in mice.

    PubMed

    Goodwin, Meagan; Sueblinvong, Viranuj; Eisenhauer, Philip; Ziats, Nicholas P; LeClair, Laurie; Poynter, Matthew E; Steele, Chad; Rincon, Mercedes; Weiss, Daniel J

    2011-07-01

    Bone marrow-derived mesenchymal stromal cells (BMSCs) mitigate inflammation in mouse models of acute lung injury. However, specific mechanisms of BMSC actions on CD4 T lymphocyte-mediated inflammation in vivo remain poorly understood. Limited data suggests promotion of Th2 phenotype in models of Th1-mediated diseases. However, whether this might alleviate or worsen Th2-mediated diseases such as allergic asthma is unknown. To ascertain the effects of systemic administration of BMSCs in a mouse model of Th2-mediated allergic airways inflammation, ovalbumin (OVA)-induced allergic airways inflammation was induced in wild-type C57BL/6 and BALB/c mice as well as in interferon-γ (IFNγ) receptor null mice. Effects of systemic administration during antigen sensitization of either syngeneic or allogeneic BMSC on airways hyperreactivity, lung inflammation, antigen-specific CD4 T lymphocytes, and serum immunoglobulins were assessed. Both syngeneic and allogeneic BMSCs inhibited airways hyperreactivity and lung inflammation through a mechanism partly dependent on IFNγ. However, contrary to existing data, BMSCs did not affect antigen-specific CD4 T lymphocyte proliferation but rather promoted Th1 phenotype in vivo as assessed by both OVA-specific CD4 T lymphocyte cytokine production and OVA-specific circulating immunoglobulins. BMSCs treated to prevent release of soluble mediators and a control cell population of primary dermal skin fibroblasts only partly mimicked the BMSC effects and in some cases worsened inflammation. In conclusion, BMSCs inhibit Th2-mediated allergic airways inflammation by influencing antigen-specific CD4 T lymphocyte differentiation. Promotion of a Th1 phenotype in antigen-specific CD4 T lymphocytes by BMSCs is sufficient to inhibit Th2-mediated allergic airways inflammation through an IFNγ-dependent process. PMID:21544902

  17. Nicotinic Acetylcholine Receptors Modulate Bone Marrow-Derived Pro-Inflammatory Monocyte Production and Survival

    PubMed Central

    St-Pierre, Stéphanie; Jiang, Wei; Roy, Patrick; Champigny, Camille; LeBlanc, Éric; Morley, Barbara J.; Hao, Junwei; Simard, Alain R.

    2016-01-01

    It is increasingly clear that nicotinic acetylcholine receptors (nAChRs) are involved in immune regulation, and that their activation can protect against inflammatory diseases. Previous data have shown that nicotine diminishes the numbers of peripheral monocytes and macrophages, especially those of the pro-inflammatory phenotype. The goal of the present study was to determine if nicotine modulates the production of bone marrow -derived monocytes/macrophages. In this study, we first found that murine bone marrow cells express multiple nAChR subunits, and that the α7 and α9 nAChRs most predominant subtypes found in immune cells and their precursors. Using primary cultures of murine bone marrow cells, we then determined the effect of nicotine on monocyte colony-stimulating factor and interferon gamma (IFNγ)-induced monocyte production. We found that nicotine lowered the overall number of monocytes, and more specifically, inhibited the IFNγ-induced increase in pro-inflammatory monocytes by reducing cell proliferation and viability. These data suggested that nicotine diminishes the ratio of pro-inflammatory versus anti-inflammatory monocyte produced in the bone marrow. We thus confirmed this hypothesis by measuring cytokine expression, where we found that nicotine inhibited the production of the pro-inflammatory cytokines TNFα, IL-1β and IL-12, while stimulating the secretion of IL-10, an anti-inflammatory cytokine. Finally, nicotine also reduced the number of pro-inflammatory monocytes in the bone marrow of LPS-challenged mice. Overall, our data demonstrate that both α7 and α9 nAChRs are involved in the regulation of pro-inflammatory M1 monocyte numbers. PMID:26925951

  18. Treatment of radiation-induced acute intestinal injury with bone marrow-derived mesenchymal stem cells

    PubMed Central

    ZHENG, KAI; WU, WEIZHEN; YANG, SHUNLIANG; HUANG, LIANGHU; CHEN, JIN; GONG, CHUNGUI; FU, ZHICHAO; LIN, RUOFEI; TAN, JIANMING

    2016-01-01

    The aim of the present study was to investigate the ability of bone marrow-derived mesenchymal stem cells (BMSCs) to repair radiation-induced acute intestinal injury, and to elucidate the underlying repair mechanism. Male Sprague-Dawley rats were subjected to whole abdominal irradiation using a single medical linear accelerator (12 Gy) and randomly assigned to two groups. Rats in the BMSC-treated group were injected with 1 ml BMSC suspension (2×106 cells/ml) via the tail vein, while the control group rats were injected with normal saline. BMSCs were identified by detecting the expression of CD29, CD90, CD34 and CD45 using flow cytometry. The expression of the cytokines stromal cell-derived factor 1 (SDF-1), prostaglandin E2 (PGE2) and interleukin (IL)-2 was detected using immunohistochemical techniques. Plasma citrulline concentrations were evaluated using an ELISA kit. Rat general conditions, including body weight, and changes in cellular morphology were also recorded. The results suggested that BMSCs exerted a protective effect on radiation-induced acute intestinal injury in rats. The histological damage was rapidly repaired in the BMSC-treated group. In addition, the BMSC-treated group showed significantly reduced radiation injury scores (P<0.01), mildly reduced body weight and plasma citrulline levels, significantly more rapid recovery (P<0.01), significantly reduced expression of the cytokines PGE2 and IL-2 (P<0.05) and significantly increased SDF-1 expression (P<0.01) compared with the control group. In summary, the present results indicate that BMSCs are able to effectively reduce inflammation and promote repair of the structure and function of intestinal tissues damaged by radiation exposure, suggesting that they may provide a promising therapeutic agent. PMID:27284330

  19. Injury mechanism dictates contribution of bone marrow-derived cells to murine hepatic vascular regeneration

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Stem and progenitor cells derived from adult marrow have been shown to regenerate vascular cells in response to injury. However, it is unclear whether the type of injury dictates the contribution of such cells to neovascularization and which subpopulations of cells contribute to vascular regeneratio...

  20. Production and Functional Characterization of Murine Osteoclasts Differentiated from ER-Hoxb8-Immortalized Myeloid Progenitor Cells.

    PubMed

    Zach, Frank; Mueller, Alexandra; Gessner, André

    2015-01-01

    In vitro differentiation into functional osteoclasts is routinely achieved by incubation of embryonic stem cells, induced pluripotent stem cells, or primary as well as cryopreserved spleen and bone marrow-derived cells with soluble receptor activator of nuclear factor kappa-B ligand and macrophage colony-stimulating factor. Additionally, osteoclasts can be derived from co-cultures with osteoblasts or by direct administration of soluble receptor activator of nuclear factor kappa-B ligand to RAW 264.7 macrophage lineage cells. However, despite their benefits for osteoclast-associated research, these different methods have several drawbacks with respect to differentiation yields, time and animal consumption, storage life of progenitor cells or the limited potential for genetic manipulation of osteoclast precursors. In the present study, we therefore established a novel protocol for the differentiation of osteoclasts from murine ER-Hoxb8-immortalized myeloid stem cells. We isolated and immortalized bone marrow cells from wild type and genetically manipulated mouse lines, optimized protocols for osteoclast differentiation and compared these cells to osteoclasts derived from conventional sources. In vitro generated ER-Hoxb8 osteoclasts displayed typical osteoclast characteristics such as multi-nucleation, tartrate-resistant acid phosphatase staining of supernatants and cells, F-actin ring formation and bone resorption activity. Furthermore, the osteoclast differentiation time course was traced on a gene expression level. Increased expression of osteoclast-specific genes and decreased expression of stem cell marker genes during differentiation of osteoclasts from ER-Hoxb8-immortalized myeloid progenitor cells were detected by gene array and confirmed by semi-quantitative and quantitative RT-PCR approaches. In summary, we established a novel method for the quantitative production of murine bona fide osteoclasts from ER-Hoxb8 stem cells generated from wild type or

  1. Production and Functional Characterization of Murine Osteoclasts Differentiated from ER-Hoxb8-Immortalized Myeloid Progenitor Cells

    PubMed Central

    Zach, Frank; Mueller, Alexandra; Gessner, André

    2015-01-01

    In vitro differentiation into functional osteoclasts is routinely achieved by incubation of embryonic stem cells, induced pluripotent stem cells, or primary as well as cryopreserved spleen and bone marrow-derived cells with soluble receptor activator of nuclear factor kappa-B ligand and macrophage colony-stimulating factor. Additionally, osteoclasts can be derived from co-cultures with osteoblasts or by direct administration of soluble receptor activator of nuclear factor kappa-B ligand to RAW 264.7 macrophage lineage cells. However, despite their benefits for osteoclast-associated research, these different methods have several drawbacks with respect to differentiation yields, time and animal consumption, storage life of progenitor cells or the limited potential for genetic manipulation of osteoclast precursors. In the present study, we therefore established a novel protocol for the differentiation of osteoclasts from murine ER-Hoxb8-immortalized myeloid stem cells. We isolated and immortalized bone marrow cells from wild type and genetically manipulated mouse lines, optimized protocols for osteoclast differentiation and compared these cells to osteoclasts derived from conventional sources. In vitro generated ER-Hoxb8 osteoclasts displayed typical osteoclast characteristics such as multi-nucleation, tartrate-resistant acid phosphatase staining of supernatants and cells, F-actin ring formation and bone resorption activity. Furthermore, the osteoclast differentiation time course was traced on a gene expression level. Increased expression of osteoclast-specific genes and decreased expression of stem cell marker genes during differentiation of osteoclasts from ER-Hoxb8-immortalized myeloid progenitor cells were detected by gene array and confirmed by semi-quantitative and quantitative RT-PCR approaches. In summary, we established a novel method for the quantitative production of murine bona fide osteoclasts from ER-Hoxb8 stem cells generated from wild type or

  2. The therapeutic potential of bone marrow-derived mesenchymal stromal cells on hepatocellular carcinoma.

    PubMed

    Bayo, Juan; Marrodán, Mariano; Aquino, Jorge B; Silva, Marcelo; García, Mariana G; Mazzolini, Guillermo

    2014-03-01

    Mesenchymal stromal cells (MSCs) are more often obtained from adult and extraembryonic tissues, with the latter sources being likely better from a therapeutic perspective. MSCs show tropism towards inflamed or tumourigenic sites. Mechanisms involved in MSC recruitment into tumours are comprehensively analysed, including chemoattractant signalling axes, endothelial adhesion and transmigration. In addition, signals derived from hepatocellular carcinoma (HCC) tumour microenvironment and their influence in MSC tropism and tumour recruitment are dissected, as well as the present controversy regarding their influence on tumour growth and/or metastasis. Finally, evidences available on the use of MSCs and other selected progenitor/stem cells as vehicles of antitumourigenic genes are discussed. A better knowledge of the mechanisms involved in progenitor/stem cell recruitment to HCC tumours is proposed in order to enhance their tumour targeting which may result in improvements in cell-based gene therapy strategies. PMID:24112437

  3. Beneficial effects of autologous bone marrow-derived mesenchymal stem cells in naturally occurring tendinopathy.

    PubMed

    Smith, Roger Kenneth Whealands; Werling, Natalie Jayne; Dakin, Stephanie Georgina; Alam, Rafiqul; Goodship, Allen E; Dudhia, Jayesh

    2013-01-01

    Tendon injuries are a common age-related degenerative condition where current treatment strategies fail to restore functionality and normal quality of life. This disease also occurs naturally in horses, with many similarities to human tendinopathy making it an ideal large animal model for human disease. Regenerative approaches are increasingly used to improve outcome involving mesenchymal stem cells (MSCs), supported by clinical data where injection of autologous bone marrow derived MSCs (BM-MSCs) suspended in marrow supernatant into injured tendons has halved the re-injury rate in racehorses. We hypothesized that stem cell therapy induces a matrix more closely resembling normal tendon than the fibrous scar tissue formed by natural repair. Twelve horses with career-ending naturally-occurring superficial digital flexor tendon injury were allocated randomly to treatment and control groups. 1X10(7) autologous BM-MSCs suspended in 2 ml of marrow supernatant were implanted into the damaged tendon of the treated group. The control group received the same volume of saline. Following a 6 month exercise programme horses were euthanized and tendons assessed for structural stiffness by non-destructive mechanical testing and for morphological and molecular composition. BM-MSC treated tendons exhibited statistically significant improvements in key parameters compared to saline-injected control tendons towards that of normal tendons and those in the contralateral limbs. Specifically, treated tendons had lower structural stiffness (p<0.05) although no significant difference in calculated modulus of elasticity, lower (improved) histological scoring of organisation (p<0.003) and crimp pattern (p<0.05), lower cellularity (p<0.007), DNA content (p<0.05), vascularity (p<0.03), water content (p<0.05), GAG content (p<0.05), and MMP-13 activity (p<0.02). Treatment with autologous MSCs in marrow supernatant therefore provides significant benefits compared to untreated tendon repair in

  4. Beneficial Effects of Autologous Bone Marrow-Derived Mesenchymal Stem Cells in Naturally Occurring Tendinopathy

    PubMed Central

    Smith, Roger Kenneth Whealands; Werling, Natalie Jayne; Dakin, Stephanie Georgina; Alam, Rafiqul; Goodship, Allen E.; Dudhia, Jayesh

    2013-01-01

    Tendon injuries are a common age-related degenerative condition where current treatment strategies fail to restore functionality and normal quality of life. This disease also occurs naturally in horses, with many similarities to human tendinopathy making it an ideal large animal model for human disease. Regenerative approaches are increasingly used to improve outcome involving mesenchymal stem cells (MSCs), supported by clinical data where injection of autologous bone marrow derived MSCs (BM-MSCs) suspended in marrow supernatant into injured tendons has halved the re-injury rate in racehorses. We hypothesized that stem cell therapy induces a matrix more closely resembling normal tendon than the fibrous scar tissue formed by natural repair. Twelve horses with career-ending naturally-occurring superficial digital flexor tendon injury were allocated randomly to treatment and control groups. 1X107 autologous BM-MSCs suspended in 2 ml of marrow supernatant were implanted into the damaged tendon of the treated group. The control group received the same volume of saline. Following a 6 month exercise programme horses were euthanized and tendons assessed for structural stiffness by non-destructive mechanical testing and for morphological and molecular composition. BM-MSC treated tendons exhibited statistically significant improvements in key parameters compared to saline-injected control tendons towards that of normal tendons and those in the contralateral limbs. Specifically, treated tendons had lower structural stiffness (p<0.05) although no significant difference in calculated modulus of elasticity, lower (improved) histological scoring of organisation (p<0.003) and crimp pattern (p<0.05), lower cellularity (p<0.007), DNA content (p<0.05), vascularity (p<0.03), water content (p<0.05), GAG content (p<0.05), and MMP-13 activity (p<0.02). Treatment with autologous MSCs in marrow supernatant therefore provides significant benefits compared to untreated tendon repair in

  5. Bone marrow-derived mesenchymal stem cells promote growth and angiogenesis of breast and prostate tumors

    PubMed Central

    2013-01-01

    Introduction Mesenchymal stem cells (MSCs) are known to migrate to tumor tissues. This behavior of MSCs has been exploited as a tumor-targeting strategy for cell-based cancer therapy. However, the effects of MSCs on tumor growth are controversial. This study was designed to determine the effect of MSCs on the growth of breast and prostate tumors. Methods Bone marrow-derived MSCs (BM-MSCs) were isolated and characterized. Effects of BM-MSCs on tumor cell proliferation were analyzed in a co-culture system with mouse breast cancer cell 4T1 or human prostate cancer cell DU145. Tumor cells were injected into nude mice subcutaneously either alone or coupled with BM-MSCs. The expression of cell proliferation and angiogenesis-related proteins in tumor tissues were immunofluorescence analyzed. The angiogenic effect of BM-MSCs was detected using a tube formation assay. The effects of the crosstalk between tumor cells and BM-MSCs on expression of angiogenesis related markers were examined by immunofluorescence and real-time PCR. Results Both co-culturing with mice BM-MSCs (mBM-MSCs) and treatment with mBM-MSC-conditioned medium enhanced the growth of 4T1 cells. Co-injection of 4T1 cells and mBM-MSCs into nude mice led to increased tumor size compared with injection of 4T1 cells alone. Similar experiments using DU145 cells and human BM-MSCs (hBM-MSCs) instead of 4T1 cells and mBM-MSCs obtained consistent results. Compared with tumors induced by injection of tumor cells alone, the blood vessel area was greater in tumors from co-injection of tumor cells with BM-MSCs, which correlated with decreased central tumor necrosis and increased tumor cell proliferation. Furthermore, both conditioned medium from hBM-MSCs alone and co-cultures of hBM-MSCs with DU145 cells were able to promote tube formation ability of human umbilical vein endothelial cells. When hBM-MSCs are exposed to the DU145 cell environment, the expression of markers associated with neovascularization (macrophage

  6. Effect of autologous bone marrow-derived cells associated with guided bone regeneration or not in the treatment of peri-implant defects.

