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Sample records for bone-marrow derived pro-angiogenic

  1. Hedgehog promotes neovascularization in pancreatic cancers by regulating Ang-1 and IGF-1 expression in bone-marrow derived pro-angiogenic cells.

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    Kazumasa Nakamura

    Full Text Available BACKGROUND: The hedgehog (Hh pathway has been implicated in the pathogenesis of cancer including pancreatic ductal adenocarcinoma (PDAC. Recent studies have suggested that the oncogenic function of Hh in PDAC involves signaling in the stromal cells rather than cell autonomous effects on the tumor cells. However, the origin and nature of the stromal cell type(s that are responsive to Hh signaling remained unknown. Since Hh signaling plays a crucial role during embryonic and postnatal vasculogenesis, we speculated that Hh ligand may act on tumor vasculature specifically focusing on bone marrow (BM-derived cells. METHODOLOGY/PRINCIPAL FINDINGS: Cyclopamine was utilized to inhibit the Hh pathway in human PDAC cell lines and their xenografts. BM transplants, co-culture systems of tumor cells and BM-derived pro-angiogenic cells (BMPCs were employed to assess the role of tumor-derived Hh in regulating the BM compartment and the contribution of BM-derived cells to angiogenesis in PDAC. Cyclopamine administration attenuated Hh signaling in the stroma rather than in the cancer cells as reflected by decreased expression of full length Gli2 protein and Gli1 mRNA specifically in the compartment. Cyclopamine inhibited the growth of PDAC xenografts in association with regression of the tumor vasculature and reduced homing of BM-derived cells to the tumor. Host-derived Ang-1 and IGF-1 mRNA levels were downregulated by cyclopamine in the tumor xenografts. In vitro co-culture and matrigel plug assays demonstrated that PDAC cell-derived Shh induced Ang-1 and IGF-1 production in BMPCs, resulting in their enhanced migration and capillary morphogenesis activity. CONCLUSIONS/SIGNIFICANCE: We identified the BMPCs as alternative stromal targets of Hh-ligand in PDAC suggesting that the tumor vasculature is an attractive therapeutic target of Hh blockade. Our data is consistent with the emerging concept that BM-derived cells make important contributions to epithelial

  2. Pro-Angiogenic Effects of Chalcone Derivatives in Zebrafish Embryos in Vivo.

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    Chen, Yau-Hung; Chang, Chao-Yuan; Chang, Chiung-Fang; Chen, Po-Chih; Lee, Ya-Ting; Chern, Ching-Yuh; Tsai, Jen-Ning

    2015-07-09

    The aim of this study was to investigate novel chalcones with potent angiogenic activities in vivo. Chalcone-based derivatives were evaluated using a transgenic zebrafish line with fluorescent vessels to real-time monitor the effect on angiogenesis. Results showed that the chalcone analogues did not possess anti-angiogenic effect on zebrafish vasculatures; instead, some of them displayed potent pro-angiogenic effects on the formation of the sub-intestinal vein. Similar pro-angiogenic effects can also be seen on wild type zebrafish embryos. Moreover, the expression of vegfa, the major regulator for angiogenesis, was also upregulated in their treatment. Taken together, we have synthesized and identified a series of novel chalcone-based derivatives as potent in vivo pro-angiogenic compounds. These novel compounds hold potential for therapeutic angiogenesis.

  3. Bone Marrow-Derived Macrophages (BMM)

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    Weischenfeldt, Joachim; Porse, Bo

    2008-01-01

    of committed myeloid progenitors into cells of the macrophage/monocyte lineage. Mice lacking functional M-CSF are deficient in macrophages and osteoclasts and suffer from osteopetrosis. In this protocol, bone marrow cells are grown in culture dishes in the presence of M-CSF, which is secreted by L929 cells...

  4. Adiponectinemia controls pro-angiogenic cell therapy.

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    Eren, Philippe; Camus, Stéphane; Matrone, Gianfranco; Ebrahimian, Téni G; François, Delphine; Tedgui, Alain; Sébastien Silvestre, Jean; Blanc-Brude, Olivier P

    2009-11-01

    Angiogenic cell therapy with the transplantation of endothelial progenitor cells (EPC) or bone marrow mononuclear cells (BM-MNC) receives considerable attention as an approach to revascularize ischemic tissues. Adiponectin is a circulating hormone produced by the apM1 gene in adipocytes. Adiponectin modulates lipid metabolism and obesity, and it was recently found to promote physiological angiogenesis in response to ischemia. Patients with multiple cardiovascular disease risk factors or myocardial infarction may benefit from progenitor cell therapy, but they display depressed adiponectinemia. We hypothesized that adiponectin stimulation of transplanted cells is critical for their pro-angiogenic function. We aimed to establish whether adiponectinemia in the cell donor or in the cell recipient determines the success of pro-angiogenic cell therapy. In vitro, we found that conditioned media derived from wild-type adipocytes (adipo-CM) or purified adiponectin strongly enhanced BM-MNC survival and proliferation and stimulated EPC differentiation, whereas adipo-CM from apM1-/- adipocytes was one-half less effective. On the other hand, wild-type and apM1-/- BM-MNC displayed similar resistance to apoptosis and proliferation rates. In vivo, wild-type, and apM1-/- BM-MNC induced similar angiogenic reactions in wild-type ischemic hindlimbs. In contrast, wild-type BM-MNC had much diminished effects in apM1-/- ischemic hindlimbs. We concluded that adiponectin enhances BM-MNC survival and proliferation, and adiponectinemia in the cell therapy recipient is essential for the pro-angiogenic benefits of cell therapy. These observations imply that progenitor cell transplantation might only induce angiogenesis in patients with high adiponectinemia.

  5. A method for generation of bone marrow-derived macrophages from cryopreserved mouse bone marrow cells.

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    Fernanda M Marim

    Full Text Available 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.

  6. Bone marrow-derived dendritic cells.

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    Roney, Kelly

    2013-01-01

    While much is understood about dendritic cells and their role in the immune system, the study of these cells is critical to gain a more complete understanding of their function. Dendritic cell isolation from mouse body tissues can be difficult and the number of cells isolated small. This protocol describes the growth of large number of dendritic cells from the culture of mouse bone marrow cells. The dendritic cells grown in culture facilitate experiments that may require large number of dendritic cells without great expense or use of large number of mice.

  7. Prostaglandin E2 promotes endothelial differentiation from bone marrow-derived cells through AMPK activation.

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    Zhenjiu Zhu

    Full Text Available Prostaglandin E2 (PGE2 has been reported to modulate angiogenesis, the process of new blood vessel formation, by promoting proliferation, migration and tube formation of endothelial cells. Endothelial progenitor cells are known as a subset of circulating bone marrow mononuclear cells that have the capacity to differentiate into endothelial cells. However, the mechanism underlying the stimulatory effects of PGE2 and its specific receptors on bone marrow-derived cells (BMCs in angiogenesis has not been fully characterized. Treatment with PGE2 significantly increased the differentiation and migration of BMCs. Also, the markers of differentiation to endothelial cells, CD31 and von Willebrand factor, and the genes associated with migration, matrix metalloproteinases 2 and 9, were significantly upregulated. This upregulation was abolished by dominant-negative AMP-activated protein kinase (AMPK and AMPK inhibitor but not protein kinase, a inhibitor. As a functional consequence of differentiation and migration, the tube formation of BMCs was reinforced. Along with altered BMCs functions, phosphorylation and activation of AMPK and endothelial nitric oxide synthase, the target of activated AMPK, were both increased which could be blocked by EP4 blocking peptide and simulated by the agonist of EP4 but not EP1, EP2 or EP3. The pro-angiogenic role of PGE2 could be repressed by EP4 blocking peptide and retarded in EP4(+/- mice. Therefore, by promoting the differentiation and migration of BMCs, PGE2 reinforced their neovascularization by binding to the receptor of EP4 in an AMPK-dependent manner. PGE2 may have clinical value in ischemic heart disease.

  8. Recruitment of bone marrow derived cells during anti-angiogenic therapy in GBM : Bone marrow derived cell in GBM

    NARCIS (Netherlands)

    Boer, Jennifer C.; Walenkamp, Annemiek M. E.; den Dunnen, Wilfred F. A.

    2014-01-01

    Glioblastoma (GBM) is a highly vascular tumor characterized by rapid and invasive tumor growth, followed by oxygen depletion, hypoxia and neovascularization, which generate a network of disorganized, tortuous and permeable vessels. Recruitment of bone marrow derived cells (BMDC) is crucial for vascu

  9. Bone marrow-derived cells are present in Mooren's ulcer.

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    Ye, Juan; Chen, Jian; Kim, Jae Chan; Yao, Ke

    2004-01-01

    To investigate whether bone marrow-derived cells are present in Mooren's ulcer and involved in its destructive and regenerative disease course, tissue specimens were collected from 3 eyes of 3 patients with Mooren's ulcer that underwent lamellar keratectomy. Three normal donor limbal corneoscleras served as controls. Immunohistochemical staining patterns were analyzed by using the following antibodies: CD34 (a marker of hematopoietic progenitor cells and endothelium), c-kit (a marker of hematopoietic and stromal progenitor cells) and STRO-1 (a differentiation antigen present on bone marrow fibroblast cells and on various nonhematopoietic progenitor cells). Strong positive CD34, c-kit and STRO-1 cells were revealed in Mooren's ulcer specimens, especially in the superficial stroma. A few weakly expressed CD34 stromal cells were seen in normal limbal cornea, but no immunoreactivity for c-kit and STRO-1 was found. Bone marrow-derived cells are present in Mooren's ulcer and contribute to its destructive and regeneration process by synergizing with other factors. Specific therapeutic strategies that target the role of these cells in Mooren's ulcer are anticipated.

  10. Contribution of bone marrow derived cells to pancreatic carcinogenesis

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    Christopher J Scarlett

    2013-03-01

    Full Text Available Pancreatic cancer is a complex, aggressive and heterogeneous malignancy driven by the multifaceted interactions within the tumor microenvironment. While it is known that the tumor microenvironment accommodates many cell types, each playing a key role in tumorigenesis, the major source of these stromal cells is not well understood. This review examines the contribution of bone marrow-derived cells (BMDC to pancreatic carcinogenesis, with respect to their role in constituting the tumor microenvironment. In particular, their role in supporting fibrosis, immunosuppression and neovascularisation will be discussed.

  11. Spine fusion using cell matrix composites enriched in bone marrow-derived cells.

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

    2003-02-01

    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.

  12. Bone-Marrow-Derived Mesenchymal Stem Cells for Organ Repair

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    Ming Li

    2013-01-01

    Full Text Available Mesenchymal stem cells (MSCs are prototypical adult stem cells with the capacity for self-renewal and differentiation with a broad tissue distribution. MSCs not only differentiate into types of cells of mesodermal lineage but also into endodermal and ectodermal lineages such as bone, fat, cartilage and cardiomyocytes, endothelial cells, lung epithelial cells, hepatocytes, neurons, and pancreatic islets. MSCs have been identified as an adherent, fibroblast-like population and can be isolated from different adult tissues, including bone marrow (BM, umbilical cord, skeletal muscle, and adipose tissue. MSCs secrete factors, including IL-6, M-CSF, IL-10, HGF, and PGE2, that promote tissue repair, stimulate proliferation and differentiation of endogenous tissue progenitors, and decrease inflammatory and immune reactions. In this paper, we focus on the role of BM-derived MSCs in organ repair.

  13. The role of bone marrow-derived cells during the bone healing process in the GFP mouse bone marrow transplantation model.

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    Tsujigiwa, Hidetsugu; Hirata, Yasuhisa; Katase, Naoki; Buery, Rosario Rivera; Tamamura, Ryo; Ito, Satoshi; Takagi, Shin; Iida, Seiji; Nagatsuka, Hitoshi

    2013-03-01

    Bone healing is a complex and multistep process in which the origin of the cells participating in bone repair is still unknown. The involvement of bone marrow-derived cells in tissue repair has been the subject of recent studies. In the present study, bone marrow-derived cells in bone healing were traced using the GFP bone marrow transplantation model. Bone marrow cells from C57BL/6-Tg (CAG-EGFP) were transplanted into C57BL/6 J wild mice. After transplantation, bone injury was created using a 1.0-mm drill. Bone healing was histologically assessed at 3, 7, 14, and 28 postoperative days. Immunohistochemistry for GFP; double-fluorescent immunohistochemistry for GFP-F4/80, GFP-CD34, and GFP-osteocalcin; and double-staining for GFP and tartrate-resistant acid phosphatase were performed. Bone marrow transplantation successfully replaced the hematopoietic cells into GFP-positive donor cells. Immunohistochemical analyses revealed that osteoblasts or osteocytes in the repair stage were GFP-negative, whereas osteoclasts in the repair and remodeling stages and hematopoietic cells were GFP-positive. The results indicated that bone marrow-derived cells might not differentiate into osteoblasts. The role of bone marrow-derived cells might be limited to adjustment of the microenvironment by differentiating into inflammatory cells, osteoclasts, or endothelial cells in immature blood vessels.

  14. CCR2 regulates the uptake of bone marrow-derived fibroblasts in renal fibrosis.

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    Yunfeng Xia

    Full Text Available Recent studies have shown that bone marrow-derived fibroblasts contribute significantly to the pathogenesis of renal fibrosis. However, the molecular mechanisms underlying the recruitment of bone marrow-derived fibroblasts into the kidney are incompletely understood. Bone marrow-derived fibroblasts express the chemokine receptor--CCR2. In this study, we tested the hypothesis that CCR2 participates in the recruitment of fibroblasts into the kidney during the development of renal fibrosis. Bone marrow-derived collagen-expressing GFP⁺ fibroblasts were detected in the obstructed kidneys of chimeric mice transplanted with donor bone marrow from collagen α1(I-GFP reporter mice. These bone marrow-derived fibroblasts expressed PDGFR-β and CCR2. CCR2 knockout mice accumulated significantly fewer bone marrow-derived fibroblast precursors expressing the hematopoietic marker-CD45 and the mesenchymal markers-PDGFR-β or procollagen I in the obstructed kidneys compared with wild-type mice. Furthermore, CCR2 knockout mice displayed fewer bone marrow-derived myofibroblasts and expressed less α-SMA or FSP-1 in the obstructed kidneys compared with wild-type mice. Consistent with these findings, genetic deletion of CCR2 inhibited total collagen deposition and suppressed expression of collagen I and fibronectin. Moreover, genetic deletion of CCR2 inhibits MCP-1 and CXCL16 gene expression associated with a reduction of inflammatory cytokine expression and macrophage infiltration, suggesting a linear interaction between two chemokines/ligand receptors in tubular epithelial cells. Taken together, our results demonstrate that CCR2 signaling plays an important role in the pathogenesis of renal fibrosis through regulation of bone marrow-derived fibroblasts. These data suggest that inhibition of CCR2 signaling could constitute a novel therapeutic approach for fibrotic kidney disease.

  15. Tumor-derived microparticles induce bone marrow-derived cell mobilization and tumor homing: a process regulated by osteopontin.

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    Fremder, Ella; Munster, Michal; Aharon, Anat; Miller, Valeria; Gingis-Velitski, Svetlana; Voloshin, Tali; Alishekevitz, Dror; Bril, Rotem; Scherer, Stefan J; Loven, David; Brenner, Benjamin; Shaked, Yuval

    2014-07-15

    Acute chemotherapy can induce rapid bone-marrow derived pro-angiogenic cell (BMDC) mobilization and tumor homing, contributing to tumor regrowth. To study the contribution of tumor cells to tumor regrowth following therapy, we focused on tumor-derived microparticles (TMPs). EMT/6 murine-mammary carcinoma cells exposed to paclitaxel chemotherapy exhibited an increased number of TMPs and significantly altered their angiogenic properties. Similarly, breast cancer patients had increased levels of plasma MUC-1(+) TMPs following chemotherapy. In addition, TMPs from cells exposed to paclitaxel induced higher BMDC mobilization and colonization, but had no increased effect on angiogenesis in Matrigel plugs and tumors than TMPs from untreated cells. Since TMPs abundantly express osteopontin, a protein known to participate in BMDC trafficking, the impact of osteopontin-depleted TMPs on BMDC mobilization, colonization, and tumor angiogenesis was examined. Although EMT/6 tumors grown in mice inoculated with osteopontin-depleted TMPs had lower numbers of BMDC infiltration and microvessel density when compared with EMT/6 tumors grown in mice inoculated with wild-type TMPs, no significant difference in tumor growth was seen between the two groups. However, when BMDCs from paclitaxel-treated mice were injected into wild-type EMT/6-bearing mice, a substantial increase in tumor growth and BMDC infiltration was detected compared to osteopontin-depleted EMT/6-bearing mice injected with BMDCs from paclitaxel-treated mice. Collectively, our results suggest that osteopontin expressed by TMPs play an important role in BMDC mobilization and colonization of tumors, but is not sufficient to enhance the angiogenic activity in tumors.

  16. Bone marrow-derived versus parenchymal sources of inducible nitric oxide synthase in experimental autoimmune encephalomyelitis

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    Zehntner, Simone P; Bourbonniere, Lyne; Hassan-Zahraee, Mina;

    2004-01-01

    . These discrepancies may reflect balance between immunoregulatory and neurocytopathologic roles for NO. We investigated selective effects of bone marrow-derived versus CNS parenchymal sources of iNOS in EAE in chimeric mice. Chimeras that selectively expressed or ablated iNOS in leukocytes both showed significant...... delay in disease onset, with no difference in disease severity. We conclude that bone marrow-derived and CNS parenchymal sources of iNOS-derived NO both play a regulatory role in EAE....

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

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    Wang FJ

    2015-12-01

    Full Text Available 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.

  18. Radiation response of mesenchymal stem cells derived from bone marrow and human pluripotent stem cells

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    Islam, Mohammad S; Stemig, Melissa E.; Takahashi, Yutaka; Hui, Susanta K.