    PubMed

    Ribeiro, F V; Suaid, F F; Ruiz, K G S; Rodrigues, T L; Carvalho, M D; Nociti, F H; Sallum, E A; Casati, M Z

    2012-01-01

    This study investigated the effect of bone marrow-derived cells associated with guided bone regeneration in the treatment of dehiscence bone defects around dental implants. Iliac-derived bone marrow cells were harvested from dogs and phenotypically characterized with regard to their osteogenic properties. After teeth extraction, three implant sites were drilled, dehiscences created and implants placed. Dehiscences were randomly assigned to: bone marrow-derived cells, bone marrow-derived cells+guided bone regeneration, and control (no treatment). After 3 months, implants with adjacent tissues were processed histologically, bone-to-implant contact, bone fill within the threads, new bone area in a zone lateral to the implant, new bone height, and new bone weight at the bottom of the defect were determined. Phenotypic characterization demonstrated that bone marrow-derived cells presented osteogenic potential. Statistically higher bone fill within the threads was observed in both bone marrow-derived cells+guided bone regeneration bone marrow-derived cell groups compared with the control group (P<0.05), with no difference between the groups treated with cells (P>0.05). For the other parameters (new bone area, bone-to-implant contact, new bone height and new bone weight), only the bone marrow-derived cells+guided bone regeneration group presented higher values compared with the non-treated control (P<0.05). Bone marrow-derived cells provided promising results for peri-implantar bone regeneration, although the combined approach seems to be relevant, especially to bone formation out of the implant threads. PMID:21924867

  7. Activation of Bone Marrow-Derived Microglia Promotes Photoreceptor Survival in Inherited Retinal Degeneration

    PubMed Central

    Sasahara, Manabu; Otani, Atsushi; Oishi, Akio; Kojima, Hiroshi; Yodoi, Yuko; Kameda, Takanori; Nakamura, Hajime; Yoshimura, Nagahisa

    2008-01-01

    The role of microglia in neurodegeneration is controversial, although microglial activation in the retina has been shown to provide an early response against infection, injury, ischemia, and degeneration. Here we show that endogenous bone marrow (BM)-derived microglia play a protective role in vascular and neural degeneration in the retinitis pigmentosa model of inherited retinal degeneration. BM-derived cells were recruited to the degenerating retina where they differentiated into microglia and subsequently localized to the degenerating vessels and neurons. Inhibition of stromal-derived factor-1 in the retina reduced the number of BM-derived microglia and accelerated the rate of neurovascular degeneration. Systemic depletion of myeloid progenitors also accelerated the degenerative process. Conversely, activation of BM-derived myeloid progenitors by systemic administration of both granulocyte colony-stimulating factor and erythropoietin resulted in the deceleration of retinal degeneration and the promotion of cone cell survival. These data indicate that BM-derived microglia may play a protective role in retinitis pigmentosa. Functional activation of BM-derived myeloid progenitors by cytokine therapy may provide a novel strategy for the treatment of inherited retinal degeneration and other neurodegenerative diseases, regardless of the underlying genetic defect. PMID:18483210

  8. Induction of prostanoid, nitric oxide, and cytokine formation in rat bone marrow derived macrophages by activin A.

    PubMed

    Nüsing, R M; Barsig, J

    1999-06-01

    1. In this study we describe that activin A, a transforming growth factor (TGF) beta-like polypeptide affects the expression of inflammatory response genes and their products. 2. In rat bone marrow derived macrophages 15 nM activin A caused the stimulation of prostaglandin (PG) E2 and thromboxane (TX) A2 formation, production of nitrite as a marker for nitric oxide (NO) and the release of the cytokines tumour necrosis factor (TNF) alpha and interleukin (IL) -1beta. As shown by mRNA analysis induction of cyclo-oxygenase-2 and inducible nitric oxide synthase by activin A gave rise to the enhanced release of prostanoids and NO. 3. Costimulation of bone marrow derived macrophages with 15 nM activin A and 100 nM 12-O-tetradecanoyl-phorbol 13-acetate (TPA) potentiated the synthesis of prostanoids in a synergistic manner. With respect to NO formation the effect of activin A and TPA was additive. 4. In contrast to the nitrite production activin A induced PGE2 synthesis was susceptible to tyrosine kinase inhibition by genistein and tyrphostin 46 (IC50 was 10 and 20 microM, respectively). This observed inhibition was caused by the selective suppression of activin A induced cyclo-oxygenase-2 mRNA expression. Further, the release of TNFalpha in the presence of activin A was potentiated by tyrosine kinase inhibition. 5. In summary, we report that activin A exerts proinflammatory activity which results in the formation of prostanoids, NO and cytokines in rat bone marrow derived macrophages. Tyrosine kinase dependent and independent signalling pathways are involved leading to the increased synthesis of these metabolites. Based upon these results, we speculate that activin A may be considered as a possible component of inflammatory processes affecting at least the haematopoietic system. PMID:10433499

  9. Small-diameter human vessel wall engineered from bone marrow-derived mesenchymal stem cells (hMSCs)

    PubMed Central

    Gong, Zhaodi; Niklason, Laura E.

    2008-01-01

    Using biodegradable scaffold and a biomimetic perfusion system, our lab has successfully engineered small-diameter vessel grafts using endothelial cells (ECs) and smooth muscle cells (SMCs) obtained from vessels in various species. However, translating this technique into humans has presented tremendous obstacles due to species and age differences. SMCs from elderly persons have limited proliferative capacity and a reduction in collagen production, which impair the mechanical strength of engineered vessels. As an alternative cell source, adult human bone marrow-derived mesenchymal stem cells (hMSCs) were studied for their ability to differentiate into SMCs in culture plates as well as in a bioreactor system. In the former setting, immunofluorescence staining showed that MSCs, after induction for 14 days, expressed smooth muscle α-actin (SMA) and calponin, early and mid-SMC phenotypic markers, respectively. In the latter setting, vessel walls were constructed with MSC-derived SMCs. Various factors (i.e., matrix proteins, soluble factors, and cyclic strain) in the engineering system were further investigated for their effects on hMSC cell proliferation and differentiation into SMCs. Based on a screening of multiple factors, the engineering system was optimized by dividing the vessel culture into proliferation and differentiation phases. The vessel walls engineered under the optimized conditions were examined histologically and molecularly, and found to be substantially similar to native vessels. In conclusion, bone marrow-derived hMSCs can serve as a new cell source of SMCs in vessel engineering. Optimization of the culture conditions to drive SMC differentiation and matrix production significantly improved the quality of the hMSC-derived engineered vessel wall.—Gong, Z., Niklason, L. E. Small-diameter human vessel wall engineered from bone marrow-derived mesenchymal stem cells (hMSCs). PMID:18199698

  10. Bone marrow-derived, neural-like cells have the characteristics of neurons to protect the peripheral nerve in microenvironment.

    PubMed

    Guo, Shi-Lei; Zhang, Zhi-Ying; Xu, Yan; Zhi, Yun-Xia; Han, Chang-Jie; Zhou, Yu-Hao; Liu, Fang; Lin, Hai-Yan; Zhang, Chuan-Sen

    2015-01-01

    Effective repair of peripheral nerve defects is difficult because of the slow growth of new axonal growth. We propose that "neural-like cells" may be useful for the protection of peripheral nerve destructions. Such cells should prolong the time for the disintegration of spinal nerves, reduce lesions, and improve recovery. But the mechanism of neural-like cells in the peripheral nerve is still unclear. In this study, bone marrow-derived neural-like cells were used as seed cells. The cells were injected into the distal end of severed rabbit peripheral nerves that were no longer integrated with the central nervous system. Electromyography (EMG), immunohistochemistry, and transmission electron microscopy (TEM) were employed to analyze the development of the cells in the peripheral nerve environment. The CMAP amplitude appeared during the 5th week following surgery, at which time morphological characteristics of myelinated nerve fiber formation were observed. Bone marrow-derived neural-like cells could protect the disintegration and destruction of the injured peripheral nerve. PMID:25861281

  11. Bone Marrow-Derived, Neural-Like Cells Have the Characteristics of Neurons to Protect the Peripheral Nerve in Microenvironment

    PubMed Central

    Guo, Shi-lei; Zhang, Zhi-ying; Zhi, Yun-xia; Han, Chang-jie; Zhou, Yu-hao; Liu, Fang; Lin, Hai-yan; Zhang, Chuan-sen

    2015-01-01

    Effective repair of peripheral nerve defects is difficult because of the slow growth of new axonal growth. We propose that “neural-like cells” may be useful for the protection of peripheral nerve destructions. Such cells should prolong the time for the disintegration of spinal nerves, reduce lesions, and improve recovery. But the mechanism of neural-like cells in the peripheral nerve is still unclear. In this study, bone marrow-derived neural-like cells were used as seed cells. The cells were injected into the distal end of severed rabbit peripheral nerves that were no longer integrated with the central nervous system. Electromyography (EMG), immunohistochemistry, and transmission electron microscopy (TEM) were employed to analyze the development of the cells in the peripheral nerve environment. The CMAP amplitude appeared during the 5th week following surgery, at which time morphological characteristics of myelinated nerve fiber formation were observed. Bone marrow-derived neural-like cells could protect the disintegration and destruction of the injured peripheral nerve. PMID:25861281

  12. Repeated autologous bone marrow-derived mesenchymal stem cell injections improve radiation-induced proctitis in pigs.

    PubMed

    Linard, Christine; Busson, Elodie; Holler, Valerie; Strup-Perrot, Carine; Lacave-Lapalun, Jean-Victor; Lhomme, Bruno; Prat, Marie; Devauchelle, Patrick; Sabourin, Jean-Christophe; Simon, Jean-Marc; Bonneau, Michel; Lataillade, Jean-Jacques; Benderitter, Marc

    2013-11-01

    The management of proctitis in patients who have undergone very-high-dose conformal radiotherapy is extremely challenging. The fibrosis-necrosis, fistulae, and hemorrhage induced by pelvic overirradiation have an impact on morbidity. Augmenting tissue repair by the use of mesenchymal stem cells (MSCs) may be an important advance in treating radiation-induced toxicity. Using a preclinical pig model, we investigated the effect of autologous bone marrow-derived MSCs on high-dose radiation-induced proctitis. Irradiated pigs received repeated intravenous administrations of autologous bone marrow-derived MSCs. Immunostaining and real-time polymerase chain reaction analysis were used to assess the MSCs' effect on inflammation, extracellular matrix remodeling, and angiogenesis, in radiation-induced anorectal and colon damages. In humans, as in pigs, rectal overexposure induces mucosal damage (crypt depletion, macrophage infiltration, and fibrosis). In a pig model, repeated administrations of MSCs controlled systemic inflammation, reduced in situ both expression of inflammatory cytokines and macrophage recruitment, and augmented interleukin-10 expression in rectal mucosa. MSC injections limited radiation-induced fibrosis by reducing collagen deposition and expression of col1a2/col3a1 and transforming growth factor-β/connective tissue growth factor, and by modifying the matrix metalloproteinase/TIMP balance. In a pig model of proctitis, repeated injections of MSCs effectively reduced inflammation and fibrosis. This treatment represents a promising therapy for radiation-induced severe rectal damage. PMID:24068742

  13. Toll-like receptor 4 in bone marrow-derived cells contributes to the progression of diabetic retinopathy.

    PubMed

    Wang, Hui; Shi, Haojun; Zhang, Jing; Wang, Guoliang; Zhang, Jinxiang; Jiang, Fagang; Xiao, Qing

    2014-01-01

    Diabetic retinopathy (DR) is a major microvascular complication in diabetics, and its mechanism is not fully understood. Toll-like receptor 4 (TLR4) plays a pivotal role in the maintenance of the inflammatory state during DR, and the deletion of TLR4 eventually alleviates the diabetic inflammatory state. To further elucidate the mechanism of DR, we used bone marrow transplantation to establish reciprocal chimeric animals of TLR4 mutant mice and TLR4 WT mice combined with diabetes mellitus (DM) induction by streptozotocin (STZ) treatment to identify the role of TLR4 in different cell types in the development of the proinflammatory state during DR. TLR4 mutation did not block the occurrence of high blood glucose after STZ injection compared with WT mice but did alleviate the progression of DR and alter the expression of the small vessel proliferation-related genes, vascular endothelial growth factor (VEGF), and hypoxia inducible factor-1α (HIF-1α). Grafting bone marrow-derived cells from TLR4 WT mice into TLR4 mutant mice increased the levels of TNF-α, IL-1β, and MIP-2 and increased the damage to the retina. Similarly, VEGF and HIF-1α expression were restored by the bone marrow transplantation. These findings identify an essential role for TLR4 in bone marrow-derived cells contributing to the progression of DR. PMID:25214718

  14. Bone marrow-derived monocytes give rise to self-renewing and fully differentiated Kupffer cells

    PubMed Central

    Scott, Charlotte L.; Zheng, Fang; De Baetselier, Patrick; Martens, Liesbet; Saeys, Yvan; De Prijck, Sofie; Lippens, Saskia; Abels, Chloé; Schoonooghe, Steve; Raes, Geert; Devoogdt, Nick; Lambrecht, Bart N.; Beschin, Alain; Guilliams, Martin

    2016-01-01

    Self-renewing tissue-resident macrophages are thought to be exclusively derived from embryonic progenitors. However, whether circulating monocytes can also give rise to such macrophages has not been formally investigated. Here we use a new model of diphtheria toxin-mediated depletion of liver-resident Kupffer cells to generate niche availability and show that circulating monocytes engraft in the liver, gradually adopt the transcriptional profile of their depleted counterparts and become long-lived self-renewing cells. Underlining the physiological relevance of our findings, circulating monocytes also contribute to the expanding pool of macrophages in the liver shortly after birth, when macrophage niches become available during normal organ growth. Thus, like embryonic precursors, monocytes can and do give rise to self-renewing tissue-resident macrophages if the niche is available to them. PMID:26813785

  15. Comparison of the osteogenic capacity of minipig and human bone marrow-derived mesenchymal stem cells.

    PubMed

    Heino, Terhi J; Alm, Jessica J; Moritz, Niko; Aro, Hannu T

    2012-07-01

    Minipigs are a recommended large animal model for preclinical testing of human orthopedic implants. Mesenchymal stem cells (MSCs) are the key repair cells in bone healing and implant osseointegration, but the osteogenic capacity of minipig MSCs is incompletely known. The aim of this study was to isolate and characterize minipig bone marrow (BM) and peripheral blood (PB) MSCs in comparison to human BM-MSCs. BM sample was aspirated from posterior iliac crest of five male Göttingen minipigs (age 15 ± 1 months). PB sample was drawn for isolation of circulating MSCs. MSCs were selected by plastic-adherence as originally described by Friedenstein. Cell morphology, colony formation, proliferation, surface marker expression, and differentiation were examined. Human BM-MSCs were isolated and cultured from adult fracture patients (n = 13, age 19-60 years) using identical techniques. MSCs were found in all minipig BM samples, but no circulating MSCs could be detected. Minipig BM-MSCs had similar morphology, proliferation, and colony formation capacities as human BM-MSCs. Unexpectedly, minipig BM-MSCs had a significantly lower ability than human BM-MSCs to form differentiated and functional osteoblasts. This observation emphasizes the need for species-specific optimization of MSC culture protocol before direct systematic comparison of MSCs between human and various preclinical large animal models can be made. PMID:22570220

  16. Characterization of subsets of bone marrow-derived macrophages by flow cytometry analysis

    SciTech Connect

    Walker, E.B.; Akporiaye, E.T.; Warner, N.L.; Stewart, C.C.

    1985-01-01

    Normal C3H bone marrow cells were grown 7 days in medium containing L cell-derived colony stimulating factor-1 (CSF-1). During the first 4 days of culture, erythroid and granulocytic cells decreased while macrophages increased exponentially with a doubling time of about 31 hr. Only 0.3% of all cells in the initial bone marrrow suspension formed discrete colonies of mononuclear phagocytes, but by day 6 60% of the nonadherent cells were capable of forming macrophage colonies, representing a 200-fold enrichment of the original progenitor population. Using flow cytometry, mononuclear phagocytes obtained after 4 days of culture were separated into two distinct phenotypes based on their autofluorescence. Nonadherent cells were a discrete population of small cells exhibiting low autofluorescence, and the adherent cells were a broad heterogenous population of large cells exhibiting high autofluorescence. A panel of currently available rat monoclonal antibodies (MABs) against murine hematopoietic cells were used to determine whether unique subsets of macrophages could be resolved. The MABs RA 31B6 and H-11 stained virtually all the nonadherent cells but not adherent cells. The MABs E-2 and 11-4.1 (anti-H-2K/sup k/) stained almost all the adherent cells and demonstrated no significant staining of nonadherent cells. Nearly all the nonadherent and adherent cells were stained by the MABs DNL 4.4 and MAC-1. Additionally, the data suggest that the epitopes for MAC-2 and MAC-3 and ..gamma..2a Fc receptors develop late in nonadherent progenitor cells as they mature into adherent macrophages. 37 references, 5 figures, 5 tables.

  17. Induction of a program gene expression during osteoblast differentiation with strontium ranelate

    SciTech Connect

    Zhu Lingling; Zaidi, Samir; Peng Yuanzhen; Zhou Hang; Moonga, Baljit S.; Blesius, Alexia; Dupin-Roger, Isabelle; Zaidi, Mone . E-mail: mone.zaidi@mssm.edu; Sun Li

    2007-04-06

    Strontium ranelate, a new agent for the treatment of osteoporosis, has been shown stimulate bone formation in various experimental models. This study examines the effect of strontium ranelate on gene expression in osteoblasts, as well as the formation of mineralized (von Kossa-positive) colony-forming unit-osteoblasts (CFU-obs). Bone marrow-derived stromal cells cultured for 21 days under differentiating conditions, when exposed to strontium ranelate, displayed a significant time- and concentration-dependent increase in the expression of the master gene, Runx2, as well as bone sialoprotein (BSP), but interestingly without effects on osteocalcin. This was associated with a significant increase in the formation of CFU-obs at day 21 of culture. In U-33 pre-osteoblastic cells, strontium ranelate significantly enhanced the expression of Runx2 and osteocalcin, but not BSP. Late, more mature osteoblastic OB-6 cells showed significant elevations in BSP and osteocalcin, but with only minimal effects on Runx2. In conclusion, strontium ranelate stimulates osteoblast differentiation, but the induction of the program of gene expression appears to be cell type-specific. The increased osteoblastic differentiation is the likely basis underlying the therapeutic bone-forming actions of strontium ranelate.

  18. Bone Marrow Stress Decreases Osteogenic Progenitors.

    PubMed

    Ng, Adeline H; Baht, Gurpreet S; Alman, Benjamin A; Grynpas, Marc D

    2015-11-01

    Age-related bone loss may be a result of declining levels of stem cells in the bone marrow. Using the Col2.3Δtk (DTK) transgenic mouse, osteoblast depletion was used as a source of marrow stress in order to investigate the effects of aging on osteogenic progenitors which reside in the marrow space. Five-month-old DTK mice were treated with one or two cycles of ganciclovir to conditionally ablate differentiated osteoblasts, whereas controls were saline-treated. Treatment cycles were two weeks in length followed by four weeks of recovery. All animals were sacrificed at 8 months of age; bone marrow stromal cells (BMSCs) were harvested for cell culture and whole bones were excised for bone quality assessment. Colony-forming unit (CFU) assays were conducted to investigate the osteogenic potential of BMSC in vitro, and RNA was extracted to assess the expression of osteoblastic genes. Bone quality assessments included bone histomorphometry, TRAP staining, microcomputed tomography, and biomechanical testing. Osteoblast depletion decreased CFU-F (fibroblast), CFU-ALP (alkaline phosphatase), and CFU-VK (von Kossa) counts and BMSC osteogenic capacity in cell culture. Ex vivo, there were no differences in bone mineral density of vertebrae or femurs between treatment groups. Histology showed a decrease in bone volume and bone connectivity with repeated osteoblast depletion; however, this was accompanied by an increase in bone formation rate. There were no notable differences in osteoclast parameters or observed bone marrow adiposity. We have developed a model that uses bone marrow stress to mimic age-related decrease in osteogenic progenitors. Our data suggest that the number of healthy BMSCs and their osteogenic potential decline with repeated osteoblast depletion. However, activity of the remaining osteoblasts increases to compensate for this loss in progenitor osteogenic potential. PMID:26220824

  19. The Macrophage Polarization Regulates MSC Osteoblast Differentiation in vitro.

    PubMed

    Gong, Lei; Zhao, Yan; Zhang, Yi; Ruan, Zhi

    2016-01-01

    Bone repair is a complex yet highly organized process involving interactions between various cell types and the extracellular environment. Macrophages are not only activated in inflammation during early phases of repair processes, but they are also present in bone throughout the whole bone repair process. Bone marrow derived mesenchymal stem cells (MSCs) represent an attractive therapeutic for bone fracture with their expansion potential, osteogenic capability, and potential for injury. However, less is known about the interaction between macrophage and MSC during bone repair and regeneration. This study was aimed to investigate whether macrophages in different statuses can regulate MSC osteoblast differentiation in vitro. Using in vitro cell coculture of macrophage and MSC, it was shown that macrophage polarization can regulate MSC osteoblast differentiation. This was evidenced by increased alkaline phosphatase (ALP), osteogenic markers, and bone mineralization in M2 macrophage cocultured MSC but decreased in M1 counterpart. These results might be mediated by pro-regenerative cytokines, such as TGF-β, VEGF, and IFG-1, produced by M2 macrophages and detrimental inflammation cytokines, such as IL-6, IL-12, and TNF-α, produced by M1 macrophages. Taken together, this shows that macrophage polarization could be crucial for maintaining bone homeostasis and promoting bone repair by regulating the MSC osteoblast differentiation. PMID:26927345

  20. Minor histocompatibility antigens on canine hemopoietic progenitor cells.

    PubMed

    Weber, Martin; Lange, Claudia; Günther, Wolfgang; Franz, Monika; Kremmer, Elisabeth; Kolb, Hans-Jochem

    2003-06-15

    Adoptive immunotherapy with CTL against minor histocompatibility Ags (mHA) provides a promising way to treat leukemia relapse in allogeneic chimeras. Here we describe the in vitro generation of CTL against mHA in the dog. We tested their inhibitory effect on the growth of hemopoietic progenitor cells stimulated by hemopoietic growth factors in a 4-day suspension culture. CTL were produced by coculture of donor PBMC with bone marrow-derived dendritic cells (DCs). These DCs were characterized by morphology, high expression of MHC class II and CD1a, and the absence of the monocyte-specific marker CD14. Characteristically these cells stimulated allogeneic lymphocytes (MLR) and, after pulsing with a foreign Ag (keyhole limpet hemocyanin), autologous T cells. CTL were generated either ex vivo by coculture with DCs of DLA-identical littermates or in vivo by immunization of the responder with DCs obtained from a DLA-identical littermate. In suspension culture assays the growth of hemopoietic progenitor cells was inhibited in 53% of DLA-identical littermate combinations. In canine families mHA segregated with DLA as restriction elements. One-way reactivity against mHA was found in five littermate combinations. In two cases mHA might be Y chromosome associated, in three cases autosomally inherited alleles were detected. We conclude that CTL can be produced in vitro and in vivo against mHA on canine hemopoietic progenitor cells using bone marrow-derived DCs. PMID:12794111

  1. Adult bone marrow-derived cells recruited during angiogenesis comprise precursors for periendothelial vascular mural cells.

    PubMed

    Rajantie, Iiro; Ilmonen, Maritta; Alminaite, Agne; Ozerdem, Ugur; Alitalo, Kari; Salven, Petri

    2004-10-01

    Bone marrow (BM)-derived cells are thought to participate in the growth of blood vessels during postnatal vascular regeneration and tumor growth, a process previously attributed to stem and precursor cells differentiating to endothelial cells. We used multichannel laser scanning confocal microscopy of whole-mounted tissues to study angiogenesis in chimeric mice created by reconstituting C57BL mice with genetically marked syngeneic BM. We show that BM-derived endothelial cells do not significantly contribute to tumor- or cytokine-induced neoangiogenesis. Instead, BM-derived periendothelial vascular mural cells were persistently detected at sites of tumor- or vascular endothelial growth factor-induced angiogenesis. Subpopulations of these cells expressed the pericyte-specific NG2 proteoglycan, or the hematopoietic markers CD11b and CD45, but did not detectably express the smooth muscle markers smooth muscle alpha-actin or desmin. Thus, the major contribution of the BM to angiogenic processes is not endothelial, but may come from progenitors for periendothelial vascular mural and hematopoietic effector cells. PMID:15191949

  2. ECM microenvironment unlocks brown adipogenic potential of adult human bone marrow-derived MSCs

    PubMed Central

    Lee, Michelle H.; Goralczyk, Anna G.; Kriszt, Rókus; Ang, Xiu Min; Badowski, Cedric; Li, Ying; Summers, Scott A.; Toh, Sue-Anne; Yassin, M. Shabeer; Shabbir, Asim; Sheppard, Allan; Raghunath, Michael

    2016-01-01

    Key to realizing the diagnostic and therapeutic potential of human brown/brite adipocytes is the identification of a renewable, easily accessible and safe tissue source of progenitor cells, and an efficacious in vitro differentiation protocol. We show that macromolecular crowding (MMC) facilitates brown adipocyte differentiation in adult human bone marrow mesenchymal stem cells (bmMSCs), as evidenced by substantially upregulating uncoupling protein 1 (UCP1) and uncoupled respiration. Moreover, MMC also induced ‘browning’ in bmMSC-derived white adipocytes. Mechanistically, MMC creates a 3D extracellular matrix architecture enshrouding maturing adipocytes in a collagen IV cocoon that is engaged by paxillin-positive focal adhesions also at the apical side of cells, without contact to the stiff support structure. This leads to an enhanced matrix-cell signaling, reflected by increased phosphorylation of ATF2, a key transcription factor in UCP1 regulation. Thus, tuning the dimensionality of the microenvironment in vitro can unlock a strong brown potential dormant in bone marrow. PMID:26883894

  3. ECM microenvironment unlocks brown adipogenic potential of adult human bone marrow-derived MSCs.

    PubMed

    Lee, Michelle H; Goralczyk, Anna G; Kriszt, Rókus; Ang, Xiu Min; Badowski, Cedric; Li, Ying; Summers, Scott A; Toh, Sue-Anne; Yassin, M Shabeer; Shabbir, Asim; Sheppard, Allan; Raghunath, Michael

    2016-01-01

    Key to realizing the diagnostic and therapeutic potential of human brown/brite adipocytes is the identification of a renewable, easily accessible and safe tissue source of progenitor cells, and an efficacious in vitro differentiation protocol. We show that macromolecular crowding (MMC) facilitates brown adipocyte differentiation in adult human bone marrow mesenchymal stem cells (bmMSCs), as evidenced by substantially upregulating uncoupling protein 1 (UCP1) and uncoupled respiration. Moreover, MMC also induced 'browning' in bmMSC-derived white adipocytes. Mechanistically, MMC creates a 3D extracellular matrix architecture enshrouding maturing adipocytes in a collagen IV cocoon that is engaged by paxillin-positive focal adhesions also at the apical side of cells, without contact to the stiff support structure. This leads to an enhanced matrix-cell signaling, reflected by increased phosphorylation of ATF2, a key transcription factor in UCP1 regulation. Thus, tuning the dimensionality of the microenvironment in vitro can unlock a strong brown potential dormant in bone marrow. PMID:26883894

  4. Characterization of Nestin, a Selective Marker for Bone Marrow Derived Mesenchymal Stem Cells

    PubMed Central

    Xie, Liang; Zeng, Xin; Hu, Jing; Chen, Qianming

    2015-01-01

    Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating into multiple cell lineages and contributing to tissue repair and regeneration. Characterization of the physiological function of MSCs has been largely hampered by lack of unique markers. Nestin, originally found in neuroepithelial stem cells, is an intermediate filament protein expressed in the early stages of development. Increasing studies have shown a particular association between Nestin and MSCs. Nestin could characterize a subset of bone marrow perivascular MSCs which contributed to bone development and closely contacted with hematopoietic stem cells (HSCs). Nestin expressing (Nes+) MSCs also play a role in the progression of various diseases. However, Nes+ cells were reported to participate in angiogenesis as MSCs or endothelial progenitor cells (EPCs) in several tissues and be a heterogeneous population comprising mesenchymal cells and endothelial cells in the developing bone marrow. In this review article, we will summarize the progress of the research on Nestin, particularly the function of Nes+ cells in bone marrow, and discuss the feasibility of using Nestin as a specific marker for MSCs. PMID:26236348

  5. [Present status of research in bone marrow-derived mesenchymal stem cells for promoting the healing of diabetic ulcer].

    PubMed

    Zheng, Shu-Juan; Jia, Chi-Yu

    2012-08-01

    The delayed healing of diabetic ulcer has been haunting the surgeons and researchers for a long time. Although we have been researching and exploring the effective therapies for many years, the progress has been limited. Bone marrow-derived mesenchymal stem cells (BMSCs) have gradually won worldwide attention for their characteristics of differentiating into tissue repair cells and secreting multiple cytokines as well as growth factors. In recent years, the role of BMSCs in the treatment of diabetic ulcer has been drawing more and more attention. This article reviewed the advancement in the research of BMSCs in promoting the healing of diabetic ulcer. Through a discussion of the treatment of diabetic ulcer, the related research in BMSCs, as well as its role in diabetic ulcer treatment, the mechanism of BMSCs in promoting healing of diabetic ulcers is discussed. We expect through further research, unified criteria for the quality of BMSCs, application approach and dosage of BMSCs could be established. PMID:23248965

  6. Quantitative and phenotypic analysis of bone marrow-derived cells in the intact and inflamed central nervous system

    PubMed Central

    Litwak, Sara

    2011-01-01

    Bone marrow has been proposed as a possible source of cells capable of replacing injured neural cells in diseases such as Multiple Sclerosis (MS). Previous studies have reported conflicting results regarding the transformation of bone marrow cells into neural cells in vivo. This study is a detailed analysis of the fate of bone marrow derived cells (BMDC) in the CNS of C57Bl/6 mice with and without experimental autoimmune encephalomyelitis using flow cytometry to identify GFP-labeled BMDC that lacked the pan-hematopoietic marker CD45 and co-expressed neural markers polysialic acid-neural cell adhesion molecule or A2B5. A small number of BMDC displaying neural markers and lacking CD45 expression was identified within both the non-inflamed and inflamed CNS. However, the majority of BMDC exhibited a hematopoietic phenotype. PMID:21975545

  7. Bone reconstruction of large defects using bone marrow derived autologous stem cells.

    PubMed

    Lucarelli, Enrico; Donati, Davide; Cenacchi, Annarita; Fornasari, Pier Maria

    2004-04-01

    Bone is a tissue that has the ability to heal itself when fractured. Occasionally, a critical defect can be formed when part of the bone is lost or excised, in this case the bone fails to heal and requires bone reconstruction to prevent a non-union defect. Autogenous cancellous bone is the current gold standard treatment in bone loss. Because the amount of autogenous cancellous bone that can be harvested is limited, the expanding need for bone reconstruction is paired by the growth of interest in the discipline of tissue engineering. Labs worldwide are working to provide the right carrier and the right set of cells that, once retransplanted, will ensure bone repair. Several investigators have focused their attention on a subset of autologous non-hematopoietic stem/progenitor cells contained in the adult bone marrow stroma, referred to as stromal stem cells (SSC), as the appropriate cells to be transplanted. The use of autologous cells is facilitated by less stringent ethical and regulatory issues and does not require the patient to be immunologically suppressed. In pre-clinical and clinical protocols of critical defects in which SSC are employed, two approaches are mainly used: in the first, SSC are derived from bone marrow and directly introduced at the lesion site, in the second, SSC are derived from several sites and are expanded ex vivo before being implanted. Both approaches, equally correct in principle, will have to demonstrate, with definitive evidence of their efficacy, their capability of solving a critical clinical problem such as non-union. In this report we outline the difficulties of working with SSC. PMID:15062758

  8. Bone marrow-derived microglia-based neurturin delivery protects against dopaminergic neurodegeneration in a mouse model of Parkinson's disease.

    PubMed

    Biju, K C; Santacruz, Rene A; Chen, Cang; Zhou, Qing; Yao, Jiemin; Rohrabaugh, Sara L; Clark, Robert A; Roberts, James L; Phillips, Kimberley A; Imam, Syed Z; Li, Senlin

    2013-02-22

    Although neurotrophic factors have long been recognized as potent agents for protecting against neuronal degeneration, clinical success in treating Parkinson's disease and other neurodegenerative disorders has been hindered by difficulties in delivery of trophic factors across the blood brain barrier (BBB). Bone marrow hematopoietic stem cell-based gene therapy is emerging as a promising tool for overcoming drug delivery problems, as myeloid cells can cross the BBB and are recruited in large numbers to sites of neurodegeneration, where they become activated microglia that can secrete trophic factors. We tested the efficacy of bone marrow-derived microglial delivery of neurturin (NTN) in protecting dopaminergic neurons against neurotoxin-induced death in mice. Bone marrow cells were transduced ex vivo with lentivirus expressing the NTN gene driven by a synthetic macrophage-specific promoter. Infected bone marrow cells were then collected and transplanted into recipient animals. Eight weeks after transplantation, the mice were injected with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropuridine (MPTP) for seven days to induce dopaminergic neurodegeneration. Microglia-mediated NTN delivery dramatically ameliorated MPTP-induced degeneration of tyrosine hydroxylase (TH)-positive neurons of the substantia nigra and their terminals in the striatum. Microglia-mediated NTN delivery also induced significant recovery of synaptic marker staining in the striatum of MPTP-treated animals. Functionally, NTN treatment restored MPTP-induced decline in general activity, rearing behavior, and food intake. Thus, bone marrow-derived microglia can serve as cellular vehicles for sustained delivery of neurotrophic factors capable of mitigating dopaminergic injury. PMID:23295906

  9. Perilipin1 Deficiency in Whole Body or Bone Marrow-Derived Cells Attenuates Lesions in Atherosclerosis-Prone Mice

    PubMed Central

    Zhao, Xiaojing; Gao, Mingming; He, Jinhan; Zou, Liangqiang; Lyu, Ying; Zhang, Ling; Geng, Bin; Liu, George; Xu, Guoheng

    2015-01-01

    Aims The objective of this study is to determine the role of perilipin 1 (Plin1) in whole body or bone marrow-derived cells on atherogenesis. Methods and Results Accumulated evidence have indicated the role of Plin1 in atherosclerosis, however, these findings are controversial. In this study, we showed that Plin1 was assembled and colocalized with CD68 in macrophages in atherosclerotic plaques of ApoE-/- mice. We further found 39% reduction of plaque size in the aortic roots of Plin1 and ApoE double knockout (Plin1-/-ApoE-/-) females compared with ApoE-/- female littermates. In order to verify whether this reduction was macrophage-specific, the bone marrow cells from wild-type or Plin1 deficient mice (Plin1-/-) were transplanted into LDL receptor deficient mice (LDLR-/-). Mice receiving Plin1-/- bone marrow cells showed also 49% reduction in aortic atherosclerotic lesions compared with LDLR-/- mice received wild-type bone marrow cells. In vitro experiments showed that Plin1-/- macrophages had decreased protein expression of CD36 translocase and an enhanced cholesterol ester hydrolysis upon aggregated-LDL loading, with unaltered expression of many other regulators of cholesterol metabolism, such as cellular lipases, and Plin2 and 3. Given the fundamental role of Plin1 in protecting LD lipids from lipase hydrolysis, it is reasonably speculated that the assembly of Plin1 in microphages might function to reduce lipolysis and hence increase lipid retention in ApoE-/- plaques, but this pro-atherosclerotic property would be abrogated on inactivation of Plin1. Conclusion Plin1 deficiency in bone marrow-derived cells may be responsible for reduced atherosclerotic lesions in the mice. PMID:25855981

  10. Role of Integrin in Mechanical Loading of Osteoblasts

    NASA Technical Reports Server (NTRS)

    Globus, Ruth; Demsky, Caroline

    2000-01-01

    Mechanical forces generated by gravity, weightbearing, and muscle contraction play a key role in the genesis and maintenance of skeletal structure. The molecular mechanisms that mediate changes in osteoblast activity in response to altered patterns of skeletal loading are not known, and a better understanding of these processes may be essential for developing effective treatment strategies to prevent disuse osteoporosis. We have elucidated specific integrin/ECM (extracellular matrix) interactions that are required for osteoblast differentiation and survival and have developed a useful loading system to further explore the molecular basis of mechano-sensitivity of osteoblasts. The long term goal of our collaborative research is to understand how the ECM and cell adhesion proteins and integrins interaction to mediate the response of osteoblasts and their progenitors to mechanical loading. We suggest that integrin/ECM interactions are crucial for basic cellular processes, including differentiation and survival, as well as to participate in detecting and mediating cellular responses to mechanical stimuli.

  11. Vegfa regulates perichondrial vascularity and osteoblast differentiation in bone development

    PubMed Central

    Duan, Xuchen; Murata, Yurie; Liu, Yanqiu; Nicolae, Claudia; Olsen, Bjorn R.; Berendsen, Agnes D.

    2015-01-01

    ABSTRACT Vascular endothelial growth factor A (Vegfa) has important roles in endochondral bone formation. Osteoblast precursors, endothelial cells and osteoclasts migrate from perichondrium into primary ossification centers of cartilage templates of future bones in response to Vegfa secreted by (pre)hypertrophic chondrocytes. Perichondrial osteolineage cells also produce Vegfa, but its function is not well understood. By deleting Vegfa in osteolineage cells in vivo, we demonstrate that progenitor-derived Vegfa is required for blood vessel recruitment in perichondrium and the differentiation of osteoblast precursors in mice. Conditional deletion of Vegfa receptors indicates that Vegfa-dependent effects on osteoblast differentiation are mediated by Vegf receptor 2 (Vegfr2). In addition, Vegfa/Vegfr2 signaling stimulates the expression and activity of Indian hedgehog, increases the expression of β-catenin and inhibits Notch2. Our findings identify Vegfa as a regulator of perichondrial vascularity and osteoblast differentiation at early stages of bone development. PMID:25977369

  12. Specific profiles of ion channels and ionotropic receptors define adipose- and bone marrow derived stromal cells.

    PubMed

    Forostyak, Oksana; Butenko, Olena; Anderova, Miroslava; Forostyak, Serhiy; Sykova, Eva; Verkhratsky, Alexei; Dayanithi, Govindan

    2016-05-01

    Adherent, fibroblastic cells from different tissues are thought to contain subsets of tissue-specific stem/progenitor cells (often called mesenchymal stem cells). These cells display similar cell surface characteristics based on their fibroblastic nature, but also exhibit differences in molecular phenotype, growth rate, and their ability to differentiate into various cell phenotypes. The mechanisms underlying these differences remain poorly understood. We analyzed Ca(2+) signals and membrane properties in rat adipose-derived stromal cells (ADSCs) and bone marrow stromal cells (BMSCs) in basal conditions, and then following a switch into medium that contains factors known to modify their character. Modified ADSCs (mADSCs) expressed L-type Ca(2+) channels whereas both L- and P/Q- channels were operational in mBMSCs. Both mADSCs and mBMSCs possessed functional endoplasmic reticulum Ca(2+) stores, expressed ryanodine receptor-1 and -3, and exhibited spontaneous [Ca(2+)]i oscillations. The mBMSCs expressed P2X7 purinoceptors; the mADSCs expressed both P2X (but not P2X7) and P2Y (but not P2Y1) receptors. Both types of stromal cells exhibited [Ca(2+)]i responses to vasopressin (AVP) and expressed V1 type receptors. Functional oxytocin (OT) receptors were, in contrast, expressed only in modified ADSCs and BMSCs. AVP and OT-induced [Ca(2+)]i responses were dose-dependent and were blocked by their respective specific receptor antagonists. Electrophysiological data revealed that passive ion currents dominated the membrane conductance in ADSCs and BMSCs. Medium modification led to a significant shift in the reversal potential of passive currents from -40 to -50mV in cells in basal to -80mV in modified cells. Hence membrane conductance was mediated by non-selective channels in cells in basal conditions, whereas in modified medium conditions, it was associated with K(+)-selective channels. Our results indicate that modification of ADSCs and BMSCs by alteration in medium