    2014-01-01

    Mesenchymal stem cells (MSCs) isolated from human pluripotent stem cells are comparable with bone marrow-derived MSCs in their function and immunophenotype. The purpose of this exploratory study was comparative evaluation of the radiation responses of mesenchymal stem cells derived from bone marrow- (BMMSCs) and from human embryonic stem cells (hESMSCs). BMMSCs and hESMSCs were irradiated at 0 Gy (control) to 16 Gy using a linear accelerator commonly used for cancer treatment. Cells were harv...

  19. Bone Marrow-Derived Stem Cell Transplantation for the Treatment of Insulin-Dependent Diabetes

    OpenAIRE

    2010-01-01

    The bone marrow is an invaluable source of adult pluripotent stem cells, as it gives rise to hematopoietic stem cells, endothelial progenitor cells, and mesenchymal cells, amongst others. The use of bone marrow-derived stem cell (BMC) transplantation (BMT) may be of assistance in achieving tissue repair and regeneration, as well as in modulating immune responses in the context of autoimmunity and transplantation. Ongoing clinical trials are evaluating the effects of BMC to preserve functiona...

  20. Human bone-marrow-derived mesenchymal stem cells

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    Kassem, Moustapha; Abdallah, Basem M

    2008-01-01

    Mesenchymal stem cells (MSC) are a group of cells present in bone-marrow stroma and the stroma of various organs with the capacity for mesoderm-like cell differentiation into, for example, osteoblasts, adipocytes, and chondrocytes. MSC are being introduced in the clinic for the treatment of a var......Mesenchymal stem cells (MSC) are a group of cells present in bone-marrow stroma and the stroma of various organs with the capacity for mesoderm-like cell differentiation into, for example, osteoblasts, adipocytes, and chondrocytes. MSC are being introduced in the clinic for the treatment...... of a variety of clinical conditions. The aim of this review is to provide an update regarding the biology of MSC, their identification and culture, and mechanisms controlling their proliferation and differentiation. We also review the current status of their clinical use. Areas in which research is needed...

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

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    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

  2. Notch pathway modulation on bone marrow-derived vascular precursor cells regulates their angiogenic and wound healing potential.

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    Caiado, Francisco; Real, Carla; Carvalho, Tânia; Dias, Sérgio

    2008-01-01

    Bone marrow (BM) derived vascular precursor cells (BM-PC, endothelial progenitors) are involved in normal and malignant angiogenesis, in ischemia and in wound healing. However, the mechanisms by which BM-PC stimulate the pre-existing endothelial cells at sites of vascular remodelling/recovery, and their contribution towards the formation of new blood vessels are still undisclosed. In the present report, we exploited the possibility that members of the Notch signalling pathway, expressed by BM-PC during endothelial differentiation, might regulate their pro-angiogenic or pro-wound healing properties. We demonstrate that Notch pathway modulates the adhesion of BM-PC to extracellular matrix (ECM) in vitro via regulation of integrin alpha3beta1; and that Notch pathway inhibition on BM-PC impairs their capacity to stimulate endothelial cell tube formation on matrigel and to promote endothelial monolayer recovery following wounding in vitro. Moreover, we show that activation of Notch pathway on BM-PC improved wound healing in vivo through angiogenesis induction. Conversely, inoculation of BM-PC pre-treated with a gamma secretase inhibitor (GSI) into wounded mice failed to induce angiogenesis at the wound site and did not promote wound healing, presumably due to a lower frequency of BM-PC at the wound area. Our data suggests that Notch pathway regulates BM-PC adhesion to ECM at sites of vascular repair and that it also regulates the capacity of BM-PC to stimulate angiogenesis and to promote wound healing. Drug targeting of the Notch pathway on BM-PC may thus represent a novel strategy to modulate neo-angiogenesis and vessel repair.

  3. Notch pathway modulation on bone marrow-derived vascular precursor cells regulates their angiogenic and wound healing potential.

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    Francisco Caiado

    Full Text Available Bone marrow (BM derived vascular precursor cells (BM-PC, endothelial progenitors are involved in normal and malignant angiogenesis, in ischemia and in wound healing. However, the mechanisms by which BM-PC stimulate the pre-existing endothelial cells at sites of vascular remodelling/recovery, and their contribution towards the formation of new blood vessels are still undisclosed. In the present report, we exploited the possibility that members of the Notch signalling pathway, expressed by BM-PC during endothelial differentiation, might regulate their pro-angiogenic or pro-wound healing properties. We demonstrate that Notch pathway modulates the adhesion of BM-PC to extracellular matrix (ECM in vitro via regulation of integrin alpha3beta1; and that Notch pathway inhibition on BM-PC impairs their capacity to stimulate endothelial cell tube formation on matrigel and to promote endothelial monolayer recovery following wounding in vitro. Moreover, we show that activation of Notch pathway on BM-PC improved wound healing in vivo through angiogenesis induction. Conversely, inoculation of BM-PC pre-treated with a gamma secretase inhibitor (GSI into wounded mice failed to induce angiogenesis at the wound site and did not promote wound healing, presumably due to a lower frequency of BM-PC at the wound area. Our data suggests that Notch pathway regulates BM-PC adhesion to ECM at sites of vascular repair and that it also regulates the capacity of BM-PC to stimulate angiogenesis and to promote wound healing. Drug targeting of the Notch pathway on BM-PC may thus represent a novel strategy to modulate neo-angiogenesis and vessel repair.

  4. Transplantation of autologous bone marrow-derived mesenchymal stem cells for traumatic brain injury

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    Jindou Jiang; Xingyao Bu; Meng Liu; Peixun Cheng

    2012-01-01

    Results from the present study demonstrated that transplantation of autologous bone marrow-derived mesenchymal stem cells into the lesion site in rat brain significantly ameliorated brain tissue pathological changes and brain edema, attenuated glial cell proliferation, and increased brain-derived neurotrophic factor expression. In addition, the number of cells double-labeled for 5-bromodeoxyuridine/glial fibrillary acidic protein and cells expressing nestin increased. Finally, blood vessels were newly generated, and the rats exhibited improved motor and cognitive functions. These results suggested that transplantation of autologous bone marrow-derived mesenchymal stem cells promoted brain remodeling and improved neurological functions following traumatic brain injury.

  5. Bone marrow aspiration

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    Iliac crest tap; Sternal tap; Leukemia - bone marrow aspiration; Aplastic anemia - bone marrow aspiration; Myelodysplastic syndrome - bone marrow aspiration; Thrombocytopenia - bone marrow aspiration; Myelofibrosis - bone marrow aspiration

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

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    Zou, He [Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto 606-8507 (Japan); Otani, Atsushi, E-mail: otan@kuhp.kyoto-u.ac.jp [Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto 606-8507 (Japan); Oishi, Akio; Yodoi, Yuko; Kameda, Takanori; Kojima, Hiroshi; Yoshimura, Nagahisa [Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto 606-8507 (Japan)

    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

  7. Tracking Intracavernously Injected Adipose-Derived Stem Cells to Bone Marrow

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    Lin, Guiting; Qiu, Xuefeng; Fandel, Thomas; Banie, Lia; Wang, Guifang; Lue, Tom F.; Lin, Ching-Shwun

    2012-01-01

    Intracavernous (IC) injection of stem cells (SCs) has been shown to improve erectile function in various erectile dysfunction (ED) animal models. However, the tissue distribution of the injected cells remains unknown. In this study we tracked IC injected adipose-derived stem cells (ADSCs) in various tissues. Rat paratesticular fat was processed for ADSC isolation and culture. The animals were then subject to cavernous nerve (CN) crush injury or sham operation, followed by IC injection of one million autologous or allogeneic ADSCs that were labeled with 5-ethynyl-2-deoxyuridine (EdU). Another group of rats received IC injection of EdU-labeled allogeneic penile smooth muscle cells (PSMCs). At 2 and 7 days post-injection, penises and femoral bone marrow were processed for histological analyses. Whole femoral bone marrows were also analyzed for EdU-positive cells by flow cytometry. The results show that ADSCs exited the penis within days of IC injection and migrated preferentially to bone marrow. Allogenicity did not affect ADSC's bone marrow appearance either at 2 or 7 days, while CN injury reduced the number of ADSCs in bone marrow significantly at 7 but not 2 days. The significance of these results in relation to SC therapy for ED is discussed. PMID:21796145

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

    Institute of Scientific and Technical Information of China (English)

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

    2014-01-01

    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.

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

    DEFF Research Database (Denmark)

    Rasmussen, Jeppe; Frøbert, Ole; Holst-Hansen, Claus

    2014-01-01

    Background: Treatment of myocardial infarction 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...... myocardial infarction 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 myocardial infarction, using a fully...... grown non-immunecompromised rat model. Methods: 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...

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

    Science.gov (United States)

    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...

  11. Immunomodulatory properties of oat and barley β-glucan populations on bone marrow derived dendritic cells

    NARCIS (Netherlands)

    Rosch, Christiane; Meijerink, Marjolein; Delahaije, Roy J.B.M.; Taverne, Nico; Gruppen, Harry; Wells, Jerry M.; Schols, Henk A.

    2016-01-01

    Specific structures of oat and barley β(1,3)(1,4)-glucans induced different in vitro immunomodulatory effects in bone marrow derived dendritic cells (BMDC) from TLR2/4 knock out mice. All barley β-glucan fractions induced larger amounts of cytokines in BMDCs than their oat equivalents. The particula

  12. Determinants of tubular bone marrow-derived cell engraftment after renal ischemia/reperfusion in rats

    NARCIS (Netherlands)

    Broekema, M; Harmsen, MC; Koerts, JA; Petersen, AH; van Luyn, MJA; Navis, G; Popa, ER

    2005-01-01

    Background. Ischemia/reperfusion (I/R) injury is a major cause of acute renal failure (ARF). ARF is reversible, due to an innate regenerative process, which is thought to depend partly on bone marrow-derived progenitor cells. The significance of these cells in the repair process has been questioned

  13. Proteinase activated receptor 1 mediated fibrosis in a mouse model of liver injury: a role for bone marrow derived macrophages.

    Directory of Open Access Journals (Sweden)

    Yiannis N Kallis

    Full Text Available Liver fibrosis results from the co-ordinated actions of myofibroblasts and macrophages, a proportion of which are of bone marrow origin. The functional effect of such bone marrow-derived cells on liver fibrosis is unclear. We examine whether changing bone marrow genotype can down-regulate the liver's fibrotic response to injury and investigate mechanisms involved. Proteinase activated receptor 1 (PAR1 is up-regulated in fibrotic liver disease in humans, and deficiency of PAR1 is associated with reduced liver fibrosis in rodent models. In this study, recipient mice received bone marrow transplantation from PAR1-deficient or wild-type donors prior to carbon tetrachloride-induced liver fibrosis. Bone marrow transplantation alone from PAR1-deficient mice was able to confer significant reductions in hepatic collagen content and activated myofibroblast expansion on wild-type recipients. This effect was associated with a decrease in hepatic scar-associated macrophages and a reduction in macrophage recruitment from the bone marrow. In vitro, PAR1 signalling on bone marrow-derived macrophages directly induced their chemotaxis but did not stimulate proliferation. These data suggest that the bone marrow can modulate the fibrotic response of the liver to recurrent injury. PAR1 signalling can contribute to this response by mechanisms that include the regulation of macrophage recruitment.

  14. Bone marrow-derived microglia infiltrate into the paraventricular nucleus of chronic psychological stress-loaded mice.

    Directory of Open Access Journals (Sweden)

    Koji Ataka

    Full Text Available BACKGROUND: Microglia of the central nervous system act as sentinels and rapidly react to infection or inflammation. The pathophysiological role of bone marrow-derived microglia is of particular interest because they affect neurodegenerative disorders and neuropathic pain. The hypothesis of the current study is that chronic psychological stress (chronic PS induces the infiltration of bone marrow-derived microglia into hypothalamus by means of chemokine axes in brain and bone marrow. METHODS AND FINDINGS: Here we show that bone marrow-derived microglia specifically infiltrate the paraventricular nucleus (PVN of mice that received chronic PS. Bone marrow derived-microglia are CX3CR1(lowCCR2(+CXCR4(high, as distinct from CX3CR1(highCCR2(-CXCR4(low resident microglia, and express higher levels of interleukin-1β (IL-1β but lower levels of tumor necrosis factor-α (TNF-α. Chronic PS stimulates the expression of monocyte chemotactic protein-1 (MCP-1 in PVN neurons, reduces stromal cell-derived factor-1 (SDF-1 in the bone marrow and increases the frequency of CXCR4(+ monocytes in peripheral circulation. And then a chemokine (C-C motif receptor 2 (CCR2 or a β3-adrenoceptor blockade prevents infiltration of bone marrow-derived microglia in the PVN. CONCLUSION: Chronic PS induces the infiltration of bone marrow-derived microglia into PVN, and it is conceivable that the MCP-1/CCR2 axis in PVN and the SDF-1/CXCR4 axis in bone marrow are involved in this mechanism.

  15. Bone Marrow-Derived Cells Contribute to Epithelial Engraftment during Wound Healing

    OpenAIRE

    Borue, Xenia; Lee, Sean; Grove, Joanna; Herzog, Erica L.; Harris, Robert; Diflo, Thomas; Glusac, Earl; Hyman, Kevin; Theise, Neil D.; Krause, Diane S.

    2004-01-01

    Recent findings suggest that bone marrow-derived cells (BMDC) may contribute to tissue maintenance throughout the body. However, it is not yet known whether marrow-derived epithelial cells are capable of undergoing proliferation. Our laboratory has shown that BMDC engraft as keratinocytes in the skin at low levels (≤ 1%) in the absence of injury. Here we show that skin damage affects the degree of engraftment of BMDC as keratinocytes and that the keratinocytes are actively cycling. Female mic...

  16. Bone marrow-derived mesenchymal stem cells increase dopamine synthesis in the injured striatum

    Institute of Scientific and Technical Information of China (English)

    Yue Huang; Cheng Chang; Jiewen Zhang; Xiaoqun Gao

    2012-01-01

    Previous studies showed that tyrosine hydroxylase or neurturin gene-modified cells transplanted into rats with Parkinson's disease significantly improved behavior and increased striatal dopamine content. In the present study, we transplanted tyrosine hydroxylase and neurturin gene-modified bone marrow-derived mesenchymal stem cells into the damaged striatum of Parkinson's disease model rats. Several weeks after cell transplantation, in addition to an improvement of motor function, tyrosine hydroxylase and neurturin proteins were up-regulated in the injured striatum, and importantly, levels of dopamine and its metabolite 3,4-dihydroxyphenylacetic acid increased significantly. Furthermore, the density of the D2 dopamine receptor in the postsynaptic membranes of dopaminergic neurons was decreased. These results indicate that transplantation of tyrosine hydroxylase and neurturin gene-modified bone marrow-derived mesenchymal stem cells increases dopamine synthesis and significantly improves the behavior of rats with Parkinson's disease.

  17. Gene expression profiles of human bone marrow derived mesenchymal stem cells and tendon cells

    Institute of Scientific and Technical Information of China (English)

    胡庆柳; 朴英杰; 邹飞

    2003-01-01

    Objective To study the gene expression profiles of human bone marrow derived mesenchymal stem cells and tendon cells.Methods Total RNA extracted from human bone marrow derived mesenchymal stem cells and tendon cells underwent reverse transcription, and the products were labeled with α-32P dCTP. The cDNA probes of total RNA were hybridized to cDNA microarray with 1176 genes, and then the signals were analyzed by AtlasImage analysis software Version 1.01a.Results Fifteen genes associated with cell proliferation and signal transduction were up-regulated, and one gene that takes part in cell-to-cell adhesion was down-regulated in tendon cells.Conclusion The 15 up-regulated and one down-regulated genes may be beneficial to the orientational differentiation of mesenchymal stem cells into tendon cells.

  18. Autologous bone marrow-derived stem cells in amyotrophic lateral sclerosis: A pilot study

    Directory of Open Access Journals (Sweden)

    Sudesh Prabhakar

    2012-01-01

    Full Text Available Background: Amyotrophic lateral sclerosis (ALS is a neurodegenerative disorder with no effective treatment. Stem cell therapy may be one of the promising treatment options for such patients. Aim: To assess the feasibility, efficacy and safety of autologous bone marrow-derived stem cells in patients of ALS. Settings and Design: We conducted an open-label pilot study of autologous bone marrow-derived stem cells in patients with ALS attending the Neurology Clinic of a tertiary care referral centre. Materials and Methods: Ten patients with ALS with mean revised ALS Functional Rating Scale (ALSFRS-R score of 30.2 (± 10.58 at baseline received intrathecal autologous bone marrow-derived stem cells. Primary end point was improvement in the ALSFRS-R score at 90, 180, 270 and 365 days post therapy. Secondary endpoints included ALSFRS-R subscores, time to 4-point deterioration, median survival and reported adverse events. Paired t-test was used to compare changes in ALSFRS-R from baseline and Kaplan-Meier analysis was used for survival calculations. Results: There was no significant deterioration in ALSFRS-R composite score from baseline at one-year follow-up (P=0.090. The median survival post procedure was 18.0 months and median time to 4-point deterioration was 16.7 months. No significant adverse events were reported. Conclusion: Autologous bone marrow-derived stem cell therapy is safe and feasible in patients of ALS. Short-term follow-up of ALSFRS-R scores suggests a trend towards stabilization of disease. However, the benefit needs to be confirmed in the long-term follow-up period.

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

    OpenAIRE

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

    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 con...