  13. The Role of KV7.3 in Regulating Osteoblast Maturation and Mineralization

    PubMed Central

    Yang, Ji Eun; Song, Min Seok; Shen, Yiming; Ryu, Pan Dong; Lee, So Yeong

    2016-01-01

    KCNQ (KV7) channels are voltage-gated potassium (KV) channels, and the function of KV7 channels in muscles, neurons, and sensory cells is well established. We confirmed that overall blockade of KV channels with tetraethylammonium augmented the mineralization of bone-marrow-derived human mesenchymal stem cells during osteogenic differentiation, and we determined that KV7.3 was expressed in MG-63 and Saos-2 cells at the mRNA and protein levels. In addition, functional KV7 currents were detected in MG-63 cells. Inhibition of KV7.3 by linopirdine or XE991 increased the matrix mineralization during osteoblast differentiation. This was confirmed by alkaline phosphatase, osteocalcin, and osterix in MG-63 cells, whereas the expression of Runx2 showed no significant change. The extracellular glutamate secreted by osteoblasts was also measured to investigate its effect on MG-63 osteoblast differentiation. Blockade of KV7.3 promoted the release of glutamate via the phosphorylation of extracellular signal-regulated kinase 1/2-mediated upregulation of synapsin, and induced the deposition of type 1 collagen. However, activation of KV7.3 by flupirtine did not produce notable changes in matrix mineralization during osteoblast differentiation. These results suggest that KV7.3 could be a novel regulator in osteoblast differentiation. PMID:26999128

  14. The Role of KV7.3 in Regulating Osteoblast Maturation and Mineralization.

    PubMed

    Yang, Ji Eun; Song, Min Seok; Shen, Yiming; Ryu, Pan Dong; Lee, So Yeong

    2016-01-01

    KCNQ (KV7) channels are voltage-gated potassium (KV) channels, and the function of KV7 channels in muscles, neurons, and sensory cells is well established. We confirmed that overall blockade of KV channels with tetraethylammonium augmented the mineralization of bone-marrow-derived human mesenchymal stem cells during osteogenic differentiation, and we determined that KV7.3 was expressed in MG-63 and Saos-2 cells at the mRNA and protein levels. In addition, functional KV7 currents were detected in MG-63 cells. Inhibition of KV7.3 by linopirdine or XE991 increased the matrix mineralization during osteoblast differentiation. This was confirmed by alkaline phosphatase, osteocalcin, and osterix in MG-63 cells, whereas the expression of Runx2 showed no significant change. The extracellular glutamate secreted by osteoblasts was also measured to investigate its effect on MG-63 osteoblast differentiation. Blockade of KV7.3 promoted the release of glutamate via the phosphorylation of extracellular signal-regulated kinase 1/2-mediated upregulation of synapsin, and induced the deposition of type 1 collagen. However, activation of KV7.3 by flupirtine did not produce notable changes in matrix mineralization during osteoblast differentiation. These results suggest that KV7.3 could be a novel regulator in osteoblast differentiation. PMID:26999128

  15. In vivo identification of periodontal progenitor cells.

    PubMed

    Roguljic, H; Matthews, B G; Yang, W; Cvija, H; Mina, M; Kalajzic, I

    2013-08-01

    The periodontal ligament contains progenitor cells; however, their identity and differentiation potential in vivo remain poorly characterized. Previous results have suggested that periodontal tissue progenitors reside in perivascular areas. Therefore, we utilized a lineage-tracing approach to identify and track periodontal progenitor cells from the perivascular region in vivo. We used an alpha-smooth muscle actin (αSMA) promoter-driven and tamoxifen-inducible Cre system (αSMACreERT2) that, in combination with a reporter mouse line (Ai9), permanently labels a cell population, termed 'SMA9'. To trace the differentiation of SMA9-labeled cells into osteoblasts/cementoblasts, we utilized a Col2.3GFP transgene, while expression of Scleraxis-GFP was used to follow differentiation into periodontal ligament fibroblasts during normal tissue formation and remodeling following injury. In uninjured three-week-old SMA9 mice, tamoxifen labeled a small population of cells in the periodontal ligament that expanded over time, particularly in the apical region of the root. By 17 days and 7 weeks after labeling, some SMA9-labeled cells expressed markers indicating differentiation into mature lineages, including cementocytes. Following injury, SMA9 cells expanded, and differentiated into cementoblasts, osteoblasts, and periodontal ligament fibroblasts. SMA9-labeled cells represent a source of progenitors that can give rise to mature osteoblasts, cementoblasts, and fibroblasts within the periodontium. PMID:23735585

  16. Osteocytes serve as a progenitor cell of osteosarcoma

    PubMed Central

    Sottnik, Joseph L; Campbell, Brittany; Mehra, Rohit; Behbahani-Nejad, Omid; Hall, Christopher L.; Keller, Evan T.

    2016-01-01

    Osteosarcoma (OSA) is the most common primary bone tumor in humans. However, the cell of origin of OSA is not clearly defined although there is evidence that osteoblasts may serve as OSA progenitors. The role of osteocytes, terminally differentiated osteoblasts, as OSA progenitors has yet to be described. Analysis of patient cDNA from publicly available microarray data revealed that patients with OSA have increased expression of dentin matrix phosphoprotein 1 (DMP1), a marker of osteocytes. Analysis of multiple murine, human, and canine OSA cell lines revealed DMP1 expression. To test the tumorigenic potential of osteocytes, MLO-Y4, an SV-40 immortalized murine osteocyte cell line, was injected into subcutaneous and orthotopic (intratibial) sites of mice. Tumor growth occurred in both locations. Orthotopic MLO-Y4 tumors produced mixed osteoblastic/osteolytic radiographic lesions; a hallmark of OSA. Together, these data demonstrate for the first time that osteocytes can serve as OSA progenitors. PMID:24700678

  17. Osteocytes serve as a progenitor cell of osteosarcoma.

    PubMed

    Sottnik, Joseph L; Campbell, Brittany; Mehra, Rohit; Behbahani-Nejad, Omid; Hall, Christopher L; Keller, Evan T

    2014-08-01

    Osteosarcoma (OSA) is the most common primary bone tumor in humans. However, the cell of origin of OSA is not clearly defined although there is evidence that osteoblasts may serve as OSA progenitors. The role of osteocytes, terminally differentiated osteoblasts, as OSA progenitors has yet to be described. Analysis of patient cDNA from publicly available microarray data revealed that patients with OSA have increased expression of dentin matrix phosphoprotein 1 (DMP1), a marker of osteocytes. Analysis of multiple murine, human, and canine OSA cell lines revealed DMP1 expression. To test the tumorigenic potential of osteocytes, MLO-Y4, a SV-40 immortalized murine osteocyte cell line, was injected into subcutaneous and orthotopic (intratibial) sites of mice. Tumor growth occurred in both locations. Orthotopic MLO-Y4 tumors produced mixed osteoblastic/osteolytic radiographic lesions; a hallmark of OSA. Together, these data demonstrate for the first time that osteocytes can serve as OSA progenitors. PMID:24700678

  18. Aerobic Glycolysis in Osteoblasts

    PubMed Central

    Esen, Emel; Long, Fanxin

    2014-01-01

    Osteoblasts, the chief bone-making cells in the body, are a focus of osteoporosis research. Although teriparatite, a synthetic fragment of the human parathyroid hormone (PTH), has been an effective bone anabolic drug, there remains a clinical need for additional therapeutics that safely stimulates osteoblast number and function. Work in the past several decades has provided unprecedented clarity about the roles of growth factors and transcription factors in regulating osteoblast differentiation and activity, but whether these factors may regulate cellular metabolism to influence cell fate and function has been largely unexplored. The past few years have witnessed a resurgence of interest in the cellular metabolism of osteoblasts, with the hope that elucidation of their metabolic profile may open new avenues for developing bone anabolic agents. Here we review the current understanding about glucose metabolism in osteoblasts. PMID:25200872

  19. Overexpression of FABP3 inhibits human bone marrow derived mesenchymal stem cell proliferation but enhances their survival in hypoxia

    SciTech Connect

    Wang, Suna Zhou, Yifu; Andreyev, Oleg; Hoyt, Robert F.; Singh, Avneesh; Hunt, Timothy; Horvath, Keith A.

    2014-04-15

    Studying the proliferative ability of human bone marrow derived mesenchymal stem cells in hypoxic conditions can help us achieve the effective regeneration of ischemic injured myocardium. Cardiac-type fatty acid binding protein (FABP3) is a specific biomarker of muscle and heart tissue injury. This protein is purported to be involved in early myocardial development, adult myocardial tissue repair and responsible for the modulation of cell growth and proliferation. We have investigated the role of FABP3 in human bone marrow derived mesenchymal stem cells under ischemic conditions. MSCs from 12 donors were cultured either in standard normoxic or modified hypoxic conditions, and the differential expression of FABP3 was tested by quantitative {sup RT}PCR and western blot. We also established stable FABP3 expression in MSCs and searched for variation in cellular proliferation and differentiation bioprocesses affected by hypoxic conditions. We identified: (1) the FABP3 differential expression pattern in the MSCs under hypoxic conditions; (2) over-expression of FABP3 inhibited the growth and proliferation of the MSCs; however, improved their survival in low oxygen environments; (3) the cell growth factors and positive cell cycle regulation genes, such as PCNA, APC, CCNB1, CCNB2 and CDC6 were all down-regulated; while the key negative cell cycle regulation genes TP53, BRCA1, CASP3 and CDKN1A were significantly up-regulated in the cells with FABP3 overexpression. Our data suggested that FABP3 was up-regulated under hypoxia; also negatively regulated the cell metabolic process and the mitotic cell cycle. Overexpression of FABP3 inhibited cell growth and proliferation via negative regulation of the cell cycle and down-regulation of cell growth factors, but enhances cell survival in hypoxic or ischemic conditions. - Highlights: • FABP3 expression pattern was studied in 12 human hypoxic-MSCs. • FABP3 mRNA and proteins are upregulated in the MSCs under hypoxic conditions.

  20. Chimeric Mouse model to track the migration of bone marrow derived cells in glioblastoma following anti-angiogenic treatments.

    PubMed

    Achyut, B R; Shankar, Adarsh; Iskander, A S M; Ara, Roxan; Knight, Robert A; Scicli, Alfonso G; Arbab, Ali S

    2016-03-01

    Bone marrow derived cells (BMDCs) have been shown to contribute in the tumor development. In vivo animal models to investigate the role of BMDCs in tumor development are poorly explored. We established a novel chimeric mouse model using as low as 5 × 10(6) GFP+ BM cells in athymic nude mice, which resulted in >70% engraftment within 14 d. In addition, chimera was established in NOD-SCID mice, which displayed >70% with in 28 d. Since anti-angiogenic therapies (AAT) were used as an adjuvant against VEGF-VEGFR pathway to normalize blood vessels in glioblastoma (GBM), which resulted into marked hypoxia and recruited BMDCs to the tumor microenvironment (TME). We exploited chimeric mice in athymic nude background to develop orthotopic U251 tumor and tested receptor tyrosine kinase inhibitors and CXCR4 antagonist against GBM. We were able to track GFP+ BMDCs in the tumor brain using highly sensitive multispectral optical imaging instrument. Increased tumor growth associated with the infiltration of GFP+ BMDCs acquiring suppressive myeloid and endothelial phenotypes was seen in TME following treatments. Immunofluorescence study showed GFP+ cells accumulated at the site of VEGF, SDF1 and PDGF expression, and at the periphery of the tumors following treatments. In conclusion, we developed a preclinical chimeric model of GBM and phenotypes of tumor infiltrated BMDCs were investigated in context of AATs. Chimeric mouse model could be used to study detailed cellular and molecular mechanisms of interaction of BMDCs and TME in cancer. PMID:26797476

  1. Galectin-9 is Involved in Immunosuppression Mediated by Human Bone Marrow-derived Clonal Mesenchymal Stem Cells.

    PubMed

    Kim, Si-Na; Lee, Hyun-Joo; Jeon, Myung-Shin; Yi, TacGhee; Song, Sun U

    2015-10-01

    Bone marrow-derived mesenchymal stem cells (MSCs) have immunomodulatory properties and can suppress exaggerated pro-inflammatory immune responses. Although the exact mechanisms remain unclear, a variety of soluble factors are known to contribute to MSC-mediated immunosuppression. However, functional redundancy in the immunosuppressive properties of MSCs indicates that other uncharacterized factors could be involved. Galectin-9, a member of the β-galactoside binding galectin family, has emerged as an important regulator of innate and adaptive immunity. We examined whether galectin-9 contributes to MSC-mediated immunosuppression. Galectin-9 was strongly induced and secreted from human MSCs upon stimulation with pro-inflammatory cytokines. An in vitro immunosuppression assay using a knockdown approach revealed that galectin-9-deficient MSCs do not exert immunosuppressive activity. We also provided evidence that galectin-9 may contribute to MSC-mediated immunosuppression by binding to its receptor, TIM-3, expressed on activated lymphocytes, leading to apoptotic cell death of activated lymphocytes. Taken together, our findings demonstrate that galectin-9 is involved in MSC-mediated immunosuppression and represents a potential therapeutic factor for the treatment of inflammatory diseases. PMID:26557808

  2. Therapeutic effects of mouse bone marrow-derived clonal mesenchymal stem cells in a mouse model of inflammatory bowel disease.

    PubMed

    Park, Jin Seok; Yi, Tac-Ghee; Park, Jong-Min; Han, Young Min; Kim, Jun-Hyung; Shin, Dong-Hee; Tak, Seon Ji; Lee, Kyuheon; Lee, Youn Sook; Jeon, Myung-Shin; Hahm, Ki-Baik; Song, Sun U; Park, Seok Hee

    2015-11-01

    Mouse bone marrow-derived clonal mesenchymal stem cells (mcMSCs), which were originated from a single cell by a subfractionation culturing method, are recognized as new paradigm for stem cell therapy featured with its homogenous cell population. Next to proven therapeutic effects against pancreatitis, in the current study we demonstrated that mcMSCs showed significant therapeutic effects in dextran sulfate sodium (DSS)-induced experimental colitis model supported with anti-inflammatory and restorative activities. mcMSCs significantly reduced the disease activity index (DAI) score, including weight loss, stool consistency, and intestinal bleeding and significantly increased survival rates. The pathological scores were also significantly improved with mcMSC. We have demonstrated that especial mucosal regeneration activity accompanied with significantly lowered level of apoptosis as beneficiary actions of mcMSCs in UC models. The levels of inflammatory cytokines including TNF-α, IFN-γ, IL-1β, IL-6, and IL-17 were all significantly concurrent with significantly repressed NF-κB activation compared to the control group and significantly decreased infiltrations of responsible macrophage and neutrophil. Conclusively, our findings provide the rationale that mcMSCs are applicable as a potential source of cell-based therapy in inflammatory bowel diseases, especially contributing either to prevent relapse or to accelerate healing as solution to unmet medical needs in IBD therapy. PMID:26566304

  3. NR2F2 regulates bone marrow-derived mesenchymal stem cell-promoted proliferation of Reh cells.

    PubMed

    Zhu, Ni; Wang, Huafang; Wei, Jieping; Wang, Binsheng; Shan, Wei; Lai, Xiaoyu; Zhao, Yanmin; Yu, Jian; Huang, He

    2016-08-01

    Bone marrow-derived mesenchymal stem cells (BM-MSCs) are pivotal components of the leukemic microenvironment. BM-MSCs have been previously reported to promote the proliferation of leukemic cells. To further understand the molecular mechanisms of BM-MSC-induced proliferation of leukemic cells, the present study co-cultured acute lymphoblastic leukemia (ALL) Reh cells with BM-MSCs. The current study used methods including shRNA, flow cytometry, MTT, reverse transcription-quantitative polymerase chain reaction, ELISA and western blotting. The data of the present study demonstrated that BM‑MSCs promote the proliferation of Reh cells and the NR2F2 mRNA and protein levels were elevated in BM‑MSCs following co‑culture. Additionally, it was demonstrated that shRNA knockdown of NR2F2 inhibited BM‑MSC‑induced proliferation of Reh cells. Furthermore, following downregulation of NR2F2, vascular endothelial growth factor A (VEGFA) secretion by BM‑MSCs was reduced. The present study demonstrated that NR2F2 mediates BM‑MSC‑induced proliferation of Reh cells, partially via regulation of VEGFA. Disrupting microenvironmental support by targeting NR2F2 may be a potential therapeutic strategy for ALL. PMID:27314877

  4. Bone Marrow-Derived Cell-Specific Chemokine (C-C motif) Receptor-2 Expression is Required for Arteriolar Remodeling

    PubMed Central

    Nickerson, Meghan M.; Song, Ji; Meisner, Joshua K.; Bajikar, Sameer; Burke, Caitlin W.; Shuptrine, Casey W.; Owens, Gary K.; Skalak, Thomas C.; Price, Richard J.

    2009-01-01

    Objective Bone marrow-derived cells (BMCs) and inflammatory chemokine receptors regulate arteriogenesis and angiogenesis. Here, we tested whether arteriolar remodeling in response to an inflammatory stimulus is dependent on BMC-specific chemokine (C-C motif) receptor 2 (CCR2) expression and whether this response involves BMC transdifferentiation into smooth muscle. Methods and Results Dorsal skinfold window chambers were implanted into C57Bl/6 wild-type (WT) mice, as well as the following bone marrow chimeras (donor-host): WT-WT, CCR2−/−-WT, WT-CCR2−/−, and EGFP+-WT. One day after implantation, tissue MCP-1 levels rose from “undetectable” to 463pg/mg, and the number of EGFP+ cells increased more than 4-fold, indicating marked inflammation. A 66% (28μm) increase in maximum arteriolar diameter was observed over 7 days in WT-WT mice. This arteriolar remodeling response was completely abolished in CCR2−/−-WT mice but largely rescued in WT-CCR2−/− mice. EGFP+ BMCs were numerous throughout the tissue, but we found no evidence that EGFP+ BMCs transdifferentiate into smooth muscle, based on examination of >800 arterioles and venules. Conclusions BMC-specific CCR2 expression is required for injury/inflammation-associated arteriolar remodeling, but this response is not characterized by the differentiation of BMCs into smooth muscle. PMID:19734197

  5. Effect of bone marrow derived mesenchymal stem cells on lung pathology and inflammation in ovalbumin-induced asthma in mouse

    PubMed Central

    Mohammadian, Maryam; Boskabady, Mohammad Hosein; Kashani, Iraj Ragerdi; Jahromi, Gila Pirzad; Omidi, Amene; Nejad, Amir Kavian; Khamse, Safoura; Sadeghipour, Hamid Reza

    2016-01-01

    Objective(s): Bone marrow-derived mesenchymal stem cells (BMSCs) have attracted significant interest to treat asthma and its complication. In this study, the effects of BMSCs on lung pathology and inflammation in an ovalbumin-induced asthma model in mouse were examined. Materials and Methods: BALB/c mice were divided into three groups: control group (animals were not sensitized), asthma group (animals were sensitized by ovalbumin), asthma+BMSC group (animals were sensitized by ovalbumin and treated with BMSCs). BMSCs were isolated and characterized and then labeled with Bromodeoxyuridine (BrdU). After that the cells transferred into asthmatic mice. Histopathological changes of the airways, BMSCs migration and total and differential white blood cell (WBC) count in bronchoalveolar lavage (BAL) fluid were evaluated. Results: A large number of BrdU-BMSCs were found in the lungs of mice treated with BMSCs. The histopathological changes, BAL total WBC counts and the percentage of neutrophils and eosinophils were increased in asthma group compared to the control group. Treatment with BMSCs significantly decreased airway pathological indices, inflammatory cell infiltration, and also goblet cell hyperplasia. Conclusion: The results of this study revealed that BMSCs therapy significantly suppressed the lung pathology and inflammation in the ovalbumin induced asthma model in mouse. PMID:27096065

  6. The temporal expression of estrogen receptor alpha-36 and runx2 in human bone marrow derived stromal cells during osteogenesis

    SciTech Connect

    Francis, W.R.; Owens, S.E.; Wilde, C.; Pallister, I.; Kanamarlapudi, V.; Zou, W.; Xia, Z.

    2014-10-24

    Highlights: • ERα36 is the predominant ERα isoform involved in bone regulation in human BMSC. • ERα36 mRNA is significantly upregulated during the process of osteogenesis. • The pattern of ERα36 and runx2 mRNA expression is similar during osteogenesis. • ERα36 appears to be co-localised with runx2 during osteogenesis. - Abstract: During bone maintenance in vivo, estrogen signals through estrogen receptor (ER)-α. The objectives of this study were to investigate the temporal expression of ERα36 and ascertain its functional relevance during osteogenesis in human bone marrow derived stromal cells (BMSC). This was assessed in relation to runt-related transcription factor-2 (runx2), a main modulatory protein involved in bone formation. ERα36 and runx2 subcellular localisation was assessed using immunocytochemistry, and their mRNA expression levels by real time PCR throughout the process of osteogenesis. The osteogenically induced BMSCs demonstrated a rise in ERα36 mRNA during proliferation followed by a decline in expression at day 10, which represents a change in dynamics within the culture between the proliferative stage and the differentiative stage. The mRNA expression profile of runx2 mirrored that of ERα36 and showed a degree subcellular co-localisation with ERα36. This study suggests that ERα36 is involved in the process of osteogenesis in BMSCs, which has implications in estrogen deficient environments.

  7. Matrine derivate MASM suppresses LPS-induced phenotypic and functional maturation of murine bone marrow-derived dendritic cells.

    PubMed

    Xu, Jing; Qi, Yang; Xu, Wei-Heng; Liu, Ying; Qiu, Lie; Wang, Ke-Qi; Hu, Hong-Gang; He, Zhi-Gao; Zhang, Jun-Ping

    2016-07-01

    Dendritic cell (DC) maturation process is a crucial step for the development of T cell immune responses and immune tolerance. In this study, we evaluated MASM, a novel derivative of the natural compound matrine that possesses a significant anti-inflammatory and immune-regulating property, for its efficacy to inhibit lipopolysaccharides (LPS)-induced maturation of murine bone marrow-derived dendritic cells. Here we show that MASM profoundly suppresses LPS-induced phenotypic and functional DC maturation. MASM inhibited LPS-induced expression of costimulatory molecules CD80 and CD86 in a concentration-dependent manner. MASM also attenuated LPS-induced IL-12p70, TNF-α, IL-6 and NO release of DCs. The MASM-treated DCs were highly efficient at antigen capture via mannose receptor-mediated endocytosis but showed weak stimulatory capacity for allogeneic T cell proliferation. Furthermore, MASM inhibited LPS-induced PI3K/Akt, MAPK and NF-κB pathways. These novel findings provide new insight into the immunopharmacological role of MASM in impacting on the DCs. PMID:27107799

  8. Regulation of Tenomodulin Expression Via Wnt/β-catenin Signaling in Equine Bone Marrow-derived Mesenchymal Stem Cells.

    PubMed

    Miyabara, Shihori; Yuda, Yohei; Kasashima, Yoshinori; Kuwano, Atsutoshi; Arai, Katsuhiko

    2014-01-01

    Tenomodulin has been recognized as a biomarker for tendon differentiation, and its gene expression is regulated by several transcription factors including Scleraxis and Mohawk. In this study, we found a novel regulatory mechanism of tenomodulin expression. Equine bone marrow-derived mesenchymal stem cells (BMSCs) in monolayer culture showed a low mRNA level of tenomodulin in comparison with the level in the tendon. When cultured in collagen gel containing a glycogen synthase kinase-3 (GSK-3) inhibitor (BIO), expression of tenomodulin in BMSCs increased up to the level in the tendon. Participation of GSK-3 in its gene expression was further demonstrated by a gene silencing experiment with small interference RNA corresponding to GSK-3, suggesting that Wnt/β-catenin signaling mediated expression of tenomodulin. These results were confirmed by nuclear translocation of β-catenin in BIO-treated BMSCs cultured in collagen gel. Under this culture condition, expression of tenomodulin-related transcription factors including Scleraxis and Mohawk was not affected, suggesting that Wnt/β-catenin signaling was independent from these transcription factors. Additionally, BIO strongly enhanced expression of type XIV collagen in collagen-embedded BMSCs up to the level in the tendon, and other tendon-related extracellular matrix components such as decorin and fibromodulin were also upregulated. Taken together, these results indicated that activation of Wnt/β-catenin signaling could induce differentiation of BMSCs into tenomodulin-expressing tendon cells in collagen gel. PMID:24834008

  9. Mitochondrial calcium uniporter inhibition attenuates mouse bone marrow-derived mast cell degranulation induced by beta-1,3-glucan

    PubMed Central

    Cuong, Dang Van; Kim, Hyoung Kyu; Marquez, Jubert; Kim, Nari; Ko, Kyung Soo; Rhee, Byoung Doo

    2016-01-01

    Mast cells are primary mediators of allergic inflammation. Beta-1,3-glucan (BG) protects against infection and shock by activating immune cells. Activation of the BG receptor induces an increase in intracellular Ca2+, which may induce exocytosis. However, little is known about the precise mechanisms underlying BG activation of immune cells and the possible role of mitochondria in this process. The present study examined whether BG induced mast cell degranulation, and evaluated the role of calcium transients during mast cell activation. Our investigation focused on the role of the mitochondrial calcium uniporter (MCU) in BG-induced degranulation. Black mouse (C57) bone marrow-derived mast cells were stimulated with 0.5 µg/ml BG, 100 µg/ml peptidoglycan (PGN), or 10 µM A23187 (calcium ionophore), and dynamic changes in cytosolic and mitochondrial calcium and membrane potential were monitored. BG-induced mast cell degranulation occurred in a time-dependent manner, and was significantly reduced under calcium-free conditions. Ruthenium red, a mitochondrial Ca2+ uniporter blocker, significantly reduced mast cell degranulation induced by BG, PGN, and A23187. These results suggest that the mitochondrial Ca2+ uniporter has an important regulatory role in BG-induced mast cell degranulation. PMID:26937218

  10. Intravenous transplantation of bone marrow-derived mononuclear cells prevents memory impairment in transgenic mouse models of Alzheimer's disease.

    PubMed

    Kanamaru, Takuya; Kamimura, Naomi; Yokota, Takashi; Nishimaki, Kiyomi; Iuchi, Katsuya; Lee, Hyunjin; Takami, Shinya; Akashiba, Hiroki; Shitaka, Yoshitsugu; Ueda, Masayuki; Katsura, Ken-Ichiro; Kimura, Kazumi; Ohta, Shigeo

    2015-04-24

    Stem cell transplantation therapy is currently in clinical trials for the treatment of ischemic stroke, and several beneficial aspects have been reported. Similarly, in Alzheimer's disease (AD), stem cell therapy is expected to provide an efficient therapeutic approach. Indeed, the intracerebral transplantation of stem cells reduced amyloid-β (Aβ) deposition and rescued memory deficits in AD model mice. Here, we show that intravenous transplantation of bone marrow-derived mononuclear cells (BMMCs) improves cognitive function in two different AD mouse models, DAL and APP mice, and prevents neurodegeneration. GFP-positive BMMCs were isolated from tibiae and femurs of 4-week-old mice and then transplanted intravenously into DAL and APP mice. Transplantation of BMMCs suppressed neuronal loss and restored memory impairment of DAL mice to almost the same level as in wild-type mice. Transplantation of BMMCs to APP mice reduced Aβ deposition in the brain. APP mice treated with BMMCs performed significantly better on behavioral tests than vehicle-injected mice. Moreover, the effects were observed even with transplantation after the onset of cognitive impairment in DAL mice. Together, our results indicate that intravenous transplantation of BMMCs has preventive effects against the cognitive decline in AD model mice and suggest a potential therapeutic effect of BMMC transplantation therapy. PMID:25698614

  11. Bone Marrow-Derived Mesenchymal Stem Cells Enhance Angiogenesis via their α6β1 Integrin Receptor

    PubMed Central

    Carrion, Bita; Kong, Yen P.; Kaigler, Darnell; Putnam, Andrew J

    2013-01-01

    Bone marrow-derived mesenchymal stem cells (BMSCs) facilitate the angiogenic response of endothelial cells (ECs) within three-dimensional (3D) matrices in vivo and in engineered tissues in vitro in part through paracrine mediators and by acting as stabilizing pericytes. However, the molecular interactions between BMSCs and nascent tubules during the process of angiogenesis are not fully understood. In this study, we have used a tractable 3D co-culture model to explore the functional role of the α6β1 integrin adhesion receptor on BMSCs in sprouting angiogenesis. We report that knockdown of the α6 integrin subunit in BMSCs significantly reduces capillary sprouting, and causes their failure to associate with the nascent vessels. Furthermore, we demonstrate that the BMSCs with attenuated α6 integrin proliferate at a significantly lower rate relative to either control cells expressing non-targeting shRNA or wild type BMSCs; however, despite adding more cells to compensate for this deficit in proliferation, deficient sprouting persists. Collectively, our findings demonstrate that the α6 integrin subunit in BMSCs is important for their ability to stimulate vessel morphogenesis. This conclusion may have important implications in the optimization of cell-based strategies to promote angiogenesis. PMID:24056178

  12. Immortalization of bone marrow-derived porcine mesenchymal stem cells and their differentiation into cells expressing cardiac phenotypic markers.

    PubMed

    Moscoso, Isabel; Rodriguez-Barbosa, Jose-Ignacio; Barallobre-Barreiro, Javier; Anon, Patricia; Domenech, Nieves

    2012-08-01

    Mesenchymal stem cells (MSCs) may be among the first stem cell types to be utilized in the clinic for cell therapy, because of their ease of isolation and extensive differentiation potential. Using a porcine model, we have established several cell lines from MSCs to facilitate in vitro and in vivo studies of their potential use for cellular therapy. Bone marrow-derived primary MSCs were immortalized using the pRNS-1 plasmid. We obtained four stable immortalized cell lines that exhibited higher proliferative capacities than the parental cells. All four cell lines displayed a common phenotype similar to that of primary mesenchymal cells, characterized by constitutively high expressions of CD90, CD29, CD44, SLA I and CD46, while CD172a, CD106 and CD56 were less expressed. Remarkably, treatment with 5-azacytidine-stimulated porcine MSCs lines to differentiate into cells that were positive for cardiac phenotypic markers, such as α-actin, connexin-43, sarcomeric actin, serca-2 and, to a lesser extent, desmin and troponin-T. These porcine MSC lines will be valuable biological tools for developing strategies for ex vivo expansion and differentiation of MSCs into a specific lineage. PMID:22162515

  13. Detection of BCR/ABL Translocation in Bone Marrow Derived Mesenchymal Stem Cells in Egyptian CML Patients

    PubMed Central

    Gaafar, Taghrid Mohamed; Raafat, Inas Ismail; Aly, Azza Ahmed; Mohamed, Nagwa Abd EL-Ghaffar; Farid, Reem Jan; Saad, Neveen Ezzat; EL-Hawary, Rabab; Mostafaa, Naglaa; Ahmed, Mirhan Mohamed

    2015-01-01

    BACKGROUND: Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder of hematopoietic stem cells. It is characterized at the cytogenetic level by Philadelphia (ph) chromosome and at the molecular level by the BCR/ABL gene rearrangement. Bone marrow derived mesenchymal stem cells (MSCs) are pluripotent stem cells that can differentiate into several mesenchymal tissues. AIM: To observe the biological characteristics of MSCS from CML patients and to determine whether MSCs harbor the abnormal BCR/ABL translocation similar to CML bone marrow cells. SUBJECTS AND METHODS: Characterized MSCs were isolated from 12 newly diagnosed Philadelphia positive untreated CML patients. RESULTS: MSCs can be readily isolated from CML marrow and exhibit major expansion. Flow cytometry analysis revealed the typical MSC phenotype. Moreover; MSCs do not harbor the BCR/ABL translocation confirmed by karyotype and real time PCR. CONCLUSION: MSCs from CML patients express the typical MSC phenotype; and do not express the BCR/ABL gene. Since; MSCs are able to support engraftment of hematopoietic stem cells in stem cell transplantation(SCT) as well as suppress alloreactive T cells causing graft versus –host disease, this current study provides evidence that in a SCT setting of CML patients, autologous MSCs could be a source of stem cell support in future cell therapy applications.

  14. Effects of Pholiota nameko polysaccharide on NF-κB pathway of murine bone marrow-derived dendritic cells.

    PubMed

    Li, Haiping; Tao, Yongqing; Zhao, Pei; Huai, Lihua; Zhi, Dexian; Liu, Jiangmei; Li, Guoliang; Dang, Chunlan; Xu, Yufeng

    2015-01-01

    This study investigated the effect of a polysaccharide purified from Pholiota nameko (PNPS-1) on the NF-κB signaling pathway of murine bone marrow-derived dendritic cells (BMDCs) and relevant mechanisms. The results showed that PNPS-1 could decrease the expression of maturation markers CD40 and CD80 on BMDCs. PNPS-1 also could decrease the mRNA expression of Myd88, TRAF6, TIRAP, IRAKI, IKBKB, NFKB1, NFKB2 and RelA in immature BMDCs determined by RT-PCR, and decreased the IKKβ and P65 production in BMDCs determined by Western blot, and decreased the NF-кB P65 production determined by ELISA. In addition, the effects of PNPS-1 on BMDCs were significantly impaired by treating the cells with anti-TLR2 antibody prior to PNPS-1 treatment, implying direct interaction between PNPS-1 and TLR2 on cell surface. These results indicate that PNPS-1 regulates BMDCs through TLR2 and downstream NF-кB signalings. PMID:25812973

  15. Malignant Transformation in Glioma Steered by an Angiogenic Switch: Defining a Role for Bone Marrow-Derived Cells.

    PubMed

    Xu, Raymond; Pisapia, David; Greenfield, Jeffrey P

    2016-01-01

    Low-grade gliomas, such as pilocytic astrocytoma and subependymoma, are often characterized as benign tumors due to their relative circumscription radiologically and typically non-aggressive biologic behavior. In contrast, low-grades that are by their nature diffusely infiltrative, such as diffuse astrocytomas and oligodendrogliomas, have the potential to transform into malignant high-grade counterparts and, given sufficient time, invariably do so. These high-grade gliomas carry very poor prognoses and are largely incurable, warranting a closer look at what causes this adverse transition. A key characteristic that distinguishes low- and high-grade gliomas is neovascularization: it is absent in low-grade gliomas, but prolific in high-grade gliomas, providing the tumor with ample blood supply for exponential growth. It has been well described in the literature that bone marrow-derived cells (BMDCs) may contribute to the angiogenic switch that is responsible for malignant transformation of low-grade gliomas. In this review, we will summarize the current literature on BMDCs and their known contribution to angiogenesis-associated tumor growth in gliomas. PMID:26973806

  16. Endoplasmic reticulum stress in bone marrow-derived cells prevents acute cardiac inflammation and injury in response to angiotensin II.

    PubMed

    Li, T-T; Jia, L-X; Zhang, W-M; Li, X-Y; Zhang, J; Li, Y-L; Li, H-H; Qi, Y-F; Du, J

    2016-01-01

    Inflammation plays an important role in hypertensive cardiac injury. The endoplasmic reticulum (ER) stress pathway is involved in the inflammatory response. However, the role of ER stress in elevated angiotensin II (Ang II)-induced cardiac injury remains unclear. In this study, we investigated the role of ER stress in Ang II-induced hypertensive cardiac injury. Transcriptome analysis and quantitative real-time PCR showed that Ang II infusion in mice increased ER stress-related genes expression in the heart. C/EBP homologous protein (CHOP) deficiency, a key mediator of ER stress, increased infiltration of inflammatory cells, especially neutrophils, the production of inflammatory cytokines, chemokines in Ang II-infused mouse hearts. CHOP deficiency increased Ang II-induced cardiac fibrotic injury: (1) Masson trichrome staining showed increased fibrotic areas, (2) immunohistochemistry staining showed increased expression of α-smooth muscle actin, transforming growth factor β1 and (3) quantitative real-time PCR showed increased expression of collagen in CHOP-deficient mouse heart. Bone marrow transplantation experiments indicated that CHOP deficiency in bone marrow cells was responsible for Ang II-induced cardiac fibrotic injury. Moreover, TUNEL staining and flow cytometry revealed that CHOP deficiency decreased neutrophil apoptosis in response to Ang II. Taken together, our study demonstrated that hypertension induced ER stress after Ang II infusion. ER stress in bone marrow-derived cells protected acute cardiac inflammation and injury in response to Ang II. PMID:27277680

  17. The role of Hibiscus sabdariffa L. (Roselle) in maintenance of ex vivo murine bone marrow-derived hematopoietic stem cells.