  20. Comparative analysis of canine monocyte- and bone-marrow-derived dendritic cells

    OpenAIRE

    Ricklin Gutzwiller, Meret Elisabeth; Moulin, Hervé Raphaël; Zurbriggen, Andreas; Roosje, Petra; Summerfield, Artur

    2010-01-01

    International audience; Dendritic cells (DC) represent a heterogeneous cell family of major importance for innate immune responses against pathogens and antigen presentation during infection, cancer, allergy and autoimmunity. The aim of the present study was to characterize canine DC generated in vitro with respect to their phenotype, responsiveness to toll-like receptor (TLR) ligands and T-cell stimulatory capacity. DC were derived from monocytes (MoDC) and from bone marrow hematopoietic cel...

  1. AI-05IMPACT OF GBM MICROENVIRONMENT ON EXPRESSION PROFILE OF BONE MARROW DERIVED PROGENITOR CELLS

    OpenAIRE

    Burrell, Kelly; Singh, Sanjay; Agnihotri, Sameer; Hill, Richard; Aldape, Kenneth; Zadeh, Gelareh

    2014-01-01

    We have recently shown that bone marrow derived cells (BMDC) provide a distinct tumor region dependent contribution to glioblastoma multiforme (GBM) neovascularization. The influence of GBM microenvironment on differentiation and modulation of expression factors by BMDC however remains unknown. In this study we establish the differential expression profile of BMDC as a consequence of recruitment and interaction with the GBM microenvironment and in response to radiation (RTx) and anti-angiogen...

  2. Effect of allogeneic bone marrow derived stromal cells on induced third-degree skin burn healing in mouse

    Directory of Open Access Journals (Sweden)

    Leyla Soleymani

    2014-10-01

    Conclusion: This experimental modulation of wound healing suggests that bone marrow-derived stromal cells can significantly enhance the rate of wound healing possibly through stimulation of granulation tissue, angiogenesis, fibroblast proliferation and collagen deposition.

  3. Review of Preclinical and Clinical Studies of Bone Marrow-Derived Cell Therapies for Intracerebral Hemorrhage

    Directory of Open Access Journals (Sweden)

    Paulo Henrique Rosado-de-Castro

    2016-01-01

    Full Text Available Stroke is the second leading cause of mortality worldwide, causing millions of deaths annually, and is also a major cause of disability-adjusted life years. Hemorrhagic stroke accounts for approximately 10 to 27% of all cases and has a fatality rate of about 50% in the first 30 days, with limited treatment possibilities. In the past two decades, the therapeutic potential of bone marrow-derived cells (particularly mesenchymal stem cells and mononuclear cells has been intensively investigated in preclinical models of different neurological diseases, including models of intracerebral hemorrhage and subarachnoid hemorrhage. More recently, clinical studies, most of them small, unblinded, and nonrandomized, have suggested that the therapy with bone marrow-derived cells is safe and feasible in patients with ischemic or hemorrhagic stroke. This review discusses the available evidence on the use of bone marrow-derived cells to treat hemorrhagic strokes. Distinctive properties of animal studies are analyzed, including study design, cell dose, administration route, therapeutic time window, and possible mechanisms of action. Furthermore, clinical trials are also reviewed and discussed, with the objective of improving future studies in the field.

  4. Review of Preclinical and Clinical Studies of Bone Marrow-Derived Cell Therapies for Intracerebral Hemorrhage

    Science.gov (United States)

    de Carvalho, Felipe Gonçalves; de Freitas, Gabriel Rodriguez

    2016-01-01

    Stroke is the second leading cause of mortality worldwide, causing millions of deaths annually, and is also a major cause of disability-adjusted life years. Hemorrhagic stroke accounts for approximately 10 to 27% of all cases and has a fatality rate of about 50% in the first 30 days, with limited treatment possibilities. In the past two decades, the therapeutic potential of bone marrow-derived cells (particularly mesenchymal stem cells and mononuclear cells) has been intensively investigated in preclinical models of different neurological diseases, including models of intracerebral hemorrhage and subarachnoid hemorrhage. More recently, clinical studies, most of them small, unblinded, and nonrandomized, have suggested that the therapy with bone marrow-derived cells is safe and feasible in patients with ischemic or hemorrhagic stroke. This review discusses the available evidence on the use of bone marrow-derived cells to treat hemorrhagic strokes. Distinctive properties of animal studies are analyzed, including study design, cell dose, administration route, therapeutic time window, and possible mechanisms of action. Furthermore, clinical trials are also reviewed and discussed, with the objective of improving future studies in the field. PMID:27698671

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

    Directory of Open Access Journals (Sweden)

    Louise A. Mesentier-Louro

    2016-01-01

    Full Text Available 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.

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

    Science.gov (United States)

    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

  7. Safety assessment of bone marrow derived MSC grown in platelet-rich plasma

    Directory of Open Access Journals (Sweden)

    Shoji Fukuda

    2015-06-01

    Full Text Available The injection of endothelial progenitor cells and mononuclear cells derived from bone marrow at the ischemic region of peripheral artery disease patients is reported to be effective for therapeutic angiogenesis; however, these cell therapies require large amounts of bone marrow to obtain sufficient numbers of cells. To solve this problem, we attempted to culture bone-marrow-derived mesenchymal stem cells (BM-MSC, which are supposed to secrete several cytokines that promote angiogenesis. We also focused on using platelet-rich plasma (PRP as a supplement for cell culture instead of fetal bovine serum. Human BM-MSC obtained from healthy volunteers expanded rapidly when cultured with 10% PRP prepared from their own blood. FACS analysis revealed that these cultured human MSC were homogeneous populations, and chromosomal analysis showed a normal karyotype. Moreover, the angiogenetic effect was apparent two weeks after human BM-MSC were injected into the ischemic muscle in SCID mice. Tumor formation was not detected three months after injection into SCID mice either subcutaneously or intramuscularly. To simulate clinical settings, canine BM-MSC were grown with canine PRP and injected into their ischemic muscles. We confirmed that donor cells existed in situ two and six weeks after operation without any side effects. These results suggest that cultured human BM-MSC can be a promising cell source for therapeutic angiogenesis.

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

    Science.gov (United States)

    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.

  9. Ex vivo expansions and transplantations of mouse bone marrow-derived hematopoietic stem/progenitor cells

    Institute of Scientific and Technical Information of China (English)

    WANG Jin-fu(王金福); WU Yi-fan(吴亦凡); HARRINTONG Jenny; McNIECE Ian K.

    2004-01-01

    To examine the effects of co-culture with bone marrow mesenchymal stem cells on expansion of hematopoietic stem/progenitor cells and the capacities of rapid neutrophil engraftment and hematopoietic reconstitution of the expanded cells, we expanded mononuclear cells (MNCs) and CD34+/c-kit+ cells from mouse bone marrow and transplanted the expanded cells into the irradiated mice. MNCs were isolated from mouse bone marrow and CD34+/c-kit+ cells were selected from MNCs by using MoFlo Cell Sorter. MNCs and CD34+/c-kit+ cells were co-cultured with mouse bone marrow-derived mesenchymal stem cells (MSCs) under a two-step expansion. The expanded cells were then transplanted into sublethally irradiated BDF1 mice. Results showed that the co-culture with MSCs resulted in expansions of median total nucleated cells,CD34+ cells, GM-CFC and HPP-CFC respectively by 10.8-, 4.8-, 65.9- and 38.8-fold for the mononuclear cell culture, and respectively by 76.1-, 2.9-, 71.7- and 51.8-fold for the CD34+/c-kit+ cell culture. The expanded cells could rapidly engraft in the sublethally irradiated mice and reconstitute their hematopoiesis. Co-cultures with MSCs in conjunction with two-step expansion increased expansions of total nucleated cells, GM-CFC and HPP-CFC, which led us to conclude MSCs may create favorable environment for expansions of hematopoietic stem/progenitor cells. The availability of increased numbers of expanded cells by the co-culture with MSCs may result in more rapid engraftment ofneutrophils following infusion to transplant recipients.

  10. Cytokines mRNA in bone marrow-derived dendritic cells in asthmatic mouse

    Institute of Scientific and Technical Information of China (English)

    YU Duowei; SUN Yun; WU Jianqing; YUAN Sheng

    2006-01-01

    By inducing and amplifying dendritic cells(DCs)derived from the bone marrow of asthma murine in vitro,cytokines mRNA were expressed,and the functions of DCS were investigated.Cells isolated from murine bone marrow have been cultured with rmGM-CSF and rmIL-4,and the expression of cytokines mRNA was determined by ribonuclease protection assay combined with multi-probe templates.Large numbers of DCS have been obtained from bone marrow,and they expressed interleukin-13(IL-13),interleukin-9(IL-9),and interleukin-3(IL-3)mRNA.Moreover.the level of IL-13 mRNA and IL-9 mRNA expressed by DCs in asthmatic mice was significantly higher than those in the control groups(P<0.05).But,the level of IL-3 mRNA showed no discrepancy between the two groups(P>0.05).Des are very important in the forming and developing of asthma,which implies that the therapy targeted at DCs will possibly become a new goal.

  11. Fusion of proinsulin-producing bone marrow-derived cells with hepatocytes in diabetes.

    Science.gov (United States)

    Fujimiya, Mineko; Kojima, Hideto; Ichinose, Masumi; Arai, Ryohachi; Kimura, Hiroshi; Kashiwagi, Atsunori; Chan, Lawrence

    2007-03-06

    We previously reported that diabetes in mice is associated with the appearance of proinsulin-producing (Proins-P) cells in the liver. It was unclear, however, whether these Proins-P bone marrow-derived cells (BMDC) merely transit through the liver or undergo fusion with hepatocytes, normally an extremely rare event. In this study, we found that, in diabetes, BMDC in the liver produce not only Proins but also TNF-alpha, suggesting that diabetes reprograms gene expression in BMDC, turning on "inappropriate" genes. Bone marrow transplantation using genetically marked donor and recipient mice showed that fusion occurs between Proins-P BMDC and hepatocytes. Cell fusion is further supported by the presence of the Y chromosome in Proins-P cells in female mice that received male bone marrow transplantation cells. Morphologically, Proins-P fusion cells are albumin-producing hepatocytes that constitute approximately 2.5% of the liver section area 5 months after diabetes induction. An extensive search failed to reveal any fusion cells in nondiabetic mice. Thus, diabetes causes fusion between Proins-P BMDC and hepatocytes in vivo, an observation that has implications for the pathophysiology of diabetes as well as the fundamental biology of heterotypic cell fusion.

  12. Rat bone marrow-derived dendritic cells generated with GM-CSF/IL-4 or FLT3L exhibit distinct phenotypical and functional characteristics.

    Science.gov (United States)

    N'diaye, Marie; Warnecke, Andreas; Flytzani, Sevasti; Abdelmagid, Nada; Ruhrmann, Sabrina; Olsson, Tomas; Jagodic, Maja; Harris, Robert A; Guerreiro-Cacais, Andre Ortlieb

    2016-03-01

    Dendritic cells are professional APCs that play a central role in the initiation of immune responses. The limited ex vivo availability of dendritic cells inspires the widespread use of bone marrow-derived dendritic cells as an alternative in research. However, the functional characteristics of bone marrow-derived dendritic cells are incompletely understood. Therefore, we compared functional and phenotypic characteristics of rat bone marrow-derived dendritic cells generated with GM-CSF/IL-4 or FLT3 ligand bone marrow-derived dendritic cells. A comparison of surface markers revealed that FLT3 ligand-bone marrow-derived dendritic cells expressed signal regulatory protein α, CD103, and CD4 and baseline levels of MHC class II, CD40, and CD86, which were highly up-regulated upon stimulation. Conversely, GM-CSF/IL-4-bone marrow-derived dendritic cells constitutively expressed signal regulatory protein α, CD11c, and CD11b but only mildly up-regulated MHC class II, CD40, or CD86 following stimulation. Expression of dendritic cell-associated core transcripts was restricted to FLT3 ligand-bone marrow-derived dendritic cells . GM-CSF/IL-4-bone marrow-derived dendritic cells were superior at phagocytosis but were outperformed by FLT3 ligand-bone marrow-derived dendritic cells at antigen presentation and T cell stimulation in vitro. Stimulated GM-CSF/IL-4-bone marrow-derived dendritic cells secreted more TNF, CCL5, CCL20, and NO, whereas FLT3 ligand-bone marrow-derived dendritic cells secreted more IL-6 and IL-12. Finally, whereas GM-CSF/IL-4-bone marrow-derived dendritic cell culture supernatants added to resting T cell cultures promoted forkhead box p3(+) regulatory T cell populations, FLT3 ligand-bone marrow-derived dendritic cell culture supernatants drove Th17 differentiation. We conclude that rat GM-CSF/IL-4-bone marrow-derived dendritic cells and FLT3 ligand-bone marrow-derived dendritic cells are functionally distinct. Our data support the current rationale that FLT3

  13. Prolonged hypoxic culture and trypsinization increase the pro-angiogenic potential of human adipose tissue-derived stem cells

    DEFF Research Database (Denmark)

    Rasmussen, Jeppe Grøndahl; Frøbert, Ole; Pilgaard, Linda;

    2011-01-01

    Transplantation of mesenchymal stromal cells (MSC), including adipose tissue-derived stem cells (ASC), is a promising option in the treatment of vascular disease. Short-term hypoxic culture of MSC augments secretion of anti-apoptotic and angiogenic cytokines. We hypothesized that prolonged hypoxic...... (1% and 5% oxygen) culture and trypsinization would augment ASC expression of anti-apoptotic and angiogenic cytokines and increase the angiogenic potential of ASC-conditioned media....

  14. Engraftment of bone marrow-derived cells after nonlethal radiation in syngeneic C57BL/6mice%Engraftment of bone marrow-derived cells after nonlethal radiation in syngeneic C57BL/6 mice

    Institute of Scientific and Technical Information of China (English)

    Wu Liao; Tan Li; Wang Yu; Liu Dengqun; Shi Chunmeng

    2015-01-01

    Objective To study the characteristics of cell engraftment in mice at a lower dose under nonlethal radiated condition.Methods A syngeneic C57BL/6 mouse model,transplanted with 1 × 107 bone marrow cells and exposed to 2.5 Gy whole body irradiation (WBI),was selected to study the chimerism of cells from green fluorescent protein positive (GFP +) transgenic mice.The control group was injected with GFP + cells without receiving irradiation.In addition,an allogenic transplantation model of BALB/c mice was also investigated which was infused by GFP + cells from C57BL/6 mice.The engraftment of bone marrow-derived cells (BMDCs) was detected by immunohistochemistry in bone marrow,liver,lung,small intestine and spleen.Results The transplanted bone marrow cells successfully grafted in the haematopoietic tissues from syngeneic GFP transgenic mice.The transplanted GFP+ cells were also detected in the non-haematopoietic tissues,such as the small intestine,liver,spleen and lung,after irradiation.However,a lethal dose irradiation of 8 Gy was required to establish successful chimerism in allogeneic transplantation model by infusing the bone marrow cells from C57BL/6 mice to BALB/c mice.Conclusions Bone marrow-derived cells can be successfully grafted into various recipient tissues receiving a 2.5 Gy dose of radiation in syngeneic mice,but not in allogeneic mice.This nonlethal model may help to further study the plasticity and mechanism of bone marrow-derived cells in tissue repair and regeneration after radiation injury.

  15. Bone marrow-derived mesenchymal stem cells protect rats from endotoxin-induced acute lung injury

    Institute of Scientific and Technical Information of China (English)

    LIANG Zhi-xin; SUN Ji-ping; WANG Ping; TIAN Qing; YANG Zhen; CHEN Liang-an

    2011-01-01

    Background Acute lung injury (ALI) is a serious and common condition for which there are currently no specific strategies for treatment.Recent studies have suggested that bone marrow-derived multipotent mesenchymal stem cells (MSCs) may have therapeutic applications in multiple clinical disorders.We explored the biological effects of MSCs during endotoxin-induced ALl and the mechanisms involved.Methods MSCs were isolated from male rat bone marrow and the ALl model was induced by intravenous endotoxin injection.Female rats were sacrificed at 6 hours,24 hours,4 days,1 week and 3 weeks post-injection of MSCs or saline and the lung tissue,bronchoalveolar lavage fluid,and serum were harvested for analysis.We further evaluated the survival of the rats and examined the effects of endotoxin-induced injury on the interaction between alveolar macrophages (AMs) and MSCs in ex vivo.Results There was a significant decrease in numbers of neutrophils in bronchoalveolar lavage fluid (P <0.05),and myeloperoxidase activity in the lung (P<0.01),and of TNF-α and IL-1β in serum (P <0.05) in the MSC treated rats at 4 days.Furthermore,MSC treated rats exhibited improved survival,lower lung injury score,higher concentration of IL-10 in the serum and a reduced hydroxyproline content,but these differences were not statistically significant.Moreover,co-cultures of MSCs and AMs had significantly reduced levels of TNF-α,IL-1β and macrophage inflammatory protein (MIP)-1α and significantly increased levels of IL-10 (P<0.05) in the culture supernatants.Conclusions Treatment with intravenous injection of bone marrow-derived MSCs have beneficial effects on endotoxin-induced ALl in rats.The beneficial effect might be achieved through the engraftment of differentiated MSCs in the lungs and appears derive more from their capacity to secrete soluble factors that modulate immune responses.

  16. Bone marrow-derived HL mitigates bone marrow-derived CETP-mediated decreases in HDL in mice globally deficient in HL and the LDLr.