    PubMed

    Abdul Hamid, Zariyantey; Lin Lin, Winnie Hii; Abdalla, Basma Jibril; Bee Yuen, Ong; Latif, Elda Surhaida; Mohamed, Jamaludin; Rajab, Nor Fadilah; Paik Wah, Chow; Wak Harto, Muhd Khairul Akmal; Budin, Siti Balkis

    2014-01-01

    Hematopoietic stem cells- (HSCs-) based therapy requires ex vivo expansion of HSCs prior to therapeutic use. However, ex vivo culture was reported to promote excessive production of reactive oxygen species (ROS), exposing HSCs to oxidative damage. Efforts to overcome this limitation include the use of antioxidants. In this study, the role of Hibiscus sabdariffa L. (Roselle) in maintenance of cultured murine bone marrow-derived HSCs was investigated. Aqueous extract of Roselle was added at varying concentrations (0-1000 ng/mL) for 24 hours to the freshly isolated murine bone marrow cells (BMCs) cultures. Effects of Roselle on cell viability, reactive oxygen species (ROS) production, glutathione (GSH) level, superoxide dismutase (SOD) activity, and DNA damage were investigated. Roselle enhanced the survival (P < 0.05) of BMCs at 500 and 1000 ng/mL, increased survival of Sca-1(+) cells (HSCs) at 500 ng/mL, and maintained HSCs phenotype as shown from nonremarkable changes of surface marker antigen (Sca-1) expression in all experimental groups. Roselle increased (P < 0.05) the GSH level and SOD activity but the level of reactive oxygen species (ROS) was unaffected. Moreover, Roselle showed significant cellular genoprotective potency against H2O2-induced DNA damage. Conclusively, Roselle shows novel property as potential supplement and genoprotectant against oxidative damage to cultured HSCs. PMID:25405216

  18. Route of delivery influences biodistribution of human bone marrow-derived mesenchymal stromal cells following experimental bone marrow transplantation

    PubMed Central

    Wang, Fangjing; Eid, Saada; Dennis, James E; Cooke, Kenneth R; Auletta, Jeffery J; Lee, Zhenghong

    2015-01-01

    Mesenchymal stromal cells (MSCs) have shown promise as treatment for graft-versus-host disease (GvHD) following allogeneic bone marrow transplantation (alloBMT). Mechanisms mediating in vivo effects of MSCs remain largely unknown, including their biodistribution following infusion. To this end, human bone-marrow derived MSCs (hMSCs) were injected via carotid artery (IA) or tail vein (TV) into allogeneic and syngeneic BMT recipient mice. Following xenogeneic transplantation, MSC biodistribution was measured by bioluminescence imaging (BLI) using hMSCs transduced with a reporter gene system containing luciferase and by scintigraphic imaging using hMSCs labeled with [99mTc]-HMPAO. Although hMSCs initially accumulated in the lungs in both transplant groups, more cells migrated to organs in alloBMT recipient as measured by in vivo BLI and scintigraphy and confirmed by ex vivo BLI imaging, immunohistochemistry and quantitative RT-PCR. IA injection resulted in persistent whole–body hMSC distribution in alloBMT recipients, while hMSCs were rapidly cleared in the syngeneic animals within one week. In contrast, TV-injected hMSCs were mainly seen in the lungs with fewer cells traveling to other organs. Summarily, these results demonstrate the potential use of IA injection to alter hMSC biodistribution in order to more effectively deliver hMSCs to targeted tissues and microenvironments. PMID:27330253

  19. Detection of organic nanoparticles in human bone marrow-derived stromal cells using ToF-SIMS and PCA.

    PubMed

    Kokesch-Himmelreich, Julia; Woltmann, Beatrice; Torger, Bernhard; Rohnke, Marcus; Arnhold, Stefan; Hempel, Ute; Müller, Martin; Janek, Jürgen

    2015-06-01

    The detection and localization of polymer-based nanoparticles in human bone marrow-derived stromal cells (hBMSC) by time-of-flight secondary ion mass spectrometry (ToF-SIMS) is reported as an example for the mass spectrometry imaging of organic nanoparticles in cell environments. Polyelectrolyte complex (PEC) nanoparticles (NP) made of polyethylenimine (PEI) and cellulose sulfate (CS), which were developed as potential drug carrier and coatings for implant materials, were chosen for the imaging experiments. To investigate whether the PEI/CS-NP were taken up by the hBMSC ToF-SIMS measurements on cross sections of the cells and depth profiling of whole, single cells were carried out. Since the mass spectra of the PEI/CS nanoparticles are close to the mass spectra of the cells principal component analysis (PCA) was performed to get specific masses of the PEI/CS-NP. Mass fragments originating from the NP compounds especially from cellulose sulfate could be used to unequivocally detect and image the PEI/CS-NP inside the hBMSC. The findings were confirmed by light and transmission electron microscopy. Graphical Abstract During ToF-SIMS analysis Bi3 (+) primary ions hit the sample surface and so called secondary ions (SI) are emitted and detected in the mass analyser. Exemplary mass images of cross sections of human mesenchymal stromal cells (red; m/z = 86.1 u) cultured with organic nanoparticles (green; m/z = 143.0 u) were obtained. PMID:25869483

  20. Effects of matrix metalloproteinase-1 on the myogenic differentiation of bone marrow-derived mesenchymal stem cells in vitro

    SciTech Connect

    Zheng, Zhenyang; Leng, Yan; Zhou, Chen; Ma, Zhenyu; Zhong, Zhigang; Shi, Xing-Ming; Zhang, Weixi

    2012-11-16

    Highlights: Black-Right-Pointing-Pointer MMP-1 is a member of the zinc-dependent endopeptidase family. Black-Right-Pointing-Pointer MMP-1 has no cytotoxic effects on BMSCs. Black-Right-Pointing-Pointer MMP-1 can promote the myogenic differentiation of BMSCs. Black-Right-Pointing-Pointer MyoD and desmin were chosen as myogenic markers in this study. -- Abstract: Matrix metalloproteinase-1 (MMP-1) is a member of the family of zinc-dependent endopeptidases that are capable of degrading extracellular matrix (ECM) and certain non-matrix proteins. It has been shown that MMP-1 can enhance muscle regeneration by improving the differentiation and migration of myoblasts. However, it is still not known whether MMP-1 can promote the myogenesis of bone marrow-derived mesenchymal stem cells (BMSCs). To address this question, we isolated BMSCs from C57BL/6J mice and investigated the effects of MMP-1 on their proliferation and myogenic differentiation. Our results showed that MMP-1 treatment, which had no cytotoxic effects on BMSCs, increased the mRNA and protein levels of MyoD and desmin in a dose-dependent manner, indicating that MMP-1 promoted myogenic differentiation of BMSCs in vitro. These results suggest that BMSCs may have a therapeutic potential for treating muscular disorders.

  1. Attachment and growth of human bone marrow derived mesenchymal stem cells on regenerated antheraea pernyi silk fibroin films.

    PubMed

    Luan, Xi-Ying; Wang, Yong; Duan, Xiang; Duan, Qiao-Yan; Li, Ming-Zhong; Lu, Shen-Zhou; Zhang, Huan-Xiang; Zhang, Xue-Guang

    2006-12-01

    Silk fibroin of the silkworm Bombyx mori has been studied extensively, while the research on Antheraea pernyi silk fibroin (A. pernyi SF) in biomaterials is only at an early stage. In this study, the attachment, morphology, growth and phenotype of human bone marrow derived mesenchymal stem cells (hBMSCs) cultured on the regenerated A. pernyi SF films were studied in vitro. The results indicated that the attachment of hBMSCs on the regenerated A. pernyi SF films was almost the same as that on the collagen films. MTT and cell counting analyses demonstrated that the growth of hBMSCs on the regenerated A. pernyi SF films was better than that on controls. Moreover, electron scanning microscopy and fluorescence-activated cell sorting assays showed that the regenerated A. pernyi SF supported hBMSCs growth and functional maintenance compared with the controls. These data suggest that the regenerated A. pernyi SF, like Bombyx mori silk fibroin (B. mori SF) and collagen, can support hBMSCs attachment, growth and phenotypic maintenance, and has better biocompatibilities for hBMSCs in vitro culture. PMID:18458403

  2. Inhibition of AQP1 Hampers Osteosarcoma and Hepatocellular Carcinoma Progression Mediated by Bone Marrow-Derived Mesenchymal Stem Cells

    PubMed Central

    Pelagalli, Alessandra; Nardelli, Anna; Fontanella, Raffaela; Zannetti, Antonella

    2016-01-01

    The complex cross-talk between tumor cells and their surrounding stromal environment plays a key role in the pathogenesis of cancer. Among several cell types that constitute the tumor stroma, bone marrow-derived mesenchymal stem cells (BM-MSCs) selectively migrate toward the tumor microenvironment and contribute to the active formation of tumor-associated stroma. Therefore, here we elucidate the involvement of BM-MSCs to promote osteosarcoma (OS) and hepatocellular carcinoma (HCC) cells migration and invasion and deepening the role of specific pathways. We analyzed the function of aquaporin 1 (AQP1), a water channel known to promote metastasis and neoangiogenes. AQP1 protein levels were analyzed in OS (U2OS) and HCC (SNU-398) cells exposed to conditioned medium from BM-MSCs. Tumor cell migration and invasion in response to BM-MSC conditioned medium were evaluated through a wound healing assay and Boyden chamber, respectively. The results showed that the AQP1 level was increased in both tumor cell lines after treatment with BM-MSC conditioned medium. Moreover, BM-MSCs-mediated tumor cell migration and invasion were hampered after treatment with AQP1 inhibitor. These data suggest that the recruitment of human BM-MSCs into the tumor microenvironment might cause OS and HCC cell migration and invasion through involvement of AQP1. PMID:27409610

  3. Nestin Positive Bone Marrow Derived Cells Responded to Injury Mobilize into Peripheral Circulation and Participate in Skin Defect Healing.

    PubMed

    Yang, Yi; Pang, Danlin; Hu, Chenghu; Lv, Yajie; He, Tao; An, Yulin; Tang, Zhangui; Deng, Zhihong

    2015-01-01

    Exogenously infused mesenchymal stem cells (MSCs) are thought to migrate to injury site through peripheral blood stream and participate in tissue repair. However, whether and how endogenous bone marrow MSCs mobilized to circulating and targeted to tissue injury has raised some controversy, and related studies were restricted by the difficulty of MSCs identifying in vivo. Nestin, a kind of intermediate filament protein initially identified in neuroepithelial stem cells, was recently reported as a credible criteria for MSCs in bone marrow. In this study, we used a green fluorescent protein (GFP) labeled bone marrow replacement model to trace the nestin positive bone marrow derived cells (BMDCs) of skin defected-mice. We found that after skin injured, numbers of nestin+ cells in peripheral blood and bone marrow both increased. A remarkable concentration of nestin+ BMDCs around skin wound was detected, while few of these cells could be observed in uninjured skin or other organs. This recruitment effect could not be promoted by granulocyte colony-stimulating factor (G-CSF), suggests a different mobilization mechanism from ones G-CSF takes effect on hematopoietic cells. Our results proposed nestin+ BMDCs as mobilized candidates in skin injury repair, which provide a new insight of endogenous MSCs therapy. PMID:26633897

  4. Mitochondrial calcium uniporter inhibition attenuates mouse bone marrow-derived mast cell degranulation induced by beta-1,3-glucan.

    PubMed

    Cuong, Dang Van; Kim, Hyoung Kyu; Marquez, Jubert; Kim, Nari; Ko, Kyung Soo; Rhee, Byoung Doo; Han, Jin

    2016-03-01

    Mast cells are primary mediators of allergic inflammation. Beta-1,3-glucan (BG) protects against infection and shock by activating immune cells. Activation of the BG receptor induces an increase in intracellular Ca(2+), which may induce exocytosis. However, little is known about the precise mechanisms underlying BG activation of immune cells and the possible role of mitochondria in this process. The present study examined whether BG induced mast cell degranulation, and evaluated the role of calcium transients during mast cell activation. Our investigation focused on the role of the mitochondrial calcium uniporter (MCU) in BG-induced degranulation. Black mouse (C57) bone marrow-derived mast cells were stimulated with 0.5 µg/ml BG, 100 µg/ml peptidoglycan (PGN), or 10 µM A23187 (calcium ionophore), and dynamic changes in cytosolic and mitochondrial calcium and membrane potential were monitored. BG-induced mast cell degranulation occurred in a time-dependent manner, and was significantly reduced under calcium-free conditions. Ruthenium red, a mitochondrial Ca(2+) uniporter blocker, significantly reduced mast cell degranulation induced by BG, PGN, and A23187. These results suggest that the mitochondrial Ca(2+) uniporter has an important regulatory role in BG-induced mast cell degranulation. PMID:26937218

  5. Homing and restorative effects of bone marrow-derived mesenchymal stem cells on cisplatin injured ovaries in rats.

    PubMed

    Liu, Jiabin; Zhang, Haiying; Zhang, Yun; Li, Nan; Wen, Yuku; Cao, Fanglei; Ai, Hao; Xue, Xiaoou

    2014-12-31

    Premature ovarian failure (POF) is a long-term adverse effect of chemotherapy treatment. However, current available treatment regimens are not optimal. Emerging evidence suggests that bone marrow-derived mesenchymal stem cells (BMSCs) could restore the structure and function of injured tissues, but the homing and restorative effects of BMSCs on chemotherapy injured ovaries are still not clear. In this study, we found that granulosa cell (GC) apoptosis induced by cisplatin was reduced when BMSCs were migrated to granulosa cells (GCs) in vitro. Chemotherapy-induced POF was induced by intraperitoneal injection of cisplatin in rats. BMSCs labeled with enhanced green fluorescent protein (EGFP) were injected into the rats via the tail vein to investigate the homing and distribution of BMSCs in vivo. The number of BMSCs in the ovarian hilum and medulla was greater than in the cortex, but no BMSCs were found in the follicles and corpus lutea. In addition, the BMSCs treatment group's antral follicle count and estradiol levels increased after 30 days, compared with the POF group. Hence, our study demonstrates that intravenously delivered BMSCs can home to the ovaries, and restore its structure and function in POF model rats. PMID:25410907

  6. The Effect of Bone-Marrow-Derived Stem Cells and Adipose-Derived Stem Cells on Wound Contraction and Epithelization

    PubMed Central

    Uysal, Cagri A.; Tobita, Morikuni; Hyakusoku, Hiko; Mizuno, Hiroshi

    2014-01-01

    Objective: The relationship between the wound contraction and levels of α-smooth muscle actin (α-SMA) has been revealed in different studies. We aimed to investigate the effects of mesenchymal stem cells (MSCs), mainly bone-marrow-derived stem cells (BSCs) and adipose-derived stem cells (ASCs), and find out the α-SMA, fibroblast growth factor (FGF), transforming growth factor beta, and vascular endothelial growth factor (VEGF) levels on an in vivo acute wound healing model after the application of MSCs. Approach: Four circular skin defects were formed on the dorsum of Fisher rats (n=20). The defects were applied phosphate-buffered saline (PBS), ASCs, BSCs, and patchy skin graft, respectively. The healing time and scar area were noted. Results: There was a statistical decrease in the healing time in ASC, BSC, and skin graft groups (p<0.05). However, the scar was smaller in the PBS group (p<0.05). The α-SMA levels were statistically lower in ASC, BSC, and graft groups (p<0.05). The FGF levels were statistically higher in ASC and BSC groups (p<0.05). The differentiation of the injected MSCs to endothelial cells and keratinocytes was observed. Innovation and Conclusion: MSCs decrease the healing time and contraction of the wound while increasing the epithelization rate by increasing angiogenesis. PMID:24940554

  7. Inhibition of AQP1 Hampers Osteosarcoma and Hepatocellular Carcinoma Progression Mediated by Bone Marrow-Derived Mesenchymal Stem Cells.

    PubMed

    Pelagalli, Alessandra; Nardelli, Anna; Fontanella, Raffaela; Zannetti, Antonella

    2016-01-01

    The complex cross-talk between tumor cells and their surrounding stromal environment plays a key role in the pathogenesis of cancer. Among several cell types that constitute the tumor stroma, bone marrow-derived mesenchymal stem cells (BM-MSCs) selectively migrate toward the tumor microenvironment and contribute to the active formation of tumor-associated stroma. Therefore, here we elucidate the involvement of BM-MSCs to promote osteosarcoma (OS) and hepatocellular carcinoma (HCC) cells migration and invasion and deepening the role of specific pathways. We analyzed the function of aquaporin 1 (AQP1), a water channel known to promote metastasis and neoangiogenes. AQP1 protein levels were analyzed in OS (U2OS) and HCC (SNU-398) cells exposed to conditioned medium from BM-MSCs. Tumor cell migration and invasion in response to BM-MSC conditioned medium were evaluated through a wound healing assay and Boyden chamber, respectively. The results showed that the AQP1 level was increased in both tumor cell lines after treatment with BM-MSC conditioned medium. Moreover, BM-MSCs-mediated tumor cell migration and invasion were hampered after treatment with AQP1 inhibitor. These data suggest that the recruitment of human BM-MSCs into the tumor microenvironment might cause OS and HCC cell migration and invasion through involvement of AQP1. PMID:27409610

  8. Activation of the germ-cell potential of human bone marrow-derived cells by a chemical carcinogen

    PubMed Central

    Liu, Chunfang; Ma, Zhan; Xu, Songtao; Hou, Jun; Hu, Yao; Yu, Yinglu; Liu, Ruilai; Chen, Zhihong; Lu, Yuan

    2014-01-01

    Embryonic/germ cell traits are common in malignant tumors and are thought to be involved in malignant tumor behaviors. The reasons why tumors show strong embryonic/germline traits (displaced germ cells or gametogenic programming reactivation) are controversial. Here, we show that a chemical carcinogen, 3-methyl-cholanthrene (3-MCA), can trigger the germ-cell potential of human bone marrow-derived cells (hBMDCs). 3-MCA promoted the generation of germ cell-like cells from induced hBMDCs that had undergone malignant transformation, whereas similar results were not observed in the parallel hBMDC culture at the same time point. The malignant transformed hBMDCs spontaneously and more efficiently generated into germ cell-like cells even at the single-cell level. The germ cell-like cells from induced hBMDCs were similar to natural germ cells in many aspects, including morphology, gene expression, proliferation, migration, further development, and teratocarcinoma formation. Therefore, our results demonstrate that a chemical carcinogen can reactivate the germline phenotypes of human somatic tissue-derived cells, which might provide a novel idea to tumor biology and therapy. PMID:24998261

  9. Induction of Spermatogenesis by Bone Marrow-derived Mesenchymal Stem Cells in Busulfan-induced Azoospermia in Hamster

    PubMed Central

    Tamadon, Amin; Mehrabani, Davood; Rahmanifar, Farhad; Jahromi, Alireza Raayat; Panahi, Mohadeseh; Zare, Shahrokh; Khodabandeh, Zahra; Jahromi, Iman Razeghian; Tanideh, Nader; Dianatpour, Mehdi; Ramzi, Mani; Koohi-Hoseinabadi, Omid

    2015-01-01

    Background Bone marrow-derived mesenchymal stem cells (BM-MSCs) have potential of differentiation and they secrete anti-inflammatory cytokines and growth factors which make them appropriate for cell therapy. Aim of the Work Were to evaluate the healing effect of BM-MSCs transplantation on germinal cells of busulfan-induced azoospermic hamsters. Material and Methods In the present experimental case control study, BM-MSCs were isolated from bone marrow of donor albino hamsters. Five mature male recipient hamsters received two doses of 10 mg/kg of busulfan with 21 days interval to stop endogenous spermatogenesis. After induction of azoospermia, right testis of hamsters was injected with 106 BM-MSCs via efferent duct and the left one remained as azoospermia control testis. Five normal mature hamsters were selected as normal intact control. After 35 days, testes and epididymis of three groups were removed for histological evaluation. Results Histomorphological analyses of BM-MSCs treated testes and epididymis showed the epithelial tissue of seminiferous tubules had normal morphology and spermatozoa were present in epididymis tubes. Spermatogenesis was observed in most cell-treated seminiferous tubules. The untreated seminiferous tubules were empty. Conclusion Transplanted BM-MSCs could successfully induce spermatogenesis in seminiferous tubules of azoospermic hamster. Therefore, BM-MSCs can be an attractive candidate in cell transplantation of azoospermia. PMID:26634062

  10. Development of a rapid culture method to induce adipocyte differentiation of human bone marrow-derived mesenchymal stem cells

    SciTech Connect

    Ninomiya, Yuichi; Sugahara-Yamashita, Yzumi; Nakachi, Yutaka; Tokuzawa, Yoshimi; Okazaki, Yasushi; Nishiyama, Masahiko

    2010-04-02

    Human mesenchymal stem cells (hMSCs) derived from bone marrow are multipotent stem cells that can regenerate mesenchymal tissues such as adipose, bone or muscle. It is thought that hMSCs can be utilized as a cell resource for tissue engineering and as human models to study cell differentiation mechanisms, such as adipogenesis, osteoblastogenesis and so on. Since it takes 2-3 weeks for hMSCs to differentiate into adipocytes using conventional culture methods, the development of methods to induce faster differentiation into adipocytes is required. In this study we optimized the culture conditions for adipocyte induction to achieve a shorter cultivation time for the induction of adipocyte differentiation in bone marrow-derived hMSCs. Briefly, we used a cocktail of dexamethasone, insulin, methylisobutylxanthine (DIM) plus a peroxisome proliferator-activated receptor {gamma} agonist, rosiglitazone (DIMRo) as a new adipogenic differentiation medium. We successfully shortened the period of cultivation to 7-8 days from 2-3 weeks. We also found that rosiglitazone alone was unable to induce adipocyte differentiation from hMSCs in vitro. However, rosiglitazone appears to enhance hMSC adipogenesis in the presence of other hormones and/or compounds, such as DIM. Furthermore, the inhibitory activity of TGF-{beta}1 on adipogenesis could be investigated using DIMRo-treated hMSCs. We conclude that our rapid new culture method is very useful in measuring the effect of molecules that affect adipogenesis in hMSCs.

  11. Autologous transplantation of CD34(+) bone marrow derived mononuclear cells in management of non-reconstructable critical lower limb ischemia.

    PubMed

    Ismail, Ahmed M; Abdou, Said M; Aty, Hassan Abdel; Kamhawy, Adel H; Elhinedy, Mohammed; Elwageh, Mohammed; Taha, Atef; Ezzat, Amal; Salem, Hoda A; Youssif, Said; Salem, Mohamed L

    2016-08-01

    Patients with a decrease in limb perfusion with a potential threat to limb viability manifested by ischemic rest pain, ischemic ulcers, and/or gangrene are considered to have critical limb ischemia (CLI). Because of this generally poor outcome, there is a strong need for attempting any procedure to save the affected limb. The aim of this work is to evaluate the possibility to use stem cell therapy as a treatment option for patients with chronic critical lower limb ischemia with no distal run off. This study includes 20 patients with chronic critical lower limb ischemia with no distal run off who are unsuitable for vascular or endovascular option. These patients underwent stem cell therapy (SCT) by autologous transplantation of bone marrow derived mononuclear cells. 55 % of patients treated with SCT showed improvement of the rest pain after the first month, 60 % continued improvement of the rest pain after 6 months, 75 % after 1 year and 80 % after 2 years and continued without any deterioration till the third year. Limb salvage rate after STC was 80 % after the first year till the end of the second and third years. SCT can result in angiogenesis in patients with no-option CLI, providing a foundation for the application of this therapy to leg ischemia. PMID:25511801

  12. Nestin Positive Bone Marrow Derived Cells Responded to Injury Mobilize into Peripheral Circulation and Participate in Skin Defect Healing

    PubMed Central

    Lv, Yajie; He, Tao; An, Yulin; Tang, Zhangui; Deng, Zhihong

    2015-01-01

    Exogenously infused mesenchymal stem cells (MSCs) are thought to migrate to injury site through peripheral blood stream and participate in tissue repair. However, whether and how endogenous bone marrow MSCs mobilized to circulating and targeted to tissue injury has raised some controversy, and related studies were restricted by the difficulty of MSCs identifying in vivo. Nestin, a kind of intermediate filament protein initially identified in neuroepithelial stem cells, was recently reported as a credible criteria for MSCs in bone marrow. In this study, we used a green fluorescent protein (GFP) labeled bone marrow replacement model to trace the nestin positive bone marrow derived cells (BMDCs) of skin defected-mice. We found that after skin injured, numbers of nestin+ cells in peripheral blood and bone marrow both increased. A remarkable concentration of nestin+ BMDCs around skin wound was detected, while few of these cells could be observed in uninjured skin or other organs. This recruitment effect could not be promoted by granulocyte colony-stimulating factor (G-CSF), suggests a different mobilization mechanism from ones G-CSF takes effect on hematopoietic cells. Our results proposed nestin+ BMDCs as mobilized candidates in skin injury repair, which provide a new insight of endogenous MSCs therapy. PMID:26633897

  13. Identification of Rorβ targets in cultured osteoblasts and in human bone

    SciTech Connect

    Roforth, Matthew M. Khosla, Sundeep Monroe, David G.

    2013-11-01

    Highlights: •We examine the gene expression patterns controlled by Rorβ in osteoblasts. •Genes involved in extracellular matrix regulation and proliferation are affected. •Rorβ mRNA levels increase in aged, human bone biopsies. •Rorβ may affect osteoblast activity by modulation of these pathways. -- Abstract: Control of osteoblastic bone formation involves the cumulative action of numerous transcription factors, including both activating and repressive functions that are important during specific stages of differentiation. The nuclear receptor retinoic acid receptor-related orphan receptor β (Rorβ) has been recently shown to suppress the osteogenic phenotype in cultured osteoblasts, and is highly upregulated in bone marrow-derived osteogenic precursors isolated from aged osteoporotic mice, suggesting Rorβ is an important regulator of osteoblast function. However the specific gene expression patterns elicited by Rorβ are unknown. Using microarray analysis, we identified 281 genes regulated by Rorβ in an MC3T3-E1 mouse osteoblast cell model (MC3T3-Rorβ-GFP). Pathway analysis revealed alterations in genes involved in MAPK signaling, genes involved in extracellular matrix (ECM) regulation, and cytokine-receptor interactions. Whereas the identified Rorβ-regulated ECM genes normally decline during osteoblastic differentiation, they were highly upregulated in this non-mineralizing MC3T3-Rorβ-GFP model system, suggesting that Rorβ may exert its anti-osteogenic effects through ECM disruption. Consistent with these in vitro findings, the expression of both RORβ and a subset of RORβ-regulated genes were increased in bone biopsies from postmenopausal women (73 ± 7 years old) compared to premenopausal women (30 ± 5 years old), suggesting a role for RORβ in human age-related bone loss. Collectively, these data demonstrate that Rorβ regulates known osteogenic pathways, and may represent a novel therapeutic target for age-associated bone loss.

  14. Id4, a new candidate gene for senile osteoporosis, acts as a molecular switch promoting osteoblast differentiation.

    PubMed

    Tokuzawa, Yoshimi; Yagi, Ken; Yamashita, Yzumi; Nakachi, Yutaka; Nikaido, Itoshi; Bono, Hidemasa; Ninomiya, Yuichi; Kanesaki-Yatsuka, Yukiko; Akita, Masumi; Motegi, Hiromi; Wakana, Shigeharu; Noda, Tetsuo; Sablitzky, Fred; Arai, Shigeki; Kurokawa, Riki; Fukuda, Toru; Katagiri, Takenobu; Schönbach, Christian; Suda, Tatsuo; Mizuno, Yosuke; Okazaki, Yasushi

    2010-07-01

    Excessive accumulation of bone marrow adipocytes observed in senile osteoporosis or age-related osteopenia is caused by the unbalanced differentiation of MSCs into bone marrow adipocytes or osteoblasts. Several transcription factors are known to regulate the balance between adipocyte and osteoblast differentiation. However, the molecular mechanisms that regulate the balance between adipocyte and osteoblast differentiation in the bone marrow have yet to be elucidated. To identify candidate genes associated with senile osteoporosis, we performed genome-wide expression analyses of differentiating osteoblasts and adipocytes. Among transcription factors that were enriched in the early phase of differentiation, Id4 was identified as a key molecule affecting the differentiation of both cell types. Experiments using bone marrow-derived stromal cell line ST2 and Id4-deficient mice showed that lack of Id4 drastically reduces osteoblast differentiation and drives differentiation toward adipocytes. On the other hand knockdown of Id4 in adipogenic-induced ST2 cells increased the expression of Ppargamma2, a master regulator of adipocyte differentiation. Similar results were observed in bone marrow cells of femur and tibia of Id4-deficient mice. However the effect of Id4 on Ppargamma2 and adipocyte differentiation is unlikely to be of direct nature. The mechanism of Id4 promoting osteoblast differentiation is associated with the Id4-mediated release of Hes1 from Hes1-Hey2 complexes. Hes1 increases the stability and transcriptional activity of Runx2, a key molecule of osteoblast differentiation, which results in an enhanced osteoblast-specific gene expression. The new role of Id4 in promoting osteoblast differentiation renders it a target for preventing the onset of senile osteoporosis. PMID:20628571

  15. Generation of mesenchymal stromal cells in the presence of platelet lysate: a phenotypic and functional comparison of umbilical cord blood- and bone marrow-derived progenitors

    PubMed Central

    Avanzini, Maria Antonietta; Bernardo, Maria Ester; Cometa, Angela Maria; Perotti, Cesare; Zaffaroni, Nadia; Novara, Francesca; Visai, Livia; Moretta, Antonia; Del Fante, Claudia; Villa, Raffaella; Ball, Lynne M.; Fibbe, Willem E.; Maccario, Rita; Locatelli, Franco

    2009-01-01

    Background Mesenchymal stromal cells are employed in various different clinical settings in order to modulate immune response. However, relatively little is known about the mechanisms responsible for their immunomodulatory effects, which could be influenced by both the cell source and culture conditions. Design and Methods We tested the ability of a 5% platelet lysate-supplemented medium to support isolation and ex vivo expansion of mesenchymal stromal cells from full-term umbilical-cord blood. We also investigated the biological/functional properties of umbilical cord blood mesenchymal stromal cells, in comparison with platelet lysate-expanded bone marrow mesenchymal stromal cells. Results The success rate of isolation of mesenchymal stromal cells from umbilical cord blood was in the order of 20%. These cells exhibited typical morphology, immunophenotype and differentiation capacity. Although they have a low clonogenic efficiency, umbilical cord blood mesenchymal stromal cells may possess high proliferative potential. The genetic stability of these cells from umbilical cord blood was demonstrated by a normal molecular karyotype; in addition, these cells do not express hTERT and telomerase activity, do express p16ink4a protein and do not show anchorage-independent cell growth. Concerning alloantigen-specific immune responses, umbilical cord blood mesenchymal stromal cells were able to: (i) suppress T- and NK-lymphocyte proliferation, (ii) decrease cytotoxic activity and (iii) only slightly increase interleukin-10, while decreasing interferon-γ secretion, in mixed lymphocyte culture supernatants. While an indoleamine 2,3-dioxygenase-specific inhibitor did not reverse mesenchymal stromal cell-induced suppressive effects, a prostaglandin E2-specific inhibitor hampered the suppressive effect of both umbilical cord blood- and bone marrow-mesenchymal stromal cells on alloantigen-induced cytotoxic activity. Mesenchymal stromal cells from both sources expressed HLA-G. Conclusions Umbilical cord blood- and bone marrow-mesenchymal stromal cells may differ in terms of clonogenic efficiency, proliferative capacity and immunomodulatory properties; these differences may be relevant for clinical applications. PMID:19773264

  16. Angiogenesis & Vasculogenesis: Inducing the growth of new blood vessels and wound healing by stimulation of Bone Marrow Derived Progenitor Cell Mobilization and Homing

    PubMed Central

    Velazquez, Omaida C.

    2009-01-01

    During embryonic development, the vasculature is among the first organs to form and is in charge of maintaining metabolic homeostasis by supplying oxygen and nutrients and removing waste products. As one would expect, blood vessels are critical not only for organ growth in the embryo, but also for repair of wounded tissue in the adult. An imbalance in ‘Angiogenesis’ (a time-honored term that globally refers to the growth of new blood vessels) contributes to the pathogenesis of numerous malignant, inflammatory, ischemic, infectious, immune, and wound healing disorders. In this review, we will focus on the central role of the growth of new blood vessels in ischemic and diabetic wound healing. We define the most current nomenclature that describes the neovascularization process in wounds. There are now two well defined, distinct, yet interrelated processes for the formation of post-natal new blood vessels, angiogenesis and vasculogenesis. We review recent new data on vasculogenesis that promises to advance the field of wound healing. PMID:17544023

  17. Brazilian minipig as a large-animal model for basic research and stem cell-based tissue engineering. Characterization and in vitro differentiation of bone marrow-derived mesenchymal stem cells

    PubMed Central

    STRAMANDINOLI-ZANICOTTI, Roberta Targa; CARVALHO, André Lopes; REBELATTO, Carmen Lúcia Kuniyoshi; SASSI, Laurindo Moacir; TORRES, Maria Fernanda; SENEGAGLIA, Alexandra Cristina; BOLDRINILEITE, Lidiane Maria; CORREA-DOMINGUEZ, Alejandro; KULIGOVSKY, Crisciele; BROFMAN, Paulo Roberto Slud

    2014-01-01

    Stem cell-based regenerative medicine is one of the most intensively researched medical issues. Pre-clinical studies in a large-animal model, especially in swine or miniature pigs, are highly relevant to human applications. Mesenchymal stem cells (MSCs) have been isolated and expanded from different sources. Objective This study aimed at isolating and characterizing, for the first time, bone marrow-derived MSCs (BM-MSCs) from a Brazilian minipig (BR1). Also, this aimed to validate a new large-animal model for stem cell-based tissue engineering. Material and Methods Bone marrow (BM) was aspirated from the posterior iliac crest of twelve adult male BR1 under general anesthesia. MSCs were selected by plastic-adherence as originally described by Friedenstein. Cell morphology, surface marker expression, and cellular differentiation were examined. The immunophenotypic profile was determined by flow cytometry. The differentiation potential was assessed by cytological staining and by RT-PCR. Results MSCs were present in all minipig BM samples. These cells showed fibroblastic morphology and were positive for the surface markers CD90 (88.6%), CD29 (89.8%), CD44 (86.9%) and negative for CD34 (1.61%), CD45 (1.83%), CD14 (1.77%) and MHC-II (2.69%). MSCs were differentiated into adipocytes, osteoblasts, and chondroblasts as demonstrated by the presence of lipidic-rich vacuoles, the mineralized extracellular matrix, and the great presence of glycosaminoglycans, respectively. The higher gene expression of adipocyte fatty-acid binding protein (AP2), alkaline phosphatase (ALP) and collagen type 2 (COLII) also confirmed the trilineage differentiation (p<0.001, p<0.001, p=0.031; respectively). Conclusions The isolation, cultivation, and differentiation of BM-MSCs from BR1 makes this animal eligible as a useful large-animal model for stem cell-based studies in Brazil. PMID:25025563

  18. Ubc9 negatively regulates BMP-mediated osteoblastic differentiation in cultured cells.

    PubMed

    Yukita, Akira; Hosoya, Akihiro; Ito, Yuzuru; Katagiri, Takenobu; Asashima, Makoto; Nakamura, Hiroaki

    2012-05-01

    SUMO (small ubiquitin-related modifier) modification (SUMOylation) has been reported to regulate various biological events such as cell-cycle progression, proliferation, and survival. Bone morphogenetic proteins (BMPs) play an important role in osteoblast differentiation and maturation. Although Smad4, which acts as a transcriptional factor in the BMP signaling, is a target of SUMOylation, the involvement of SUMOylation in osteoblast differentiation remains unclear. In this report, we demonstrated spatial expression patterns of SUMO proteins and Ubc9 (ubiquitin conjugating enzyme 9), which is a unique E2-SUMOylation enzyme, in mouse tibia. Furthermore, siRNA knockdown of Ubc9 enhanced osteoblastic differentiation induced by BMP2 in C2C12 mouse myoblasts and ST2 mouse bone-marrow derived stromal cells. Ubc9 knockdown elevated the BMP signaling transduction and reduced the expression of muscle-related genes in cooperation with BMP2. Finally, a luciferase assay using an Id1 (target gene of BMP signaling) reporter revealed that Smad4 mutants prevented from SUMOylation at their Lys158 possessed more potent transcriptional activity than wild-type Smad4. Taken together, these findings suggest that Ubc9 negatively regulates osteoblastic differentiation induced by BMP via, at least in part, SUMOylation of Smad4. PMID:22366399

  19. Inhibition of cyclooxygenase (COX)-2 affects endothelial progenitor cell proliferation

    SciTech Connect

    Colleselli, Daniela; Bijuklic, Klaudija; Mosheimer, Birgit A.; Kaehler, Christian M. . E-mail: C.M.Kaehler@uibk.ac.at

    2006-09-10

    Growing evidence indicates that inducible cyclooxygenase-2 (COX-2) is involved in the pathogenesis of inflammatory disorders and various types of cancer. Endothelial progenitor cells recruited from the bone marrow have been shown to be involved in the formation of new vessels in malignancies and discussed for being a key point in tumour progression and metastasis. However, until now, nothing is known about an interaction between COX and endothelial progenitor cells (EPC). Expression of COX-1 and COX-2 was detected by semiquantitative RT-PCR and Western blot. Proliferation kinetics, cell cycle distribution and rate of apoptosis were analysed by MTT test and FACS analysis. Further analyses revealed an implication of Akt phosphorylation and caspase-3 activation. Both COX-1 and COX-2 expression can be found in bone-marrow-derived endothelial progenitor cells in vitro. COX-2 inhibition leads to a significant reduction in proliferation of endothelial progenitor cells by an increase in apoptosis and cell cycle arrest. COX-2 inhibition leads further to an increased cleavage of caspase-3 protein and inversely to inhibition of Akt activation. Highly proliferating endothelial progenitor cells can be targeted by selective COX-2 inhibition in vitro. These results indicate that upcoming therapy strategies in cancer patients targeting COX-2 may be effective in inhibiting tumour vasculogenesis as well as angiogenic processes.

  20. Prostacyclin Suppresses Twist Expression in the Presence of Indomethacin in Bone Marrow-Derived Mesenchymal Stromal Cells

    PubMed Central

    Kemper, Oliver; Herten, Monika; Fischer, Johannes; Haversath, Marcel; Beck, Sascha; Classen, Tim; Warwas, Sebastian; Tassemeier, Tjark; Landgraeber, Stefan; Lensing-Höhn, Sabine; Krauspe, Rüdiger; Jäger, Marcus

    2014-01-01

    Background Iloprost, a stable prostacyclin I2 analogue, seems to have an osteoblast-protective potential, whereas indomethacin suppresses new bone formation. The aim of this study was to investigate human bone marrow stromal cell (BMSC) proliferation and differentiation towards the osteoblastic lineage by administration of indomethacin and/or iloprost. Material/Methods Human bone marrow cells were obtained from 3 different donors (A=26 yrs/m; B=25 yrs/f, C=35 yrs/m) via vacuum aspiration of the iliac crest followed by density gradient centrifugation and flow cytometry with defined antigens (CD105+/73+/45−/14−). The cells were seeded and incubated as follows: without additives (Group 0; donor A/B/C), with 10−7 M iloprost only (Group 0+ilo; A/B), with indomethacin only in concentrations of 10−6 M (Group 1, A), 10−5 M (Group 2, B), 10−4 M (Group 3, A/B), and together with 10−7 M iloprost (Groups 4–6, A/B/C). On Day 10 and 28, UV/Vis spectrometric and immunocytochemical assays (4 samples per group and donor) were performed to investigate cell proliferation (cell count measurement) and differentiation towards the osteoblastic lineage (CD34−, CD45−, CD105+, type 1 collagen (Col1), osteocalcin (OC), alkaline phosphatase (ALP), Runx2, Twist, specific ALP-activity). Results Indomethacin alone suppressed BMSC differentiation towards the osteoblastic lineage by downregulation of Runx2, Col1, and ALP. In combination with indomethacin, iloprost increased cell proliferation and differentiation and it completely suppressed Twist expression at Day 10 and 28. Iloprost alone did not promote cell proliferation, but moderately enhanced Runx2 and Twist expression. However, the proliferative effects and the specific ALP-activity varied donor-dependently. Conclusions Iloprost partially antagonized the suppressing effects of indomethacin on BMSC differentiation towards the osteoblast lineage. It enhanced the expression of Runx2 and, only in the presence of indomethacin

  1. Bone marrow-derived CD11b+Jagged2+ cells promote epithelial-to-mesenchymal transition and metastasization in colorectal cancer.