    Science.gov (United States)

    Hime, Neil J; Black, Audrey S; Bonnet, David J; Curtiss, Linda K

    2014-09-01

    The objective of this study was to determine the combined effects of HL and cholesteryl ester transfer protein (CETP), derived exclusively from bone marrow (BM), on plasma lipids and atherosclerosis in high-fat-fed, atherosclerosis-prone mice. We transferred BM expressing these proteins into male and female double-knockout HL-deficient, LDL receptor-deficient mice (HL(-/-)LDLr(-/-)). Four BM chimeras were generated, where BM-derived cells expressed 1) HL but not CETP, 2) CETP and HL, 3) CETP but not HL, or 4) neither CETP nor HL. After high-fat feeding, plasma HDL-cholesterol (HDL-C) was decreased in mice with BM expressing CETP but not HL (17 ± 4 and 19 ± 3 mg/dl, female and male mice, respectively) compared with mice with BM expressing neither CETP nor HL (87 ± 3 and 95 ± 4 mg/dl, female and male mice, respectively, P HL mitigated this CETP-mediated decrease in HDL-C. BM-derived CETP decreased the cholesterol component of HDL particles and increased plasma cholesterol. BM-derived HL mitigated these effects of CETP. Atherosclerosis was not significantly different between BM chimeras. These results suggest that BM-derived HL mitigates the HDL-lowering, HDL-modulating, and cholesterol-raising effects of BM-derived CETP and warrant further studies to characterize the functional properties of these protein interactions.

  17. A PEDF-Derived Peptide Inhibits Retinal Neovascularization and Blocks Mobilization of Bone Marrow-Derived Endothelial Progenitor Cells

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    Richard Longeras

    2012-01-01

    Full Text Available Proliferative diabetic retinopathy is characterized by pathological retinal neovascularization, mediated by both angiogenesis (involving mature endothelial cells and vasculogenesis (involving bone marrow-derived circulating endothelial progenitor cells (EPCs. Pigment epithelium-derived factor (PEDF contains an N-terminal 34-amino acid peptide (PEDF-34 that has antiangiogenic properties. Herein, we present a novel finding that PEDF-34 also possesses antivasculogenic activity. In the oxygen-induced retinopathy (OIR model using transgenic mice that have Tie2 promoter-driven GFP expression, we quantified Tie2GFP+ cells in bone marrow and peripheral blood by fluorescence-activated cell sorting (FACS. OIR significantly increased the number of circulating Tie2-GFP+ at P16, correlating with the peak progression of neovascularization. Daily intraperitoneal injections of PEDF-34 into OIR mice decreased the number of Tie2-GFP+ cells in the circulation at P16 by 65% but did not affect the number of Tie2-GFP+ cells in the bone marrow. These studies suggest that PEDF-34 attenuates EPC mobilization from the bone marrow into the blood circulation during retinal neovascularization.

  18. The proteomic dataset for bone marrow derived human mesenchymal stromal cells: Effect of in vitro passaging

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    Samuel T. Mindaye

    2015-12-01

    Full Text Available Bone-marrow derived mesenchymal stromal cells (BMSCs have been in clinical trials for therapy. One major bottleneck in the advancement of BMSC-based products is the challenge associated with cell isolation, characterization, and ensuring cell fitness over the course of in vitro cell propagation steps. The data in this report is part of publications that explored the proteomic changes following in vitro passaging of BMSCs [4] and the molecular heterogeneity in cultures obtained from different human donors [5,6].The methodological details involving cell manufacturing, proteome harvesting, protein identification and quantification as well as the bioinformatic analyses were described to ensure reproducibility of the results.

  19. EXPRESSION OF rhBMP-7 GENE IN TRANSDUCED BONE MARROW DERIVED STROMAL CELLS

    Institute of Scientific and Technical Information of China (English)

    段德宇; 杜靖远; 王洪; 刘勇; 郭晓东

    2002-01-01

    Objective. To explore the possibility of expression of exogenous gene in transduced bone marrow derived stromal cells(BMSCs). Methods. The marker gene , pbLacZ, was transferred into cultured BMSCs and the expression of transduced gene by X-gal staining was examined. Then plasmid pcDNA3-rhBMP7 was delivered to cultured BMSCs. Through immunohistochemical staining and RT-PCR assay, the expression of rhBMP7 gene was detected. Results. The exogenous gene could be expressed efficiently in transduced BMSCs. Conculsion. The present study provided a theoretical basis to gene therapy on the problems of bone and cartilage tissue.

  20. Caveolin-1 and migration of bone-marrow derived cells in the mouse eye.

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    May, Christian-Albrecht

    2013-12-01

    Bone marrow derived cells (BMDCs) can be found in almost every tissue showing a distinct turnover and density. Since caveolin-1 regulates junction-associated proteins in endothelial and epithelial cells, its role for BMDC was investigated in the eyes of caveolin-1 knock-out mice transplanted with GFP-marked BMDC. Distribution and turnover of BMDC in connective tissues (cornea, iris, ciliary body and choroid) was not altered. The absence of caveolin-1, however, caused a significant decrease of BMDC turnover in cornea epithelium, ciliary epithelium, and in the retina. This finding emphasizes an important, hitherto unknown role of caveolin-1 in neuronal and epithelial tissues.

  1. Pharmacologically active microcarriers delivering BDNF within a hydrogel: Novel strategy for human bone marrow-derived stem cells neural/neuronal differentiation guidance and therapeutic secretome enhancement.

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    Kandalam, Saikrishna; Sindji, Laurence; Delcroix, Gaëtan J-R; Violet, Fabien; Garric, Xavier; André, Emilie M; Schiller, Paul C; Venier-Julienne, Marie-Claire; des Rieux, Anne; Guicheux, Jérôme; Montero-Menei, Claudia N

    2017-02-01

    Stem cells combined with biodegradable injectable scaffolds releasing growth factors hold great promises in regenerative medicine, particularly in the treatment of neurological disorders. We here integrated human marrow-isolated adult multilineage-inducible (MIAMI) stem cells and pharmacologically active microcarriers (PAMs) into an injectable non-toxic silanized-hydroxypropyl methylcellulose (Si-HPMC) hydrogel. The goal is to obtain an injectable non-toxic cell and growth factor delivery device. It should direct the survival and/or neuronal differentiation of the grafted cells, to safely transplant them in the central nervous system, and enhance their tissue repair properties. A model protein was used to optimize the nanoprecipitation conditions of the neuroprotective brain-derived neurotrophic factor (BDNF). BDNF nanoprecipitate was encapsulated in fibronectin-coated (FN) PAMs and the in vitro release profile evaluated. It showed a prolonged, bi-phasic, release of bioactive BDNF, without burst effect. We demonstrated that PAMs and the Si-HPMC hydrogel increased the expression of neural/neuronal differentiation markers of MIAMI cells after 1week. Moreover, the 3D environment (PAMs or hydrogel) increased MIAMI cells secretion of growth factors (b-NGF, SCF, HGF, LIF, PlGF-1, SDF-1α, VEGF-A & D) and chemokines (MIP-1α & β, RANTES, IL-8). These results show that PAMs delivering BDNF combined with Si-HPMC hydrogel represent a useful novel local delivery tool in the context of neurological disorders. It not only provides neuroprotective BDNF but also bone marrow-derived stem cells that benefit from that environment by displaying neural commitment and an improved neuroprotective/reparative secretome. It provides preliminary evidence of a promising pro-angiogenic, neuroprotective and axonal growth-promoting device for the nervous system.

  2. Robust growth of avirulent phase II Coxiella burnetii in bone marrow-derived murine macrophages

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    Cockrell, Diane C.; Long, Carrie M.; Robertson, Shelly J.; Shannon, Jeffrey G.; Miller, Heather E.; Myers, Lara; Larson, Charles L.; Starr, Tregei; Beare, Paul A.

    2017-01-01

    Published data show that murine bone marrow-derived macrophages (BMDM) restrict growth of avirulent phase II, but not virulent phase I, Coxiella burnetii. Growth restriction of phase II bacteria is thought to result from potentiated recognition of pathogen-associated molecular patterns, which leads to production of inhibitory effector molecules. Past studies have used conditioned medium from L-929 murine fibroblasts as a source of macrophage-colony stimulating factor (M-CSF) to promote differentiation of bone marrow-derived myeloid precursors into macrophages. However, uncharacterized components of conditioned medium, such as variable amounts of type I interferons, can affect macrophage activation status and their permissiveness for infection. In the current study, we show that the C. burnetii Nine Mile phase II (NMII) strain grows robustly in primary macrophages from C57BL/6J mice when bone marrow cells are differentiated with recombinant murine M-CSF (rmM-CSF). Bacteria were readily internalized by BMDM, and replicated within degradative, LAMP1-positive vacuoles to achieve roughly 3 logs of growth over 6 days. Uninfected BMDM did not appreciably express CD38 or Egr2, markers of classically (M1) and alternatively (M2) activated macrophages, respectively, nor did infection change the lack of polarization. In accordance with an M0 phenotype, infected BMDM produced moderate amounts of TNF and nitric oxide. Similar NMII growth results were obtained using C57BL/6J myeloid progenitors immortalized with an estrogen-regulated Hoxb8 (ER-Hoxb8) oncogene. To demonstrate the utility of the ER-Hoxb8 system, myeloid progenitors from natural resistance-associated macrophage protein 1 (Nramp1) C57BL/6J knock-in mice were transduced with ER-Hoxb8, and macrophages were derived from immortalized progenitors using rmM-CSF and infected with NMII. No difference in growth was observed when compared to macrophages from wild type mice, indicating depletion of metal ions by the Nramp1

  3. Neuronal-like cell differentiation of non-adherent bone marrow cell-derived mesenchymal stem cells*

    Institute of Scientific and Technical Information of China (English)

    Yuxin Wu; Jinghan Zhang; Xiaoming Ben

    2013-01-01

    Non-adherent bone marrow cel-derived mesenchymal stem cel s from C57BL/6J mice were sepa-rated and cultured using the “pour-off” method. Non-adherent bone marrow cel-derived mesen-chymal stem cel s developed colony-forming unit-fibroblasts, and could be expanded by supple-mentation with epidermal growth factor. Immunocytochemistry showed that the non-adherent bone marrow cel-derived mesenchymal stem cel s exposed to basic fibroblast growth factor/epidermal growth factor/nerve growth factor expressed the neuron specific markers, neurofilament-200 and NeuN, in vitro. Non-adherent bone marrow cel-derived mesenchymal stem cel s fromβ-galactosidase transgenic mice were also transplanted into focal ischemic brain (right corpus striatum) of C57BL/6J mice. At 8 weeks, cel s positive for LacZ andβ-galactosidase staining were observed in the ischemic tissues, and cel s co-labeled with both β-galactosidase and NeuN were seen by double immunohistochemical staining. These findings suggest that the non-adherent bone marrow cel-derived mesenchymal stem cel s could differentiate into neuronal-like cel s in vitro and in vivo.

  4. Passage of bone-marrow-derived liver stem cells in a proliferating culture system

    Institute of Scientific and Technical Information of China (English)

    Yun-Feng Cai; Ji-Sheng Chen; Shu-Ying Su; Zuo-Jun Zhen; Huan-Wei Chen

    2009-01-01

    AIM: To explore the feasibility of passage of bonemarrow-derived liver stem cells (BDLSCs) in culture systems that contain cholestatic serum. METHODS: Whole bone marrow cells of rats were purified with conditioning selection media that contained 50 mL/L cholestatic serum. The selected BDLSCs were grown in a proliferating culture system and a differentiating culture system. The culture systems contained factors that stimulated the proliferation and differentiation of BDLSCs. Each passage of the proliferated stem cells was subjected to flow cytometry to detect stem cell markers. The morphology and phenotypic markers of BDLSCs were characterized using immunohistochemistry, reverse transcription polymerase chain reaction (RT-PCR) and electron microscopy. The metabolic functions of differentiated cells were also determined by glycogen staining and urea assay. RESULTS: The conditioning selection medium isolated BDLSCs directly from cultured bone marrow cells. The selected BDLSCs could be proliferated for six passages and maintained stable markers in our proliferating system. When the culture system was changed to a differentiating system, hepatocyte-like colony-forming units (H-CFUs) were formed. H-CFUs expressed markers of embryonic hepatocytes (alpha-fetoprotein, albumin and cytokeratin 8/18), biliary cells (cytokeratin 19), hepatocyte functional proteins (transthyretin and cytochrome P450-2b1), and hepatocyte nuclear factors 1α and -3β). They also had glycogen storage and urea synthesis functions, two of the critical features of hepatocytes. CONCLUSION: BDLSCs can be selected directly from bone marrow cells, and pure BDLSCs can be proliferated for six passages. The differentiated cells have hepatocyte-like phenotypes and functions. BDLSCs represent a new method to provide a readily available alternate source of cells for clinical hepatocyte therapy.

  5. Bone marrow transplant

    Science.gov (United States)

    Transplant - bone marrow; Stem cell transplant; Hematopoietic stem cell transplant; Reduced intensity nonmyeloablative transplant; Mini transplant; Allogenic bone marrow transplant; Autologous bone marrow transplant; Umbilical ...

  6. Differentiation of mouse bone marrow derived stem cells toward microglia-like cells

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    Stolzing Alexandra

    2011-08-01

    Full Text Available Abstract Background Microglia, the macrophages of the brain, have been implicated in the causes of neurodegenerative diseases and display a loss of function during aging. Throughout life, microglia are replenished by limited proliferation of resident microglial cells. Replenishment by bone marrow-derived progenitor cells is still under debate. In this context, we investigated the differentiation of mouse microglia from bone marrow (BM stem cells. Furthermore, we looked at the effects of FMS-like tyrosine kinase 3 ligand (Flt3L, astrocyte-conditioned medium (ACM and GM-CSF on the differentiation to microglia-like cells. Methods We assessed in vitro-derived microglia differentiation by marker expression (CD11b/CD45, F4/80, but also for the first time for functional performance (phagocytosis, oxidative burst and in situ migration into living brain tissue. Integration, survival and migration were assessed in organotypic brain slices. Results The cells differentiated from mouse BM show function, markers and morphology of primary microglia and migrate into living brain tissue. Flt3L displays a negative effect on differentiation while GM-CSF enhances differentiation. Conclusion We conclude that in vitro-derived microglia are the phenotypic and functional equivalents to primary microglia and could be used in cell therapy.

  7. Bone marrow-derived progenitor cells augment venous remodeling in a mouse dorsal skinfold chamber model.

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    Megan E Doyle

    Full Text Available The delivery of bone marrow-derived cells (BMDCs has been widely used to stimulate angiogenesis and arteriogenesis. We identified a progenitor-enriched subpopulation of BMDCs that is able to augment venular remodeling, a generally unexplored area in microvascular research. Two populations of BMDCs, whole bone marrow (WBM and Lin(-/Sca-1(+ progenitor cells, were encapsulated in sodium alginate and delivered to a mouse dorsal skinfold chamber model. Upon observation that encapsulated Sca-1(+ progenitor cells enhance venular remodeling, the cells and tissue were analyzed on structural and molecular levels. Venule walls were thickened and contained more nuclei after Sca-1(+ progenitor cell delivery. In addition, progenitors expressed mRNA transcript levels of chemokine (C-X-C motif ligand 2 (CXCL2 and interferon gamma (IFNγ that are over 5-fold higher compared to WBM. Tissues that received progenitors expressed significantly higher protein levels of vascular endothelial growth factor (VEGF, monocyte chemotactic protein-1 (MCP-1, and platelet derived growth factor-BB (PDGF-BB compared to tissues that received an alginate control construct. Nine days following cell delivery, tissue from progenitor recipients contained 39% more CD45(+ leukocytes, suggesting that these cells may enhance venular remodeling through the modulation of the local immune environment. Results show that different BMDC populations elicit different microvascular responses. In this model, Sca-1(+ progenitor cell-derived CXCL2 and IFNγ may mediate venule enlargement via modulation of the local inflammatory environment.

  8. Possible mechanisms of retinal function recovery with the use of cell therapy with bone marrow-derived stem cells

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    Rubens Camargo Siqueira

    2010-10-01

    Full Text Available Bone marrow has been proposed as a potential source of stem cells for regenerative medicine. In the eye, degeneration of neural cells in the retina is a hallmark of such widespread ocular diseases as age-related macular degeneration (AMD and retinitis pigmentosa. Bone marrow is an ideal tissue for studying stem cells mainly because of its accessibility. Furthermore, there are a number of well-defined mouse models and cell surface markers that allow effective study of hematopoiesis in healthy and injured mice. Because of these characteristics and the experience of bone marrow transplantation in the treatment of hematological disease such as leukemia, bone marrow-derived stem cells have also become a major tool in regenerative medicine. Those cells may be able to restore the retina function through different mechanisms: A cellular differentiation, B paracrine effect, and C retinal pigment epithelium repair. In this review, we described these possible mechanisms of recovery of retinal function with the use of cell therapy with bone marrow-derived stem cells.

  9. Nonhematopoietic cells are the primary source of bone marrow-derived lung epithelial cells.

    Science.gov (United States)

    Kassmer, Susannah H; Bruscia, Emanuela M; Zhang, Ping-Xia; Krause, Diane S

    2012-03-01

    Previous studies have demonstrated that bone marrow (BM)-derived cells differentiate into nonhematopoietic cells of multiple tissues. To date, it remains unknown which population(s) of BM cells are primarily responsible for this engraftment. To test the hypothesis that nonhematopoietic stem cells in the BM are the primary source of marrow-derived lung epithelial cells, either wild-type hematopoietic or nonhematopoietic BM cells were transplanted into irradiated surfactant-protein-C (SPC)-null mice. Donor-derived, SPC-positive type 2 pneumocytes were predominantly detected in the lungs of mice receiving purified nonhematopoietic cells and were absent from mice receiving purified hematopoietic stem and progenitor cells. We conclude that cells contained in the nonhematopoietic fraction of the BM are the primary source of marrow-derived lung epithelial cells. These nonhematopoietic cells may represent a primitive stem cell population residing in adult BM.

  10. Impaired phagocytosis of apoptotic cells causes accumulation of bone marrow-derived macrophages in aged mice

    Science.gov (United States)

    Kim, Ok-Hee; Kim, Hyojung; Kang, Jinku; Yang, Dongki; Kang, Yu-Hoi; Lee, Dae Ho; Cheon, Gi Jeong; Park, Sang Chul; Oh, Byung-Chul

    2017-01-01

    Accumulation of tissue macrophages is a significant characteristic of disease-associated chronic inflammation, and facilitates the progression of disease pathology. However, the functional roles of these bone marrow-derived macrophages (BMDMs) in aging are unclear. Here, we identified age-dependent macrophage accumulation in the bone marrow, showing that aging significantly increases the number of M1 macrophages and impairs polarization of BMDMs. We found that age-related dysregulation of BMDMs is associated with abnormal overexpression of the anti-inflammatory interleukin-10. BMDM dysregulation in aging impairs the expression levels of pro-inflammatory cytokines and genes involved in B-cell maturation and activation. Phagocytosis of apoptotic Jurkat cells by BMDMs was reduced because of low expression of phagocytic receptor CD14, indicating that increased apoptotic cells may result from defective phagocytosis of apoptotic cells in the BM of aged mice. Therefore, CD14 may represent a promising target for preventing BMDM dysregulation, and macrophage accumulation may provide diagnostic and therapeutic clues. PMID:27866511

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

    Science.gov (United States)

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

    2011-05-12

    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-1(op/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.

  12. Activation of Cannabinoid Receptor 2 Enhances Osteogenic Differentiation of Bone Marrow Derived Mesenchymal Stem Cells

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    Yong-Xin Sun

    2015-01-01

    Full Text Available Bone marrow derived mesenchymal stem cells (BM-MSCs are considered as the most promising cells source for bone engineering. Cannabinoid (CB receptors play important roles in bone mass turnover. The aim of this study is to test if activation of CB2 receptor by chemical agonist could enhance the osteogenic differentiation and mineralization in bone BM-MSCs. Alkaline phosphatase (ALP activity staining and real time PCR were performed to test the osteogenic differentiation. Alizarin red staining was carried out to examine the mineralization. Small interference RNA (siRNA was used to study the role of CB2 receptor in osteogenic differentiation. Results showed activation of CB2 receptor increased ALP activity, promoted expression of osteogenic genes, and enhanced deposition of calcium in extracellular matrix. Knockdown of CB2 receptor by siRNA inhibited ALP activity and mineralization. Results of immunofluorescent staining showed that phosphorylation of p38 MAP kinase is reduced by knocking down of CB2 receptor. Finally, bone marrow samples demonstrated that expression of CB2 receptor is much lower in osteoporotic patients than in healthy donors. Taken together, data from this study suggested that activation of CB2 receptor plays important role in osteogenic differentiation of BM-MSCs. Lack of CB2 receptor may be related to osteoporosis.

  13. Characterization of common marmoset (Callithrix jacchus) bone marrow-derived mesenchymal stem cells.

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    Kanda, Akifumi; Sotomaru, Yusuke; Nobukiyo, Asako; Yamaoka, Emi; Hiyama, Eiso

    2013-01-01

    Mesenchymal stem cells (MSCs) could be useful for regenerative medicine because they can beharvested easily from the bone marrow of living donors and the cells can be differentiated into adipogenic, osteogenic, and chondrogenic lineages in vitro. To apply MSCs for the medical treatment of human diseases as regenerative medicine, detailed experimental characterization of the cells is required. Recently, a New World primate, the common marmoset (Callithrix jacchus), has been widely used as a new human disease model because of its ease of handling and breeding. Although common marmoset MSCs have been established and will be used in preclinical studies of regenerative medicine, the characteristics of these cells remain unclear. Aiming to characterize common marmoset MSCs further, we harvested common marmoset bone marrow-derived cells (cmBMDCs) from the femurs of newborn males. We revealed that the morphology of the cells was similar to common marmoset fibroblasts, and extracellular matrix components, such as gelatin and fibronectin, were effective for their proliferation and formation of colony-forming unit fibroblasts. Furthermore, we were able to differentiate cmBMDCs into adipocytes, osteocytes, and chondrocytes in vitro, and they expressed the MSCmarkers CD44, CD73, CD90, and CD105, but their expression decreased with increasing passage number. The data demonstrate that cmBMDCs exhibit characteristics of MSCs and thus it would be beneficial to use these cells in preclinical studies.

  14. IFITM1 increases osteogenesis through Runx2 in human alveolar-derived bone marrow stromal cells.

    Science.gov (United States)

    Kim, Beom-Su; Kim, Hyung-Jin; Kim, Jin Seong; You, Yong-Ouk; Zadeh, Homa; Shin, Hong-In; Lee, Seung-Jin; Park, Yoon-Jeong; Takata, Takashi; Pi, Sung-Hee; Lee, Jun; You, Hyung-Keun

    2012-09-01

    The exact molecular mechanisms governing the differentiation of bone marrow stromal stem/progenitor cells (BMSCs) into osteoblasts remain largely unknown. In this study, a highly expressed protein that had a high degree of homology with interferon-induced transmembrane protein 1 (IFITM1) was identified using differentially expressed gene (DEG) screening. We sought to determine whether IFITM1 influenced osteoblast differentiation. During differentiation, IFITM1 expression gradually increased from 5 to 10days and subsequently decreased at 15 days in culture. Analysis of IFITM1 protein expression in several cell lines as well as in situ studies on human tissues revealed its selective expression in bone cells and human bone. Proliferation of human alveolar-derived bone marrow stromal cells (hAD-BMSCs) was significantly inhibited by IFITM1 knockdown by using short hairpin RNA, as were bone specific markers such as alkaline phosphatase, collagen type I α 1, bone sialoprotein, osteocalcin, and osterix were decreased. Calcium accumulation also decreased following IFITM1 knockdown. Moreover, IFITM1 knockdown in hAD-BMSCs was associated with inhibition of Runx2 mRNA and protein expression. Collectively, the present data provide evidence for the role of IFITM1 in osteoblast differentiation. The exact mechanisms of IFITM1's involvement in osteoblast differentiation are still under investigation.

  15. The Comparison of Biologic Characteristics between Mice Embryonic Stem Cells and Bone Marrow Derived Dendritic Cells

    Institute of Scientific and Technical Information of China (English)

    Junfeng Liu; Zhixu He; Dong Shen; Jin Huang; Haowen Wang

    2009-01-01

    OBJECTIVE This research was to induce dendritic cells (DCs)from mice embryonic stem cells and bone marrow mononuclear cells in vitro, and then compare the biologic characteristics of them.METHODS Embryonic stem cells (ESCs) suspending cultured in petri dishes were induced to generate embryonic bodies (EBs).Fourteen-day well-developed EBs were transferred to histological culture with the same medium and supplemented 25 ng/ml GM-CSF and 25 ng/ml IL-3. In the next 2 weeks, there were numerous immature DCs outgrown. Meantime, mononuclear cells isolated from mice bone marrow were induced to derive dendritic cells by supplementing 25 ng/ml GM-CSF and 25 ng/ml IL-4, and then the morphology, phenotype and function of both dendritic cells from different origins were examined.RESULTS Growing mature through exposure to lipopolysaccharide (LPS), both ESC-DCs and BM-DCs exhibited dramatic veils of cytoplasm and extensive dendrites on their surfaces, highly expressed CD11c, MHC-Ⅱ and CD86 with strong capacity to stimulate primary T cell responses in mixed leukocyte reaction (MLR).CONCLUSION ESC-DC has the same biologic characteristics as BM-DC, and it provides a new, reliable source for the functional research of DC and next produce corresponding anti-tumor vaccine.

  16. Migration of bone marrow-derived cells into the central nervous system in models of neurodegeneration.

    Science.gov (United States)

    Lampron, Antoine; Pimentel-Coelho, Pedro M; Rivest, Serge

    2013-12-01

    Microglia are the brain-resident macrophages tasked with the defense and maintenance of the central nervous system (CNS). The hematopoietic origin of microglia has warranted a therapeutic potential for the hematopoietic system in treating diseases of the CNS. However, migration of bone marrow-derived cells (BMDC) into the CNS is a marginal event under normal, healthy conditions. A busulfan-based chemotherapy regimen was used for bone marrow transplantation in wild-type mice before subjecting them to a hypoxic-ischemic brain injury or in APP/PS1 mice prior to the formation of amyloid plaques. The cells were tracked and analyzed throughout the development of the pathology. The efficacy of a preventive macrophage colony-stimulating factor (M-CSF) treatment was also studied to highlight the effects of circulating monocytes in hypoxic-ischemic brain injury. Such an injury induces a strong migration of BMDC into the CNS, without the need for irradiation. These migrating cells do not replace the entire microglial pool but rather are confined to the sites of injury for several weeks, suggesting that they could perform specific functions. M-CSF showed neuroprotective effects as a preventive treatment. In APP/PS1 mice, the formation of amyloid plaques was sufficient to induce the entry of cells into the parenchyma, though in low numbers. This study confirms that BMDC infiltrate the CNS in animal models for stroke and Alzheimer's disease and that peripheral cells can be targeted to treat affected regions of the CNS.

  17. Selective retention of bone marrow-derived cells to enhance spinal fusion.

    Science.gov (United States)

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

    2005-03-01

    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.

  18. Bone marrow-derived myofibroblasts recruited to the upper dermis appear beneath regenerating epidermis after deep dermal burn injury.

    Science.gov (United States)

    Yamaguchi, Ryo; Takami, Yoshihiro; Yamaguchi, Yoshihiro; Shimazaki, Syuji

    2007-01-01

    Fibroblasts and myofibroblasts migrating to sites of tissue repair after injury may not only be locally recruited but could also be recruited from the bone marrow. However, the characteristics and functional roles, if any, of these cells in wound healing are poorly understood. Here, we show unequivocally that bone marrow-derived fibroblasts do contribute to deep dermal burn wound healing. Bone-marrow stromal cells were collected from femurs of male Lewis rats, cultured for a week, and then the adherent cells were labeled with the fluorescent marker PKH-26. These cells stained positive for alpha-smooth muscle actin and prolyl 4-hydroxylase, but did not express RM-4 (a macrophage marker), CD34, or cytokeratin, characteristic of myofibroblastic differentiation. When injected intravenously into Lewis rats, they homed to the bone marrow. Five days after transplantation, a deep dermal burn was made on the back of the rat, and biopsies were taken 7, 10, and 14 days later. PKH-positive cells were not found at day 7, but by day 10, they were easily detected mainly in the upper dermis close beneath the regenerating epidermis. These PKH-positive cells still stained for alpha-SMA and prolyl 4-hydroxylase, but not RM4. Thus, it is suggested that myofibroblasts originating in the bone marrow contribute not only to promotion of granulation but also enhancement of dermal-epidermal interaction after thermal injury.

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

    Science.gov (United States)

    Short, Martin A; Campanale, Naomi; Litwak, Sara; Bernard, Claude C A

    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.

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

    Science.gov (United States)

    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 (P0.05). 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 (Pregeneration, although the combined approach seems to be relevant, especially to bone formation out of the implant threads.

  1. Bone Marrow Stem Cell Derived Paracrine Factors for Regenerative Medicine: Current Perspectives and Therapeutic Potential

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    Tom J. Burdon

    2011-01-01

    Full Text Available During the past several years, there has been intense research in the field of bone marrow-derived stem cell (BMSC therapy to facilitate its translation into clinical setting. Although a lot has been accomplished, plenty of challenges lie ahead. Furthermore, there is a growing body of evidence showing that administration of BMSC-derived conditioned media (BMSC-CM can recapitulate the beneficial effects observed after stem cell therapy. BMSCs produce a wide range of cytokines and chemokines that have, until now, shown extensive therapeutic potential. These paracrine mechanisms could be as diverse as stimulating receptor-mediated survival pathways, inducing stem cell homing and differentiation or regulating the anti-inflammatory effects in wounded areas. The current review reflects the rapid shift of interest from BMSC to BMSC-CM to alleviate many logistical and technical issues regarding cell therapy and evaluates its future potential as an effective regenerative therapy.

  2. Mouse bone marrow-derived mesenchymal stem cells inhibit leukemia/lymphoma cell proliferation in vitro and in a mouse model of allogeneic bone marrow transplant.

    Science.gov (United States)

    Song, Ningxia; Gao, Lei; Qiu, Huiying; Huang, Chongmei; Cheng, Hui; Zhou, Hong; Lv, Shuqing; Chen, Li; Wang, Jianmin

    2015-07-01

    The allogeneic hematopoietic stem cell (HSC) transplantation of mesenchymal stem cells (MSCs) contributes to the reconstitution of hematopoiesis by ameliorating acute graft‑versus‑host disease (aGVHD). However, the role of MSCs in graft‑versus‑leukemia remains to be determined. In the present study, we co‑cultured C57BL/6 mouse bone marrow (BM)‑derived MSCs with A20 murine B lymphoma, FBL3 murine erythroleukemia and P388 murine acute lymphocytic leukemia cells. Cell proliferation, apoptosis, cell cycle progression and the amount of cytokine secretion were then measured using a Cell Counting kit‑8, Annexin V/propidium iodide staining, flow cytometry and ELISA, respectively. We also established a model of allogeneic bone marrow transplantation (BMT) using BALB/c mice. Following the administration of A20 cells and MSCs, we recorded the symptoms and the survival of the mice for 4 weeks, assessed the T cell subsets present in peripheral blood, and, after the mice were sacrifice, we determined the infiltration of MSCs into the organs by histological staining. Our results revealed that the MSCs inhibited the proliferation of the mouse lymphoma and leukemia cells in vitro, leading to cell cycle arrest and reducing the secretion of interleukin (IL)‑10. In our model of allogeneic BMT, the intravenous injection of MSCs into the mice injected wth A20 cells decreased the incidence of lymphoma, improved survival, increased the fraction of CD3+CD8+ T cells, decreased the fraction of CD3+CD4+ T cells and CD4+CD25+ T cells in peripheral blood, and ameliorated the manifestation of aGVHD. The results from the present study indicate that MSCs may be safe and effective when used in allogeneic BMT for the treatment of hemotological malignancies.

  3. Reconstruction of the adenosine system by bone marrow-derived mesenchymal stem cell transplantation

    Institute of Scientific and Technical Information of China (English)

    Huicong Kang; Qi Hu; Xiaoyan Liu; Yinhe Liu; Feng Xu; Xiang Li; Suiqiang Zhu

    2012-01-01

    In the present study, we transplanted bone marrow-derived mesenchymal stem cells into the CA3 area of the hippocampus of chronic epilepsy rats kindled by lithium chloride-pilocarpine. Immunofluorescence and western blotting revealed an increase in adenosine A1 receptor expression and a decrease in adenosine A2a receptor expression in the brain tissues of epileptic rats 3 months after transplantation. Moreover, the imbalance in the A1 adenosine receptor/A2a adenosine receptor ratio was improved. Electroencephalograms showed that frequency and amplitude of spikes in the hippocampus and frontal lobe were reduced. These results suggested that mesenchymal stem cell transplantation can reconstruct the normal function of the adenosine system in the brain and greatly improve epileptiform discharges.

  4. Lithium Chloride Modulates Adipogenesis and Osteogenesis of Human Bone Marrow-Derived Mesenchymal Stem Cells

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    Linjun Tang

    2015-08-01

    Full Text Available Background/Aims: Lithium chloride (LiCl has long been used as a psychiatric medication; however, its role in the differentiation of bone marrow-derived mesenchymal stem cells (BMSCs remains largely unknown. The aim of this study is to explore the effect of LiCl on the differentiation of BMSCs. Methods: The roles of LiCl in osteogenic and adipogenic processes were observed using alizarin red staining and oil red O staining, respectively. The effects of LiCl on the Wnt and Hedgehog (Hh pathways were investigated. Results: Our data showed that LiCl effectively promoted osteogenesis and inhibited adipogenesis by simultaneously affecting the Wnt and Hh pathways. Conclusion: These results suggest that LiCl influences the differentiation of BMSCs directly through the Wnt and Hh pathways and thus may be a candidate drug for the treatment of osteoporosis.

  5. Bone Marrow-Derived Mesenchymal Cell Differentiation toward Myogenic Lineages: Facts and Perspectives

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    Daniela Galli

    2014-01-01

    Full Text Available Bone marrow-derived mesenchymal stem cells (BM-MSCs are valuable platforms for new therapies based on regenerative medicine. BM-MSCs era is coming of age since the potential of these cells is increasingly demonstrated. In fact, these cells give origin to osteoblasts, chondroblasts, and adipocyte precursors in vitro, and they can also differentiate versus other mesodermal cell types like skeletal muscle precursors and cardiomyocytes. In our short review, we focus on the more recent manipulations of BM-MSCs toward skeletal and heart muscle differentiation, a growing field of obvious relevance considering the toll of muscle disease (i.e., muscular dystrophies, the heavier toll of heart disease in developed countries, and the still not completely understood mechanisms of muscle differentiation and repair.

  6. Bone marrow derived stem cells in regenerative medicine as advanced therapy medicinal products.

    Science.gov (United States)

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

    2010-05-15

    Bone marrow derived stem cells administered after minimal manipulation represent an important cell source for cell-based 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 cell-based 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 cell-based 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.

  7. EXPRESSION OF rhBMP—7 GENE IN TRANSDUCED BONE MARROW DERIVED STROMAL CELLS

    Institute of Scientific and Technical Information of China (English)

    段德宇; 杜靖远

    2002-01-01

    Objective:To explore the possibility of expression of exogenous gene in transduced bone marrow derived stromal cells(BMSCs).Methods:The marker gene,pbLacZ,was transferred into cultured BMSCs and the expression of transduced gene by x-gal staining was examined.Then plasmid pcDNA3-rhBMP7 was delivered to cultured BMSCs.Through immunohistochemical staining and RT-PCR assay,the expression of rhBMP7 gene was detected.Results:The exogenous gene could be expressed efficiently in transduced BMSCs.Conculsion:The present study provided a theoretical basis to gene therapy on the problems of bone and cartilage tissue.