    PubMed

    Caiado, Francisco; Carvalho, Tânia; Rosa, Isadora; Remédio, Leonor; Costa, Ana; Matos, João; Heissig, Beate; Yagita, Hideo; Hattori, Koichi; da Silva, João Pereira; Fidalgo, Paulo; Pereira, António Dias; Dias, Sérgio

    2013-07-15

    Timely detection of colorectal cancer metastases may permit improvements in their clinical management. Here, we investigated a putative role for bone marrow-derived cells in the induction of epithelial-to-mesenchymal transition (EMT) as a marker for onset of metastasis. In ectopic and orthotopic mouse models of colorectal cancer, bone marrow-derived CD11b(Itgam)(+)Jagged2 (Jag2)(+) cells infiltrated primary tumors and surrounded tumor cells that exhibited diminished expression of E-cadherin and increased expression of vimentin, 2 hallmarks of EMT. In vitro coculture experiments showed that the bone marrow-derived CD11b(+)Jag2(+) cells induced EMT through a Notch-dependent pathway. Using neutralizing antibodies, we imposed a blockade on CD11b(+) cells' recruitment to tumors, which decreased the tumor-infiltrating CD11b(+)Jag2(+) cell population of interest, decreasing tumor growth, restoring E-cadherin expression, and delaying EMT. In support of these results, we found that peripheral blood levels of CD11b(+)Jag2(+) cells in mouse models of colorectal cancer and in a cohort of untreated patients with colorectal cancer were indicative of metastatic disease. In patients with colorectal cancer, the presence of circulating CD11b(+)Jag2(+) cells was accompanied by loss of E-cadherin in the corresponding patient tumors. Taken together, our results show that bone marrow-derived CD11b(+)Jag2(+) cells, which infiltrate primary colorectal tumors, are sufficient to induce EMT in tumor cells, thereby triggering onset of metastasis. Furthermore, they argue that quantifying circulating CD11b(+)Jag2(+) cells in patients may offer an indicator of colorectal cancer progression to metastatic levels of the disease. PMID:23722542

  2. Increased recruitment of bone marrow-derived cells into the brain associated with altered brain cytokine profile in senescence-accelerated mice.

    PubMed

    Hasegawa-Ishii, Sanae; Inaba, Muneo; Li, Ming; Shi, Ming; Umegaki, Hiroyuki; Ikehara, Susumu; Shimada, Atsuyoshi

    2016-04-01

    Bone marrow-derived cells enter the brain in a non-inflammatory condition through the attachments of choroid plexus and differentiate into ramified myeloid cells. Neurodegenerative conditions may be associated with altered immune-brain interaction. The senescence-accelerated mouse prone 10 (SAMP10) undergoes earlier onset neurodegeneration than C57BL/6 (B6) strain. We hypothesized that the dynamics of immune cells migrating from the bone marrow to the brain is perturbed in SAMP10 mice. We created 4 groups of radiation chimeras by intra-bone marrow-bone marrow transplantation using 2-month-old (2 mo) and 10 mo SAMP10 and B6 mice as recipients with GFP transgenic B6 mice as donors, and analyzed histologically 4 months later. In the [B6 → 10 mo SAMP10] chimeras, more ramified marrow-derived cells populated a larger number of discrete brain regions than the other chimeras, especially in the diencephalon. Multiplex cytokine assays of the diencephalon prepared from non-treated 3 mo and 12 mo SAMP10 and B6 mice revealed that 12 mo SAMP10 mice exhibited higher tissue concentrations of CXCL1, CCL11, G-CSF, CXCL10 and IL-6 than the other groups. Immunohistologically, choroid plexus epithelium and ependyma produced CXCL1, while astrocytic processes in the attachments of choroid plexus expressed CCL11 and G-CSF. The median eminence produced CXCL10, hypothalamic neurons G-CSF and tanycytes CCL11 and G-CSF. These brain cytokine profile changes in 12 mo SAMP10 mice were likely to contribute to acceleration of the dynamics of marrow-derived cells to the diencephalon. Further studies on the functions of ramified marrow-derived myeloid cells would enhance our understanding of the brain-bone marrow interaction. PMID:25577138

  3. Fibroblast Growth Factor 2 Regulates High Mobility Group A2 Expression in Human Bone Marrow-Derived Mesenchymal Stem Cells.

    PubMed

    Kalomoiris, Stefanos; Cicchetto, Andrew C; Lakatos, Kinga; Nolta, Jan A; Fierro, Fernando A

    2016-09-01

    Mesenchymal stem cells (MSCs) are an excellent source for numerous cellular therapies due to their simple isolation, low immunogenicity, multipotent differentiation potential and regenerative secretion profile. However, over-expanded MSCs show decreased therapeutic efficacy. This shortcoming may be circumvented by identifying methods that promote self-renewal of MSCs in culture. HMGA2 is a DNA-binding protein that regulates self-renewal in multiple types of stem cells through chromatin remodeling, but its impact on human bone marrow-derived MSCs is not known. Using an isolation method to obtain pure MSCs within 9 days in culture, we show that expression of HMGA2 quickly decreases during early expansion of MSCs, while let-7 microRNAs (which repress HMGA2) are simultaneously increased. Remarkably, we demonstrate that FGF-2, a growth factor commonly used to promote self-renewal in MSCs, rapidly induces HMGA2 expression in a time- and concentration-dependent manner. The signaling pathway involves FGF-2 receptor 1 (FGFR1) and ERK1/2, but acts independent from let-7. By silencing HMGA2 using shRNAs, we demonstrate that HMGA2 is necessary for MSC proliferation. However, we also show that over-expression of HMGA2 does not increase cell proliferation, but rather abrogates the mitogenic effect of FGF-2, possibly through inhibition of FGFR1. In addition, using different methods to assess in vitro differentiation, we show that modulation of HMGA2 inhibits adipogenesis, but does not affect osteogenesis of MSCs. Altogether, our results show that HMGA2 expression is associated with highly proliferating MSCs, is tightly regulated by FGF-2, and is involved in both proliferation and adipogenesis of MSCs. J. Cell. Biochem. 117: 2128-2137, 2016. © 2016 Wiley Periodicals, Inc. PMID:26888666

  4. Population doubling level-dependent change of secreted glycosaminoglycan in equine bone marrow-derived mesenchymal stem cells

    PubMed Central

    SASAO, Takafumi; FUKUDA, Yuki; YOSHIDA, Sayako; MIYABARA, Shihori; KASASHIMA, Yoshinori; KUWANO, Atsutoshi; ARAI, Katsuhiko

    2015-01-01

    ABSTRACT In regenerative medicine using transplantation of mesenchymal stem cells (MSCs), the importance of regulating the quality of MSCs has been well recognized; however, there is little information concerning the relationship between the population doubling level (PDL) and the stemness of MSCs in equine medicine. In this study, we showed that the amount of glycosaminoglycan (GAG) secreted by bone marrow-derived MSCs (BMSCs) decreases with increase of PDL. Enzymatic digestion and two-dimensional electrophoresis revealed that a main component of GAG produced by BMSCs was hyaluronan with a small amount of chondroitin sulfate. Increase of PDL downregulated the expression of MSC CD markers, including CD44, CD73, CD90, CD105, and CD146, along with loss of differentiation capacity. Thus, the effect of hyaluronan supplement to the growth medium on both expression of CD markers and the tri-lineage potential of BMSCs was evaluated. Expression of CD73 and CD90 was preserved by continuous addition of hyaluronan to the growth medium, whereas mRNA levels corresponding to CD44, CD105 and CD146 were not preserved by supplementation of hyaluronan. BMSCs subcultured with hyaluronan-supplemented growth medium to PDL-12 showed osteogenic capacity, however adipogenic and chondrogenic activities at PDL-12 were not preserved by exogenous hyaluronan. These results suggest that downregulation of CD44, CD105 and CD146 might not affect the osteogenic capacity. Taken together, the results suggested that supplementation of hyaluronan to the growth medium might be effective at maintaining the osteogenic capacity of equine BMSCs. PMID:26435680

  5. Intervertebral disc regeneration using platelet-rich plasma-containing bone marrow-derived mesenchymal stem cells: A preliminary investigation

    PubMed Central

    WANG, SHAN-ZHENG; JIN, JI-YANG; GUO, YU-DONG; MA, LIANG-YU; CHANG, QING; PENG, XIN-GUI; GUO, FANG-FANG; ZHANG, HAI-XIANG; HU, XIN-FENG; WANG, CHEN

    2016-01-01

    Platelet-rich plasma (PRP) is a promising strategy for intervertebral disc degeneration (IDD). However, the short half-life of growth factors released from PRP cannot continuously stimulate the degenerated discs. Thus, the present study hypothesized that the combined use of PRP and bone marrow-derived mesenchymal stem cells (BMSCs) may repair the early degenerated discs in the long term for their synergistic reparative effect. In the present study, following the induction of early IDD by annular puncture in rabbits, PRP was prepared and mixed with BMSCs (PRP-BMSC group) for injection into the early degenerated discs. As controls, phosphate-buffered saline (PBS; PBS group) and PRP (PRP group) were similarly injected. Rabbits without any intervention served as a control group. At 8 weeks following treatment, histological changes of the injected discs were assessed. Magnetic resonance imaging (MRI) was used to detect the T2-weighted signal intensity of the targeted discs at weeks 1, 2 and 8 following treatment. Annular puncture resulted in disc narrowing and decreased T2-weighted signal intensity. At weeks 1 and 3, MRI examinations showed regenerative changes in the PRP-BMSC group and PRP group, whereas the PBS group exhibited a continuous degenerative process of the discs. At 8 weeks post-injection, the PRP-BMSCs induced a statistically significant restoration of discs, as shown by MRI (PRP-BMSCs, vs.PRP and PBS; P<0.05), which was also confirmed by histological evaluations. Thus, compared with PRP, the administration of PRP-containing BMSCs resulted in a superior regenerative effect on the early degenerated discs, which may be a promising therapeutic strategy for the restoration of early degenerated discs. PMID:26956080

  6. [Differentiation of rat bone marrow-derived mesenchymal stem cells into cardiomyocyte-like cells induced by cyclic stretching strain].

    PubMed

    Kuang, Wei; Tang, Min; He, Xueling; Wu, Wenchao; Liu, Xiaojing; Li, Liang

    2014-06-01

    Bone marrow-derived mesenchymal stem cells (BMSCs) are multipotent stem cells that differentiate into a variety of cell types and widely used in tissue regeneration engineering. The purpose of this study is to investigate whether the cyclic biaxial stretching strain could promote the rat BMSCs (rBMSCs) to differentiate into cardiomyocyte-like cells in vitro. The second or third generation of rBMSCs were randomly divided into the cyclic stretching stain group, the control group and the blank group. Those rBMSCs in the cyclic stretching strain group were seeded on a silicone membrane with complete medium were exposed to biaxial stretching strain of 10% of membrane at a frequency of 1 Hz lasting for 6 h, 12 h and 24 h. Those in the control group were seeded on silicone membrane with complete medium. Those in the blank group were seeded in the 6-wells plates with complete medium. The mRNA expression of GATA4 and myocyte-specific enhancer factor 2C (MEF-2C) were detected by the real-time fluorescent quantification PCR and the protein expression of connexin 43 (Cx43) was detected by using the Western blot method. The results showed that the mRNA expression level of the GATA4 and MEF-2C, and the protein expression level of Cx43 were significantly higher in the cyclic stretching strain groups, compared with those in the relative control groups (P < 0.05). It suggests that cyclic biaxial stretching strain could play a part in the induction of rBMSCs to differentiate into cardiomyocyte-like cells in vitro, but the differentiation mechanism is still unclear. PMID:25219242

  7. Role of stress-inducible protein-1 in recruitment of bone marrow derived cells into the ischemic brains

    PubMed Central

    Lee, Shin-Da; Lai, Ted Weita; Lin, Shinn-Zong; Lin, Chen-Huan; Hsu, Yung-Hsiang; Li, Chi-Yuan; Wang, Hsiao-Jung; Lee, Wei; Su, Ching-Yuan; Yu, Yung-Luen; Shyu, Woei-Cherng

    2013-01-01

    Stress-inducible protein-1 (STI-1) is the proposed ligand for the cellular prion protein (PrPC), which is thought to facilitate recovery following stroke. Whether STI-1 expression is affected by stroke and how its signalling facilitates recovery remain elusive. Brain slices from patients that died of ischemic stroke were collected for STI-1 immunohistochemistry. These findings were compared to results from cell cultures, mice with or without the PrPC knockout, and rats. Based on these findings, molecular and pharmacological interventions were administered to investigate the underlying mechanisms and to test the possibility for therapy in experimental stroke models. STI-1 was upregulated in the ischemic brains from humans and rodents. The increase in STI-1 expression in vivo was not cell-type specific, as it was found in neurons, glia and endothelial cells. Likewise, this increase in STI-1 expression can be mimicked by sublethal hypoxia in primary cortical cultures (PCCs) in vitro, and appear to have resulted from the direct binding of the hypoxia inducible factor-1α (HIF-1α) to the STI-1 promoter. Importantly, this STI-1 signalling promoted bone marrow derived cells (BMDCs) proliferation and migration in vitro and recruitment to the ischemic brain in vivo, and augmenting its signalling facilitated neurological recovery in part by recruiting BMDCs to the ischemic brain. Our results thus identified a novel mechanism by which ischemic insults can trigger a self-protective mechanism to facilitate recovery. This work identifies HIF-1α-mediated transcription of STI-1 and PrPc interaction as leading to BMDCs recruitment into ischemic brains following stroke in both patients and animal models of stroke, highlighting novel neuroprotective possibilities. PMID:23836498

  8. Promoting effect of small molecules in cardiomyogenic and neurogenic differentiation of rat bone marrow-derived mesenchymal stem cells

    PubMed Central

    Khanabdali, Ramin; Saadat, Anbarieh; Fazilah, Maizatul; Bazli, Khairul Fidaa’ Khairul; Qazi, Rida-e-Maria; Khalid, Ramla Sana; Hasan Adli, Durriyyah Sharifah; Moghadamtousi, Soheil Zorofchian; Naeem, Nadia; Khan, Irfan; Salim, Asmat; Shamsuddin, ShamsulAzlin Ahmad; Mohan, Gokula

    2016-01-01

    Small molecules, growth factors, and cytokines have been used to induce differentiation of stem cells into different lineages. Similarly, demethylating agents can trigger differentiation in adult stem cells. Here, we investigated the in vitro differentiation of rat bone marrow mesenchymal stem cells (MSCs) into cardiomyocytes by a demethylating agent, zebularine, as well as neuronal-like cells by β-mercaptoethanol in a growth factor or cytokines-free media. Isolated bone marrow-derived MSCs cultured in Dulbecco’s Modified Eagle’s Medium exhibited a fibroblast-like morphology. These cells expressed positive markers for CD29, CD44, and CD117 and were negative for CD34 and CD45. After treatment with 1 μM zebularine for 24 hours, the MSCs formed myotube-like structures after 10 days in culture. Expression of cardiac-specific genes showed that treated MSCs expressed significantly higher levels of cardiac troponin-T, Nkx2.5, and GATA-4 compared with untreated cells. Immunocytochemical analysis showed that differentiated cells also expressed cardiac proteins, GATA-4, Nkx 2.5, and cardiac troponin-T. For neuronal differentiation, MSCs were treated with 1 and 10 mM β-mercaptoethanol overnight for 3 hours in complete and serum-free Dulbecco’s Modified Eagle’s Medium, respectively. Following overnight treatment, neuron-like cells with axonal and dendritic-like projections originating from the cell body toward the neighboring cells were observed in the culture. The mRNA expression of neuronal-specific markers, Map2, Nefl, Tau, and Nestin, was significantly higher, indicating that the treated cells differentiated into neuronal-like cells. Immunostaining showed that differentiated cells were positive for the neuronal markers Flk, Nef, Nestin, and β-tubulin. PMID:26766903

  9. High Physiological Concentrations of Progesterone Reverse Estradiol-Mediated Changes in Differentiation and Functions of Bone Marrow Derived Dendritic Cells.

    PubMed

    Xiu, Fangming; Anipindi, Varun C; Nguyen, Philip V; Boudreau, Jeanette; Liang, Hong; Wan, Yonghong; Snider, Denis P; Kaushic, Charu

    2016-01-01

    Female sex steroids, estradiol (E2) and progesterone (P4), play a key role in regulating immune responses in women, including dendritic cell (DC) development, and functions. Although the two hormones co-occur in the body of women throughout the reproductive years, no studies have explored their complex combinatorial effects on DCs, given their ability to regulate each other's actions. We examined murine bone marrow derived dendritic cells (BMDC) differentiation and functions, in the presence of a wide range of physiological concentrations of each hormone, as well as the combination of the two hormones. E2 (10-12 to 10-8M) enhanced the differentiation of CD11b+CD11c+ DCs from BM precursor cells, and promoted the expression of CD40 and MHC Class-II, in a dose-dependent manner. In contrast, P4 (10-9 to 10-5M) inhibited DC differentiation, but only at the highest concentrations. These effects on BMDCs were observed both in the presence or absence of LPS. When both hormones were combined, higher concentrations of P4, at levels seen in pregnancy (10-6M) reversed the E2 effects, regardless of the concentration of E2, especially in the absence of LPS. Functionally, antigen uptake was decreased and pro-inflammatory cytokines, IL-12, IL-1 and IL-6 production by CD11b+CD11c+ DCs, was increased in the presence of E2 and these effects were reversed by high concentrations of P4. Our results demonstrate the distinct effects of E2 and P4 on differentiation and functions of bone marrow myeloid DCs. The dominating effect of higher physiological concentrations of P4 provides insight into how DC functions could be modulated during pregnancy. PMID:27064901

  10. Isolation of Mature (Peritoneum-Derived) Mast Cells and Immature (Bone Marrow-Derived) Mast Cell Precursors from Mice

    PubMed Central

    Meurer, Steffen K.; Neß, Melanie; Weiskirchen, Sabine; Kim, Philipp; Tag, Carmen G.; Kauffmann, Marlies; Huber, Michael; Weiskirchen, Ralf

    2016-01-01

    Mast cells (MCs) are a versatile cell type playing key roles in tissue morphogenesis and host defence against bacteria and parasites. Furthermore, they can enhance immunological danger signals and are implicated in inflammatory disorders like fibrosis. This granulated cell type originates from the myeloid lineage and has similarities to basophilic granulocytes, both containing large quantities of histamine and heparin. Immature murine mast cells mature in their destination tissue and adopt either the connective tissue (CTMC) or mucosal (MMC) type. Some effector functions are executed by activation/degranulation of MCs which lead to secretion of a typical set of MC proteases (MCPT) and of the preformed or newly synthesized mediators from its granules into the local microenvironment. Due to the potential accumulation of mutations in key signalling pathway components of corresponding MC cell-lines, primary cultured MCs are an attractive mean to study general features of MC biology and aspects of MC functions relevant to human disease. Here, we describe a simple protocol for the simultaneous isolation of mature CTMC-like murine MCs from the peritoneum (PMCs) and immature MC precursors from the bone marrow (BM). The latter are differentiated in vitro to yield BM-derived MCs (BMMC). These cells display the typical morphological and phenotypic features of MCs, express the typical MC surface markers, and can be propagated and kept in culture for several weeks. The provided protocol allows simple amplification of large quantities of homogenous, non-transformed MCs from the peritoneum and bone marrow-derived mast cells for cell- and tissue-based biomedical research. PMID:27337047