  8. Artemisinin-derived sesquiterpene lactones as potential antitumour compounds : Cytotoxic action against bone marrow and tumour cells

    NARCIS (Netherlands)

    Beekman, AC; Wierenga, PK; Woerdenbag, HJ; Van Uden, W; Pras, N; Konings, AWT; El-Feraly, FS; Galal, AM; Wikstrom, HV

    1998-01-01

    We determined the in vitro cytotoxic activity of the sesquiterpene lactone endoperoxide artemisinin (1) and some chemically prepared derivatives, which have been found to display cytotoxicity to cloned murine Ehrlich ascites tumour (EAT) cells and human HeLa cells and against murine bone marrow usin

  9. Ciclosporin Does Not Influence Bone Marrow-Derived Cell Differentiation to Myofibroblasts Early after Renal Ischemia/Reperfusion

    NARCIS (Netherlands)

    Broekema, Martine; Harmsen, Martin C.; Koerts, Jasper A.; van Kooten, Theo G.; Uges, Donald R. A.; Petersen, Arjen H.; van Luyn, Marja J. A.; Navis, Gerjan; Popa, Eliane R.

    2009-01-01

    Background: Ischemia/reperfusion injury (IRI) is a risk factor for the development of interstitial fibrosis. Previously we had shown that after renal IRI, bone marrow-derived cells (BMDC) can differentiate to interstitial myofibroblasts. Here we hypothesized that the immunosuppressant ciclosporin A

  10. Autotransplantation of bone marrow-derived stem cells as a therapy for neurodegenerative diseases.

    Science.gov (United States)

    Kan, I; Melamed, E; Offen, D

    2007-01-01

    Neurodegenerative diseases are characterized by a progressive degeneration of selective neural populations. This selective hallmark pathology and the lack of effective treatment modalities make these diseases appropriate candidates for cell therapy. Bone marrow-derived mesenchymal stem cells (MSCs) are self-renewing precursors that reside in the bone marrow and may further be exploited for autologous transplantation. Autologous transplantation of MSCs entirely circumvents the problem of immune rejection, does not cause the formation of teratomas, and raises very few ethical or political concerns. More than a few studies showed that transplantation of MSCs resulted in clinical improvement. However, the exact mechanisms responsible for the beneficial outcome have yet to be defined. Possible rationalizations include cell replacement, trophic factors delivery, and immunomodulation. Cell replacement theory is based on the idea that replacement of degenerated neural cells with alternative functioning cells induces long-lasting clinical improvement. It is reasoned that the transplanted cells survive, integrate into the endogenous neural network, and lead to functional improvement. Trophic factor delivery presents a more practical short-term approach. According to this approach, MSC effectiveness may be credited to the production of neurotrophic factors that support neuronal cell survival, induce endogenous cell proliferation, and promote nerve fiber regeneration at sites of injury. The third potential mechanism of action is supported by the recent reports claiming that neuroinflammatory mechanisms play an important role in the pathogenesis of neurodegenerative disorders. Thus, inhibiting chronic inflammatory stress might explain the beneficial effects induced by MSC transplantation. Here, we assemble evidence that supports each theory and review the latest studies that have placed MSC transplantation into the spotlight of biomedical research.

  11. Bone marrow derived mesenchymal stem cells incorporate into the prostate during regrowth.

    Directory of Open Access Journals (Sweden)

    Veronica R Placencio

    Full Text Available BACKGROUND: Prostate cancer recurrence involves increased growth of cancer epithelial cells, as androgen dependent prostate cancer progresses to castrate resistant prostate cancer (CRPC following initial therapy. Understanding CRPC prostate regrowth will provide opportunities for new cancer therapies to treat advanced disease. METHODOLOGY/PRINCIPAL FINDINGS: Elevated chemokine expression in the prostate stroma of a castrate resistant mouse model, Tgfbr2(fspKO, prompted us to look at the involvement of bone marrow derived cells (BMDCs in prostate regrowth. We identified bone marrow cells recruited to the prostate in GFP-chimeric mice. A dramatic increase in BMDC recruitment for prostate regrowth occurred three days after exogenous testosterone implantation. Recruitment led to incorporation of BMDCs within the prostate epithelia. Immunofluorescence staining suggested BMDCs in the prostate coexpressed androgen receptor; p63, a basal epithelial marker; and cytokeratin 8, a luminal epithelial marker. A subset of the BMDC population, mesenchymal stem cells (MSCs, were specifically found to be incorporated in the prostate at its greatest time of remodeling. Rosa26 expressing MSCs injected into GFP mice supported MSC fusion with resident prostate epithelial cells through co-localization of β-galactosidase and GFP during regrowth. In a human C4-2B xenograft model of CRPC, MSCs were specifically recruited. Injection of GFP-labeled MSCs supported C4-2B tumor progression by potentiating canonical Wnt signaling. The use of MSCs as a targeted delivery vector for the exogenously expressed Wnt antagonist, secreted frizzled related protein-2 (SFRP2, reduced tumor growth, increased apoptosis and potentiated tumor necrosis. CONCLUSIONS/SIGNIFICANCE: Mesenchymal stem cells fuse with prostate epithelia during the process of prostate regrowth. MSCs recruited to the regrowing prostate can be used as a vehicle for transporting genetic information with potential

  12. Human gingiva-derived mesenchymal stem cells are superior to bone marrow-derived mesenchymal stem cells for cell therapy in regenerative medicine

    Energy Technology Data Exchange (ETDEWEB)

    Tomar, Geetanjali B.; Srivastava, Rupesh K.; Gupta, Navita; Barhanpurkar, Amruta P.; Pote, Satish T. [National Center for Cell Science, University of Pune Campus, Pune 411 007 (India); Jhaveri, Hiral M. [Department of Periodontics and Oral Implantology, Dr. D.Y. Patil Dental College and Hospital, Pune (India); Mishra, Gyan C. [National Center for Cell Science, University of Pune Campus, Pune 411 007 (India); Wani, Mohan R., E-mail: mohanwani@nccs.res.in [National Center for Cell Science, University of Pune Campus, Pune 411 007 (India)

    2010-03-12

    Mesenchymal stem cells (MSCs) are capable of self-renewal and differentiation into multiple cell lineages. Presently, bone marrow is considered as a prime source of MSCs; however, there are some drawbacks and limitations in use of these MSCs for cell therapy. In this study, we demonstrate that human gingival tissue-derived MSCs have several advantages over bone marrow-derived MSCs. Gingival MSCs are easy to isolate, homogenous and proliferate faster than bone marrow MSCs without any growth factor. Importantly, gingival MSCs display stable morphology and do not loose MSC characteristic at higher passages. In addition, gingival MSCs maintain normal karyotype and telomerase activity in long-term cultures, and are not tumorigenic. Thus, we reveal that human gingiva is a better source of MSCs than bone marrow, and large number of functionally competent clinical grade MSCs can be generated in short duration for cell therapy in regenerative medicine and tissue engineering.

  13. Toll-Like Receptor 4 in Bone Marrow-Derived Cells Contributes to the Progression of Diabetic Retinopathy

    Directory of Open Access Journals (Sweden)

    Hui Wang

    2014-01-01

    Full Text Available 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.

  14. Selective resistance of bone marrow-derived hemopoietic progenitor cells to gliotoxin

    Energy Technology Data Exchange (ETDEWEB)

    Muellbacher, A.; Hume, D.; Braithwaite, A.W.; Waring, P.; Eichner, R.D.

    1987-06-01

    The fungal metabolite gliotoxin at low concentrations prevents mitogen stimulation of mature lymphocytes as a result of gliotoxin-induced genomic DNA degradation. Bone marrow, on the other hand, contains a subpopulation of cells resistant to gliotoxin at similar concentrations. This population includes the hemopoietic progenitor cells that grow in vitro in response to appropriate colony-stimulating factors and cells that form colonies in the spleens of lethally irradiated recipients. Gliotoxin treatment of lymph node cell-enriched bone marrow significantly delayed the onset of graft-versus-host disease in fully allogeneic bone marrow chimeras.

  15. Perilipin1 deficiency in whole body or bone marrow-derived cells attenuates lesions in atherosclerosis-prone mice.

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    Xiaojing Zhao

    Full Text Available The objective of this study is to determine the role of perilipin 1 (Plin1 in whole body or bone marrow-derived cells on atherogenesis.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.Plin1 deficiency in bone marrow-derived cells may be responsible for reduced atherosclerotic lesions in the mice.

  16. Bone marrow-derived cells contribute to epithelial engraftment during wound healing.

    Science.gov (United States)

    Borue, Xenia; Lee, Sean; Grove, Joanna; Herzog, Erica L; Harris, Robert; Diflo, Thomas; Glusac, Earl; Hyman, Kevin; Theise, Neil D; Krause, Diane S

    2004-11-01

    Recent findings suggest that bone marrow-derived cells (BMDC) may contribute to tissue maintenance throughout the body. However, it is not yet known whether marrow-derived epithelial cells are capable of undergoing proliferation. Our laboratory has shown that BMDC engraft as keratinocytes in the skin at low levels (BMDC as keratinocytes and that the keratinocytes are actively cycling. Female mice reconstituted with sex-mismatched BM were wounded by punch biopsy and incision. At the wound site, engraftment of BMDC as epidermal cells increased within 1 day, and continued to increase to approximately 4% by 3 weeks after injury. Using a Cre-lox system, fusion of BMDC with epithelial cells was ruled out. BMDC-derived epithelial cells at the wound edges expressed Ki67, a marker for actively cycling cells, and this proliferation correlated with an increase in the number of donor-derived cells within the wound. Donor-derived cytokeratin 5-expressing cells were rare, suggesting that BMDC do not engraft as epidermal stem cells, and the level of engraftment peaked and then decreased over time, further suggesting that BMDC may assist in early wound healing by engrafting as transit-amplifying cells, which then differentiate into keratinocytes.

  17. Bone marrow derived cells and alternative pathways of oogenesis in adult rodents.

    Science.gov (United States)

    Bukovsky, Antonin; Ayala, Maria E; Dominguez, Roberto; Svetlikova, Marta; Selleck-White, Rachel

    2007-09-15

    Oocyte generation in adult mouse ovaries by putative germ cells (PGCs) in bone marrow and peripheral blood has recently been proposed. It, however, remains unclear whether in laboratory rodents the PGCs reside in BM or the BM cells stimulate oogenesis from ovarian stem cells. We utilized immunoperoxidase staining to localize PGCs, oocytes, and BM derived cells in ovaries of adult (age 45-60 days) control and neonatally estrogenized rat females. In controls, BM derived cells accompanied emergence of PGCs from the ovarian surface epithelium (OSE) cells. The PGCs divided symmetrically, separated, and formed primordial follicles. A proportion (50%) of adult neonatally estrogenized rats lacked OSE. They exhibited occurrence of numerous BM derived cells and appearance of PGC precursors in the medulla. In juxtaposed deep ovarian cortex the emerging PGCs exhibited distinct pseudopodia and apparently migrated toward the mid cortex, where numerous primordial follicles were found. These observations indicate that BM derived cells accompany origination of PGCs from the OSE stem cells in normal adult rat females and from the medullary precursors in the adult neonatally estrogenized rats lacking OSE. An alternative origin of PGCs from the medullary region may explain why ovaries with destructed OSE are still capable of forming new primordial follicles.

  18. Characterization and Comparison of Canine Multipotent Stromal Cells Derived from Liver and Bone Marrow

    Science.gov (United States)

    Malagola, Ermanno; Teunissen, Michelle; van der Laan, Luc J.W.; Verstegen, Monique M.A.; Schotanus, Baukje A.; van Steenbeek, Frank G.; Penning, Louis C.; van Wolferen, Monique E.; Tryfonidou, Marianna A.

    2016-01-01

    Liver-derived multipotent stromal cells (L-MSCs) may prove preferable for treatment strategies of liver diseases, in comparison to the widely studied bone marrow-derived MSCs (BM-MSCs). Canines are a large animal model, in which the pathologies of liver diseases are similar to man. This study further promotes the implementation of canine models in MSC-based treatments of liver diseases. L-MSCs were characterized and compared to BM-MSCs from the same individual. Both cell types demonstrated a spindle-shaped fibroblast-like morphology, possessed the same growth potential, and demonstrated similar immunomodulation gene expression of CD274, PTGS-1, and PTGS-2. Marked differences in cell surface markers, CD105 and CD146, distinguished these two cell populations, and L-MSCs retained a liver-specific imprinting, observed by expression of CK18 and CK19. Finally, both populations differentiated toward the osteogenic and adipogenic lineage; however, L-MSCs failed to differentiate into the chondrogenic lineage. In conclusion, characterization of canine L-MSCs and BM-MSCs demonstrated that the two cell type populations are highly comparable. Although it is still unclear which cell source is preferred for clinical application in liver treatment strategies, this study provides a foundation for future controlled studies with MSC therapy in various liver diseases in dogs before their application in man. PMID:26462417

  19. Advances of mesenchymal stem cells derived from bone marrow and dental tissue in craniofacial tissue engineering.

    Science.gov (United States)

    Yang, Maobin; Zhang, Hongming; Gangolli, Riddhi

    2014-05-01

    Bone and dental tissues in craniofacial region work as an important aesthetic and functional unit. Reconstruction of craniofacial tissue defects is highly expected to ensure patients to maintain good quality of life. Tissue engineering and regenerative medicine have been developed in the last two decades, and been advanced with the stem cell technology. Bone marrow derived mesenchymal stem cells are one of the most extensively studied post-natal stem cell population, and are widely utilized in cell-based therapy. Dental tissue derived mesenchymal stem cells are a relatively new stem cell population that isolated from various dental tissues. These cells can undergo multilineage differentiation including osteogenic and odontogenic differentiation, thus provide an alternative source of mesenchymal stem cells for tissue engineering. In this review, we discuss the important issues in mesenchymal stem cell biology including the origin and functions of mesenchymal stem cells, compare the properties of these two types of mesenchymal cells, update recent basic research and clinic applications in this field, and address important future challenges.

  20. Bone Marrow-Derived c-kit+ Cells Attenuate Neonatal Hyperoxia-Induced Lung Injury

    Science.gov (United States)

    Ramachandran, Shalini; Suguihara, Cleide; Drummond, Shelley; Chatzistergos, Konstantinos; Klim, Jammie; Torres, Eneida; Huang, Jian; Hehre, Dorothy; Rodrigues, Claudia O.; McNiece, Ian K.; Hare, Joshua M.; Young, Karen C.

    2016-01-01

    Recent studies suggest that bone marrow (BM)-derived stem cells have therapeutic efficacy in neonatal hyperoxia-induced lung injury (HILI). c-kit, a tyrosine kinase receptor that regulates angiogenesis, is expressed on several populations of BM-derived cells. Preterm infants exposed to hyperoxia have decreased lung angiogenesis. Here we tested the hypothesis that administration of BM-derived c-kit+ cells would improve angiogenesis in neonatal rats with HILI. To determine whether intratracheal (IT) administration of BM-derived c-kit+ cells attenuates neonatal HILI, rat pups exposed to either normobaric normoxia (21% O2) or hyperoxia (90% O2) from postnatal day (P) 2 to P15 were randomly assigned to receive either IT BM-derived green fluorescent protein (GFP)+ c-kit− cells (PL) or BM-derived GFP+ c-kit+ cells on P8. The effect of cell therapy on lung angiogenesis, alveolarization, pulmonary hypertension, vascular remodeling, cell proliferation, and apoptosis was determined at P15. Cell engraftment was determined by GFP immunostaining. Compared to PL, the IT administration of BM-derived c-kit+ cells to neonatal rodents with HILI improved alveolarization as evidenced by increased lung septation and decreased mean linear intercept. This was accompanied by an increase in lung vascular density, a decrease in lung apoptosis, and an increase in the secretion of proangiogenic factors. There was no difference in pulmonary vascular remodeling or the degree of pulmonary hypertension. Confocal microscopy demonstrated that 1% of total lung cells were GFP+ cells. IT administration of BM-derived c-kit+ cells improves lung alveolarization and angiogenesis in neonatal HILI, and this may be secondary to an improvement in the lung angiogenic milieu. PMID:23759597

  1. Turnover of bone marrow-derived cells in the irradiated mouse cornea

    Science.gov (United States)

    Chinnery, Holly R; Humphries, Timothy; Clare, Adam; Dixon, Ariane E; Howes, Kristen; Moran, Caitlin B; Scott, Danielle; Zakrzewski, Marianna; Pearlman, Eric; McMenamin, Paul G

    2008-01-01

    In light of an increasing awareness of the presence of bone marrow (BM)-derived macrophages in the normal cornea and their uncertain role in corneal diseases, it is important that the turnover rate of these resident immune cells be established. The baseline density and distribution of macrophages in the corneal stroma was investigated in Cx3cr1gfp transgenic mice in which all monocyte-derived cells express enhanced green fluorescent protein (eGFP). To quantify turnover, BM-derived cells from transgenic eGFP mice were transplanted into whole-body irradiated wild-type recipients. Additionally, wild-type BM-derived cells were injected into irradiated Cx3cr1+/gfp recipients, creating reverse chimeras. At 2, 4 and 8 weeks post-reconstitution, the number of eGFP+ cells in each corneal whole mount was calculated using epifluorescence microscopy, immunofluorescence staining and confocal microscopy. The total density of myeloid-derived cells in the normal Cx3cr1+/gfp cornea was 366 cells/mm2. In BM chimeras 2 weeks post-reconstitution, 24% of the myeloid-derived cells had been replenished and were predominantly located in the anterior stroma. By 8 weeks post-reconstitution 75% of the myeloid-derived cells had been replaced and these cells were distributed uniformly throughout the stroma. All donor eGFP+ cells expressed low to moderate levels of CD45 and CD11b, with approximately 25% coexpressing major histocompatibility complex class II, a phenotype characteristic of previous descriptions of corneal stromal macrophages. In conclusion, 75% of the myeloid-derived cells in the mouse corneal stroma are replenished after 8 weeks. These data provide a strong basis for functional investigations of the role of resident stromal macrophages versus non-haematopoietic cells using BM chimeric mice in models of corneal inflammation. PMID:18540963

  2. Radix Astragali-induced differentiation of rat bone marrow-derived mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Xinsheng Wang; Haifeng Li; Ying Zhao; Xiaoli Zhang; Aihua Bo

    2009-01-01

    BACKGROUND: Chemical induction has been shown to be effective at promoting the differentiation of bone marrow-derived mesenchymal stem cells (MSCs). However, these inductors have cytotoxicity side effects that may damage cells over time. Traditional Chinese medicines avoid this disadvantage while still producing effective induction.OBJECTIVE: To investigate the influence of Radix Astragali (Huangqi) on the differentiation of MSCs.DESIGN, TIME AND SETTING: In vitro study of traditional Chinese medicine in neural stem cell differentiation. The experiment was performed at the Central Laboratory of Hebei North University between April and June 2007.MATERIALS: Radix Astragafi solution (lot No. 060105; license No. Z53021585) was purchased from Daii Pharmaceutical Co., Ltd., China; rabbit anti-rat nestin, rabbit anti-rat neuron-specific enolase (NSE), mouse anti-rat microtubule-associated protein 2, and rabbit anti-rat glial fibrillary acidic protein were purchased from Wuhan Boster, China.METHODS: Whole bone marrow was isolated from the femur and tibia of 6-week-old male Wistar rats and subcultured. The fourth passage of MSCs were harvested and induced by different concentrations (50, 100, 200, 400 g/L) of Radix Astragali.MAIN OUTCOME MEASURES: Hematoxylin-eosin staining was used to observe MSC morphology after 24 hours of induction. Immunocytochemistry was employed to observe the expression of NSE (specific neuronal marker), nestin (marker of neural stem cell), glial fibrillary acidic protein and microtubule-associated protein 2 (markers of astrocytes).RESULTS: Following Radix Astragafi treatment, changes occurred in cell morphology including: cell body pyknosis; thin and long processes formed in some cells, with growth corresponding to drug concentration and induction time; and the formation of network-like connections between some cells.With increasing drug concentration and induction time, nestin expression was upregulated, and the number of positive cells increased

  3. Osteogenic potential: comparison between bone marrow and adipose-derived mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Han-Tsung; Liao; Chien-Tzung; Chen

    2014-01-01

    Bone tissue engineering(BTE) is now a promising re-search issue to improve the drawbacks from traditional bone grafting procedure such as limited donor sources and possible complications. Stem cells are one of the major factors in BTE due to the capability of self re-newal and multi-lineage differentiation. Unlike embry-onic stem cells, which are more controversial in ethical problem, adult mesenchymal stem cells are considered to be a more appropriate cell source for BTE. Bone marrow mesenchymal stem cells(BMSCs) are the ear-liest-discovered and well-known stem cell source using in BTE. However, the low stem cell yield requiring long expansion time in vitro, pain and possible morbidities during bone marrow aspiration and poor proliferation and osteogenic ability at old age impede its’ clinical ap-plication. Afterwards, a new stem cell source coming from adipose tissue, so-called adipose-derived stemcells(ASCs), is found to be more suitable in clinical ap-plication because of high stem cells yield from lipoaspi-rates, faster cell proliferation and less discomfort and morbidities during harvesting procedure. However, the osteogenic capacity of ASCs is now still debated be-cause most papers described the inferior osteogenesis of ASCs than BMSCs. A better understanding of the osteogenic differences between ASCs and BMSCs is crucial for future selection of cells in clinical application for BTE. In this review, we describe the commonality and difference between BMSCs and ASCs by cell yield, cell surface markers and multiple-differentiation poten-tial. Then we compare the osteogenic capacity in vitro and bone regeneration ability in vivo between BMSCs and ASCs based on the literatures which utilized both BMSCs and ASCs simultaneously in their articles. The outcome indicated both BMSCs and ASCs exhibited the osteogenic ability to a certain extent both in-vitro and in-vivo. However, most in-vitro study papers verified the inferior osteogenesis of ASCs; conversely, in

  4. Cranial irradiation induces bone marrow-derived microglia in adult mouse brain tissue.

    Science.gov (United States)

    Okonogi, Noriyuki; Nakamura, Kazuhiro; Suzuki, Yoshiyuki; Suto, Nana; Suzue, Kazutomo; Kaminuma, Takuya; Nakano, Takashi; Hirai, Hirokazu

    2014-07-01

    Postnatal hematopoietic progenitor cells do not contribute to microglial homeostasis in adult mice under normal conditions. However, previous studies using whole-body irradiation and bone marrow (BM) transplantation models have shown that adult BM cells migrate into the brain tissue and differentiate into microglia (BM-derived microglia; BMDM). Here, we investigated whether cranial irradiation alone was sufficient to induce the generation of BMDM in the adult mouse brain. Transgenic mice that express green fluorescent protein (GFP) under the control of a murine stem cell virus (MSCV) promoter (MSCV-GFP mice) were used. MSCV-GFP mice express GFP in BM cells but not in the resident microglia in the brain. Therefore, these mice allowed us to detect BM-derived cells in the brain without BM reconstitution. MSCV-GFP mice, aged 8-12 weeks, received 13.0 Gy irradiation only to the cranium, and BM-derived cells in the brain were quantified at 3 and 8 weeks after irradiation. No BM-derived cells were detected in control non-irradiated MSCV-GFP mouse brains, but numerous GFP-labeled BM-derived cells were present in the brain stem, basal ganglia and cerebral cortex of the irradiated MSCV-GFP mice. These BM-derived cells were positive for Iba1, a marker for microglia, indicating that GFP-positive BM-derived cells were microglial in nature. The population of BMDM was significantly greater at 8 weeks post-irradiation than at 3 weeks post-irradiation in all brain regions examined. Our results clearly show that cranial irradiation alone is sufficient to induce the generation of BMDM in the adult mouse.

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

    Science.gov (United States)

    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.

  6. Effects of murine and human bone marrow-derived mesenchymal stem cells on cuprizone induced demyelination.

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    Jasmin Nessler

    Full Text Available For the treatment of patients with multiple sclerosis there are no regenerative approaches to enhance remyelination. Mesenchymal stem cells (MSC have been proposed to exert such regenerative functions. Intravenous administration of human MSC reduced the clinical severity of experimental autoimmune encephalomyelitis (EAE, an animal model mimicking some aspects of multiple sclerosis. However, it is not clear if this effect was achieved by systemic immunomodulation or if there is an active neuroregeneration in the central nervous system (CNS. In order to investigate remyelination and regeneration in the CNS we analysed the effects of intravenously and intranasally applied murine and human bone marrow-derived MSC on cuprizone induced demyelination, a toxic animal model which allows analysis of remyelination without the influence of the peripheral immune system. In contrast to EAE no effects of MSC on de- and remyelination and glial cell reactions were found. In addition, neither murine nor human MSC entered the lesions in the CNS in this toxic model. In conclusion, MSC are not directed into CNS lesions in the cuprizone model where the blood-brain-barrier is intact and thus cannot provide support for regenerative processes.

  7. Targeting gallbladder carcinoma: bone marrow-derived stem cells as therapeutic delivery vehicles of myxoma virus

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    Weng Mingzhe; Zhang Mingdi; Qin Yiyu; Gong Wei; Tang Zhaohui; Quan Zhiwei; Wu Kejin

    2014-01-01

    Background Gallbladder carcinoma (GBC) has a high mortality rate,requiring synergistic anti-tumor management for effective treatment.The myxoma virus (MYXV) exhibits a modest clinical value through its oncolytic potential and narrow host tropism.Methods We performed viral replication assays,cell viability assays,migration assays,and xenograft tumor models to demonstrate that bone marrow-derived stem cells (BMSCs) may enhance efficiency of intravenous MYXV delivery.Results We examined the permissiveness of various GBC cell lines towards MYXV infection and found two supported single and multiple rounds of MYXV replication,leading to an oncolytic effect.Furthermore,we found that BMSCs exhibited tropism for GBC cells within a Matrigel migration system.BMSCs failed to affect the growth of GBC cells,in terms of tumor volume and survival time.Finally,we demonstrated in vivo that intravenous injection of MYXV-infected BMSCs significantly improves the oncolytic effect of MYXV alone,almost to the same extent as intratumoral injection of MYXV.Conclusion This study indicates that BMSCs are a promising novel vehicle for MYXV to clinically address gallbladder tumors.

  8. Comparison of immature and mature bone marrow-derived dendritic cells by atomic force microscopy

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    Xing, Feiyue; Wang, Jiongkun; Hu, Mingqian; Yu, Yu; Chen, Guoliang; Liu, Jing

    2011-07-01

    A comparative study of immature and mature bone marrow-derived dendritic cells (BMDCs) was first performed through an atomic force microscope (AFM) to clarify differences of their nanostructure and adhesion force. AFM images revealed that the immature BMDCs treated by granulocyte macrophage-colony stimulating factor plus IL-4 mainly appeared round with smooth surface, whereas the mature BMDCs induced by lipopolysaccharide displayed an irregular shape with numerous pseudopodia or lamellapodia and ruffles on the cell membrane besides becoming larger, flatter, and longer. AFM quantitative analysis further showed that the surface roughness of the mature BMDCs greatly increased and that the adhesion force of them was fourfold more than that of the immature BMDCs. The nano-features of the mature BMDCs were supported by a high level of IL-12 produced from the mature BMDCs and high expression of MHC-II on the surface of them. These findings provide a new insight into the nanostructure of the immature and mature BMDCs.

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

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    Eren Çerman

    Full Text Available 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.

  10. Isolation of Murine Bone Marrow Derived Mesenchymal Stem Cells using Twist2 Cre Transgenic Mice

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    Liu, Yaling; Wang, Liping; Fatahi, Reza; Kronenberg, Mark; Kalajzic, Ivo; Rowe, David; Li, Yingcui; Maye, Peter

    2010-01-01

    While human bone marrow derived mesenchymal stem cells (BMSCs) are of great interest for their potential therapeutic value, its murine equivalent remains an important basic research model that can provide critical insights into the biology of this progenitor cell population. Here we present a novel transgenic strategy that allowed for the selective identification and isolation of murine BMSCs at the early stages of stromal cell culture. This strategy involved crossing Twist2 –Cre mice with Cre reporter mice such as Z/EG or Ai9, which express EGFP or Tomato fluorescent protein, respectively, upon Cre mediated excision of a stop sequence. Using this approach, we identified an adherent fluorescent protein+ cell population (T2C+) that is present during the earliest stages of colony formation and by day 5 of culture represents ~20% of the total cell population. Cell surface profiling by flow cytometry showed that T2C+ cells are highly positive for SCA1 and CD29 and negative for CD45, CD117, TIE2, and TER119. Isolation of T2C+ cells by FACS selected for a cell population with skeletal potential that can be directed to differentiate into osteoblasts, adipocytes, or chondrocytes. We also demonstrated in a calvarial bone defect model that T2C+ cells retain a strong efficacy for osteogenic repair and can support a hematopoietic environment. Collectively, these studies provide evidence that the Twist2-Cre x Cre reporter breeding strategy can be used to positively identify and isolate multipotent murine BMSCs. PMID:20673822

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

    Science.gov (United States)

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

    2015-09-01

    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.

  12. Multiple Tumor Types May Originate from Bone Marrow-Derived Cells

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    Chunfang Liu

    2006-09-01

    Full Text Available 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.

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

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    Paweł Czubak

    2014-01-01

    Full Text Available 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.

  14. Extracellular Vesicles Derived from Osteogenically Induced Human Bone Marrow Mesenchymal Stem Cells Can Modulate Lineage Commitment

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    Margarida Martins

    2016-03-01

    Full Text Available The effective osteogenic commitment of human bone marrow mesenchymal stem cells (hBMSCs is critical for bone regenerative therapies. Extracellular vesicles (EVs derived from hBMSCs have a regenerative potential that has been increasingly recognized. Herein, the osteoinductive potential of osteogenically induced hBMSC-EVs was examined. hBMSCs secreted negatively charged nanosized vesicles (∼35 nm with EV-related surface markers. The yield of EVs over 7 days was dependent on an osteogenic stimulus (standard chemical cocktail or RUNX2 cationic-lipid transfection. These EVs were used to sequentially stimulate homotypic uncommitted cells during 7 days, matching the seeding density of EV parent cells, culture time, and stimuli. Osteogenically committed hBMSC-EVs induced an osteogenic phenotype characterized by marked early induction of BMP2, SP7, SPP1, BGLAP/IBSP, and alkaline phosphatase. Both EV groups outperformed the currently used osteoinductive strategies. These data show that naturally secreted EVs can guide the osteogenic commitment of hBMSCs in the absence of other chemical or genetic osteoinductors.

  15. Effects of intermittent negative pressure on osteogenesis in human bone marrow-derived stroma cells

    Institute of Scientific and Technical Information of China (English)

    Zhi YANG; Miao LIU; Yin-gang ZHANG; Xiong GUO; Peng XU

    2009-01-01

    Objective: We investigated the effects of intermittent negative pressure on osteogenesis in human bone marrow-derived stroma cells (BMSCs) in vitro. Methods: BMSCs were isolated from adult marrow donated by a hip osteoarthritis patient with prosthetic replacement and cultured in vitro. The third passage cells were divided into negative pressure treatment group and control group. The treatment group was induced by negative pressure intermittently (pressure: 50 kPa, 30 rain/times, and twice daily). The control was cultured in conventional condition. The osteogenesis of BMSCs was examined by phase-contrast mi-croscopy, the determination of alkaline phosphatase (ALP) activities, and the immunohistochemistry of collagen type I. The mRNA expressions of osteoprotegerin (OPG) and osteoprotegerin ligand (OPGL) in BMSCs were analyzed by real-time poly-merase chain reaction (PCR). Results: BMSCs showed a typical appearance of osteoblast after 2 weeks of induction by intermit-tent negative pressure, the activity of ALP increased significantly, and the expression of collagen type 1 was positive. In the treatment group, the mRNA expression of OPG increased significantly (P<0.05) and the mRNA expression of OPGL decreased significantly (P<0.05) after 2 weeks, compared with the control. Conclusion: Intermittent negative pressure could promote os-teogenesis in human BMSCs in vitro.

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

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    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

  17. Effect of Different Titanium Surfaces on Maturation of Murine Bone Marrow-Derived Dendritic Cells

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    Zheng, Xiaofei; Zhou, Fengjuan; Gu, Yifei; Duan, Xiaobo; Mo, Anchun

    2017-01-01

    Dendritic cells (DCs) play a pivotal role in the host response to implanted biomaterials. Osseointegration of titanium (Ti) implant is an immunological and inflammatory-driven process. However, the role of DCs in this complex process is largely unknown. This study aimed to investigate the effect of different Ti surfaces on DC maturation, and evaluate its subsequent potential on osteogenic differentiation of preosteoblasts. Murine bone marrow-derived DCs were seeded on Ti disks with different surface treatments, including pretreatment (PT), sandblasted/acid-etched (SLA) and modified SLA (modSLA) surface. Compared with DCs cultured on PT and SLA surfaces, the cells seeded on modSLA surface demonstrated a more round morphology with lower expression of CD86 and MHC-II, the DC maturation markers. Those cells also secreted high levels of anti-inflammatory cytokine IL-10 and TGF-β. Notably, addition of conditioned medium (CM) from modSLA-induced DCs significantly increased the mRNA expression of Runx2 and ALP as well as ALP activity by murine preosteoblast MC3T3-E1 cells. Our data demonstrated that Ti disks with different surfaces lead to differential DCs responses. PT and SLA surfaces induce DCs mature, while DCs seeded on modSLA-Ti surface maintain an immature phenotype and exhibit a potential of promoting osteogenic differentiation of MC3T3-E1 cells. PMID:28157196

  18. Burkholderia pseudomallei enhances maturation of bone marrow-derived dendritic cells.

    Science.gov (United States)

    Williams, Natasha L; Kloeze, Eveline; Govan, Brenda L; Körner, Heinrich; Ketheesan, Natkunam

    2008-12-01

    T-cell activation is essential for protection against Burkholderia pseudomallei infection. Using bone marrow-derived dendritic cells (BMDC) isolated from partially resistant C57BL/6 and susceptible BALB/c mice, the degree of BMDC activation in the presence of B. pseudomallei was investigated. Maturation, cytokine production and internalization of B. pseudomallei by BMDC was assessed in response to infection with a highly virulent and a low-virulent clinical isolate. Maturation was determined by identifying the up-regulation of cell-surface markers CD11c and CD86. IL-1beta and IL-12p40 expression were assessed by reverse-transcriptase PCR. The uptake of B. pseudomallei by BMDC was measured using an internalization assay. This study demonstrated that B. pseudomallei isolates stimulate the maturation of BMDC to the same degree regardless of virulence. However, maturation of BMDC was significantly increased in BALB/c mice compared with C57BL/6 mice. Additionally, the uptake of B. pseudomallei by BMDC was significantly greater with the highly virulent isolate compared with the low-virulent isolate. Expression of IL-12 and IL-1beta following infection with B. pseudomallei was up-regulated. The differences observed may have implications in the development of an effective immune response to B. pseudomallei.

  19. Repeated Administration of Bone Marrow-Derived Cells Prevents Disease Progression in Experimental Silicosis

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    Miquéias Lopes-Pacheco

    2013-12-01

    Full Text Available Background/Aims: Bone marrow-derived cells (BMDCs reduced mechanical and histologic changes in the lung in a murine model of silicosis, but these beneficial effects did not persist in the course of lung injury. We hypothesized that repeated administration of BMDCs may decrease lung inflammation and remodeling thus preventing disease progression. Methods: One hundred and two C57BL/6 mice were randomly divided into SIL (silica, 20 mg intratracheally [IT] and control (C groups (saline, IT. C and SIL groups were further randomized to receive BMDCs (2×106 cells or saline IT 15 and 30 days after the start of the protocol. Results: By day 60, BMDCs had decreased the fractional area of granuloma and the number of polymorphonuclear cells, macrophages (total and M1 phenotype, apoptotic cells, the level of transforming growth factor (TGF-β‚ and types I and III collagen fiber content in the granuloma. In the alveolar septa, BMDCs reduced the amount of collagen and elastic fibers, TGF-β, and the number of M1 and apoptotic cells. Furthermore, interleukin (IL-1β, IL-1R1, caspase-3 mRNA levels decreased and the level of IL-1RN mRNA increased. Lung mechanics improved after BMDC therapy. The presence of male donor cells in lung tissue was not observed using detection of Y chromosome DNA. Conclusion: repeated administration of BMDCs reduced inflammation, fibrogenesis, and elastogenesis, thus improving lung mechanics through the release of paracrine factors.

  20. Rapamycin Modulates the Maturation of Rat Bone Marrow-derived Dendritic Cells

    Institute of Scientific and Technical Information of China (English)

    Yingjun DING; Xiang CHENG; Tingting TANG; Rui YAO; Yong CHEN; Jiangjiao XIE; Xian YU; Yuhua LIAO

    2008-01-01

    The purpose of the study was to observe the effect of rapamycin (RAPA) on the differentiation and maturation of rat bone marrow-derived dendritic cells (BMDCs) in vitro. BMDCs from Wistar rats were cultured with granulocyte-macrophage colony-stimulating factor plus interleukin-4in the presence or absence of RAPA (20 ng/mL), and stimulated with lipopolysaccharide (LPS) for 24h before cells and supernatants were collected. Surface phenotype of BMDCs was flow-cytometrically detected to determine the expression of maturation markers, MHC class Ⅱ and CD86. Supematants were analyzed for the production of IL-12 and IFN-γ cytokines by using ELISA.BMDCs were co-cultured with T cells from Lewis rats and mixed lymphocyte reaction was assessed by MTT method. The morphology of BMDCs stimulated with LPS remained immature after RAPA pretreatment. RAPA significantly decreased the CD86 expression, impaired the IL-12 and IFN-γproduction of BMDCs stimulated with LPS, and inhibited the proliferation of allogeneic T cells. In conclusion, RAPA can inhibit the maturation of BMDCs stimulated with LPS in terms of the morphology, surface phenotype, cytokine production, and ability of BMDCs to stimulate the proliferation of allogeneic T cells in vitro.

  1. Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow.

    Science.gov (United States)

    Dong, Yifei; Arif, Arif A; Poon, Grace F T; Hardman, Blair; Dosanjh, Manisha; Johnson, Pauline

    2016-06-25

    Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

  2. OPTIMIZATION OF ELECTROPORATION PARAMETERS FOR TRANSFECTION OF PLASMID DNA INTO MURINE BONE MARROW-DERIVED DENDRITIC CELL

    Institute of Scientific and Technical Information of China (English)

    KE Shan; CHEN Xue-hua; LI Hao; LI Jian-fang; GU Qin-long; ZHU Zheng-gang; LIU Bing-ya

    2006-01-01

    Objective To explore the optimal electroporation parameters for transfection of plasmid DNA into murine bone marrow-derived dendritic cells. Methods Murine bone marrow-derived dendritic cells (DCs) were electroporated with plasmid DNA in varied conditions, such as electrical voltage, pulse time,pre-electroporation cell condition and serum concentration in electrical buffer. Inverted fluorescence microscope and flow cytometer were used to determine the transfection efficiency. Some of the DCs genetically modified under different conditions were stained with trypan-blue and its viability was observed microscopically 48h after electroporation. Results Highest transfection efficiency (22.10%) could be reached when electrical voltage was 250V and pulse time was 20ms. Refreshing the culture medium pre-electroporation may help the cells recover more easily from gene transfer.Besides, electrical buffer containing serum may benefit the viability of DC after electroporation and temperature may has little influence on transfection efficiency. Conclusion Our observations demonstrated plasmid DNA could be efficiently transferred into murine bone marrow-derived DCs by electroporation. These data may helpful for cancer research related to murine DC transfection.

  3. Conditioned medium from hypoxic bone marrow-derived mesenchymal stem cells enhances wound healing in mice.

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    Lei Chen

    Full Text Available Growing evidence indicates that bone marrow-derived mesenchymal stem cells (BM-MSCs enhance wound repair via paracrine. Because the extent of environmental oxygenation affects the innate characteristics of BM-MSCs, including their stemness and migration capacity, the current study set out to elucidate and compare the impact of normoxic and hypoxic cell-culture conditions on the expression and secretion of BM-MSC-derived paracrine molecules (e.g., cytokines, growth factors and chemokines that hypothetically contribute to cutaneous wound healing in vivo. Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR and enzyme-linked immunosorbent assay (ELISA analyses of normoxic and hypoxic BM-MSCs and their conditioned medium fractions showed that the stem cells expressed and secreted significantly higher amounts of basic fibroblast growth factor (bFGF,vascular endothelial growth factor A (VEGF-A interleukin 6 (IL-6 and interleukin 8 (IL-8 under hypoxic conditions. Moreover, hypoxic BM-MSC-derived conditioned medium (hypoCM vs. normoxic BM-MSC-derived conditioned medium (norCM or vehicle control medium significantly enhanced the proliferation of keratinocytes, fibroblasts and endothelial cells, the migration of keratinocytes, fibroblasts, endothelial cells and monocytes, and the formation of tubular structures by endothelial cells cultured on Matrigel matrix. Consistent with these in vitro results, skin wound contraction was significantly accelerated in Balb/c nude mice treated with topical hypoCM relative to norCM or the vehicle control. Notably increased in vivo cell proliferation, neovascularization as well as recruitment of inflammatory macrophages and evidently decreased collagen I, and collagen III were also found in the hypoCM-treated group. These findings suggest that BM-MSCs promote murine skin wound healing via hypoxia-enhanced paracrine.

  4. Use of bone marrow derived stem cells in a fracture non-union

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    Binod C. Raulo

    2012-01-01

    Full Text Available This is an attempt of using in vitro cultured mesenchymal stem cells (MSCs from bone marrow in joining of a fracture non-union. Bone marrow cells were obtained and differentially centrifuged for MSCs that were grown in vitro in mesenchymal stem cell basal medium aseptically, for 10 d. The cell mass was injected around the fracture non-union. Healthy conditions of development of tissue regeneration at the trauma site and due bone joining were recorded. It is concluded that in vitro cultured MSCs had a blithesome effect on the fracture non-union.

  5. Use of bone marrow derived stem cells in a fracture non-union

    Institute of Scientific and Technical Information of China (English)

    Binod C Raulo; Chidananda Dash; Shakti Rath; Sukumar Chakrabarty; Padmanav Rautray; Jagannath Sahoo; Rabindra N Padhy

    2012-01-01

    This is an attempt of using in vitro cultured mesenchymal stem cells (MSCs) from bone marrow in joining of a fracture non-union. Bone marrow cells were obtained and differentially centrifuged for MSCs that were grown in vitro in mesenchymal stem cell basal medium aseptically, for 10 d. The cell mass was injected around the fracture non-union. Healthy conditions of development of tissue regeneration at the trauma site and due bone joining were recorded. It is concluded that in vitro cultured MSCs had a blithesome effect on the fracture non-union.

  6. Differentiation of bone marrow derived Thy-1+β2M-cells into hepatocytes induced by coculture with transgenic CFSCs

    Institute of Scientific and Technical Information of China (English)

    WANG Yunfang; NAN Xue; ZHANG Rui; LI Yanhua; YUE Wen; YAN Fang; PEI Xuetao

    2004-01-01

    Studies of transplantation in vivo indicted that bone marrow derived stem cells had a potential to differentiate into mature hepatocytes. However, there are lots of doubts and uncertainties in the influencing factors and control agents of effectively inducing stem cell differentiation in vitro, the efficiency of stem cells' differentiation into hepatocytes and differentiated cells' life-span and functional state,etc. In this study, rat bone marrow derived Thy-1+β2M- cells (BDTCs) were induced to differentiate into hepatocytes by co-culturing with CFSC/HGF feeder layers which expressed hHGF efficiently and stably. RT-PCR and immunofluorescent texts proved induced BDTCs expressed infant and adult hepatocyte specific genes. Further more, these cells displayed functions of indocyanine green (ICG) uptake, ammonium metabolism and albumin production. It was shown that growth factors together with hepatic nonparenchyma cells provided a feasible microenvironment for differentiation of bone marrow stem cells into hepatocytes. The studies not only provided a significant biological model for going deep into the mechanism of stem cell plasticity, but also offered a theoretical and technical foundation of gene and stem cell engineering-based regenerative medicine for end-stage liver diseases.

  7. Effects of bone marrow-derived endothelial progenitor cell transplantation on vein microenvironment in a rat model of chronic thrombosis

    Institute of Scientific and Technical Information of China (English)

    LI Xiao-qiang; MENG Qing-you; WU Hao-rong

    2007-01-01

    Background Endothelial progenitor cells(EPCs) have been used in both experimental studies and clinical treatments of limb ischemia,as well as in the construction of engineered vascular tissue.The objective of this study was to investigate the effects of transplanted bone marrow-derived EPCs on the vein microenvironment in a rat model of chronic vein thrombosis.Methods Mononuclear cells were isolated from the bone marrow of immature rats by density gradient centrifugation,cultured,and then transplanted into experimentally induced thrombi into inferior vena cava through the femoral vein.Vascular endothelial growth factor(VEGF),angiopoietin-1(ANG-1) and monocyte chemotactic protein-1(MCP-1) mRNA and protein expression levels were measured by real-time quantitative polymerase chain reaction and Western blotting of thrombi and adjacent caval walls 28 days post-transplantation.Results Levels of VEGF,ANG-1,and MCP-1 mRNA in EPC-transplanted thrombi were 100%,230.7%,and 212.5% of levels detected in the sham-operated group(P<0.01),and 99.9%,215.4%,and 177.8% of levels detected in the experimental control group(P<0.01).VEGF,ANG-1 and MCP-1 protein levels exhibited a similar trend.Conclusions Transplanted bone marrow-derived EPCs appear to alter the vein microenvironment in experimentally induced chronic vein thrombosis by upregulating cytokines associated with thrombic organization and recanalization.

  8. Migration and Differentiation of GFP-transplanted Bone Marrow-derived Cells into Experimentally Induced Periodontal Polyp in Mice.

    Science.gov (United States)

    Matsuda, Saeka; Shoumura, Masahito; Osuga, Naoto; Tsujigiwa, Hidetsugu; Nakano, Keisuke; Okafuji, Norimasa; Ochiai, Takanaga; Hasegawa, Hiromasa; Kawakami, Toshiyuki

    2016-01-01

    Perforation of floor of the dental pulp is often encountered during root canal treatment in routine clinical practice of dental caries. If perforation were large, granulation tissue would grow to form periodontal polyp. Granulation tissue consists of proliferating cells however their origin is not clear. It was shown that the cells in granulation tissue are mainly from migration of undifferentiated mesenchymal cells of the bone marrow. Hence, this study utilized GFP bone marrow transplantation mouse model. The floor of the pulp chamber in maxillary first molar was perforated using ½ dental round bur. Morphological assessment was carried out by micro CT and microscopy and GFP cell mechanism was further assessed by immunohistochemistry using double fluorescent staining with GFP-S100A4; GFP-Runx2 and GFP-CD31. Results of micro CT revealed alveolar bone resorption and widening of periodontal ligament. Histopathological examination showed proliferation of fibroblasts with some round cells and blood vessels in the granulation tissue. At 2 weeks, the outermost layer of the granulation tissue was lined by squamous cells with distinct intercellular bridges. At 4 weeks, the granulation tissue became larger than the perforation and the outermost layer was lined by relatively typical stratified squamous epithelium. Double immunofluorescent staining of GFP and Runx2 revealed that both proteins were expressed in spindle-shaped cells. Double immunofluorescent staining of GFP and CD31 revealed that both proteins were expressed in vascular endothelial cells in morphologically distinct vessels. The results suggest that fibroblasts, periodontal ligament fibroblasts and blood vessels in granulation tissue were derived from transplanted-bone marrow cells. Thus, essential growth of granulation tissue in periodontal polyp was caused by the migration of undifferentiated mesenchymal cells derived from bone marrow, which differentiated into fibroblasts and later on differentiated into

  9. Impaired function of bone marrow-derived endothelial progenitor cells in murine liver fibrosis.

    Science.gov (United States)

    Shirakura, Katsuya; Masuda, Haruchika; Kwon, Sang-Mo; Obi, Syotaro; Ito, Rie; Shizuno, Tomoko; Kurihara, Yusuke; Mine, Tetsuya; Asahara, Takayuki

    2011-01-01

    Liver fibrosis (LF) caused by chronic liver damage has been considered as an irreversible disease. As alternative therapy for liver transplantation, there are high expectations for regenerative medicine of the liver. Bone marrow (BM)- or peripheral blood-derived stem cells, including endothelial progenitor cells (EPCs), have recently been used to treat liver cirrhosis. We investigated the biology of BM-derived EPC in a mouse model of LF. C57BL/6J mice were subcutaneously injected with carbon tetrachloride (CCl(4)) every 3 days for 90 days. Sacrificed 2 days after final injection, whole blood (WB) was collected for isolation of mononuclear cells (MNCs) and biochemical examination. Assessments of EPC in the peripheral blood and BM were performed by flow cytometry and EPC colony-forming assay, respectively, using purified MNCs and BM c-KIT(+), Sca-1(+), and Lin(-) (KSL) cells. Liver tissues underwent histological analysis with hematoxylin/eosin/Azan staining, and spleens were excised and weighed. CCl(4)-treated mice exhibited histologically bridging fibrosis, pseudolobular formation, and splenomegaly, indicating successful induction of LF. The frequency of definitive EPC-colony-forming-units (CFU) as well as total EPC-CFU at the equivalent cell number of 500 BM-KSL cells decreased significantly (p changes in primitive EPC-CFU occurred in LF mice. The frequency of WB-MNCs of definitive EPC-CFU decreased significantly (p < 0.01) in LF mice compared with control mice. Together, these findings indicated the existence of impaired EPC function and differentiation in BM-derived EPCs in LF mice and might be related to clinical LF.

  10. Targeting eradication of malignant cells derived from human bone marrow mesenchymal stromal cells

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Yingbin [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); School of Life Science, Southwest University, Chongqing 400715 (China); Cai, Shaoxi, E-mail: sxcai@cqu.edu.cn [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); Yang, Li [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); College of Pharmacy, Jinan University, Guangzhou 510632 (China); Yu, Shuhui [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); Library of Southwest University, Chongqing 400715 (China); Jiang, Jiahuan; Yan, Xiaoqing [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); Zhang, Haoxing [School of Life Science, Southwest University, Chongqing 400715 (China); Liu, Lan [Department of Laboratory of Medicine, Children' s Hospital of Chongqin Medical University, Chongqing 400014 (China); Liu, Qun [College of Life Science and Technology, Southwest University for Nationalities, Chengdu 610041 (China); Du, Jun [Center of Microbiology, Biochemistry, and Pharmacology, School of Pharmaceutical Science, Sun Yat-Sen University, Guangzhou 510080 (China); Cai, Shaohui [College of Pharmacy, Jinan University, Guangzhou 510632 (China); Sung, K.L. Paul [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); Departments of Orthopaedic Surgery and Bioengineering, University of California, SD 0412 (United States)

    2010-12-10

    Human bone marrow mesenchymal stromal cells (hBMSC) have been shown to participate in malignant transformation. However, hampered by the low frequency of malignant transformation of hBMSC, we do not yet know how to prevent malignant transformation of implanted hBMSC. In this study, in order to establish a model for the eradication of hBMSC-derived malignant cells, a gene fusion consisting of a human telomerase (hTERT) promoter modified with both c-Myc and myeloid zinc finger protein2 (MZF-2) binding elements and followed by the E. coli cytosine deaminase (CD) and luciferase genes was stably transferred into hBMSC via lentiviral transduction; n-phosphonacelyl-L-aspartic acid (PALA) selection was used to generate malignant cell colonies derived from transduced hBMSC after treatment with the carcinogenic reagent BPDE. Cells that were amplified after PALA selection were used for transplantation and 5-FC pro-drug cytotoxicity tests. The results showed that PALA-resistant malignant cells could be generated from hBMSC co-induced with lentiviral transduction and treatment with Benzo(a)pyrene Diol Epoxide (BPDE); the modification of c-Myc and MZF-2 binding elements could remarkably enhance the transcriptional activities of the hTERT promoter in malignant cells, whereas transcriptional activity was depressed in normal hBMSC; malignant cells stably expressing CD under the control of the modified hTERT promoter could be eliminated by 5-FC administration. This study has provided a method for targeted eradication of malignant cells derived from hBMSC.

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

    Science.gov (United States)

    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 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.

  12. Cell viability and dopamine secretion of 6-hydroxydopamine-treated PC12 cells co-cultured with bone marrow-derived mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Yue Tang; Yongchun Cui; Fuliang Luo; Xiaopeng Liu; Xiaojuan Wang; Aili Wu; Junwei Zhao; Zhong Tian; Like Wu

    2012-01-01

    In the present study, PC12 cells induced by 6-hydroxydopamine as a model of Parkinson's Disease, were used to investigate the protective effects of bone marrow-derived mesenchymal stem cells bone marrow-derived mesenchymal stem cells against 6-hydroxydopamine-induced neurotoxicity and to verify whether the mechanism of action relates to abnormal α-synuclein accumulation in cells. Results showed that co-culture with bone marrow-derived mesenchymal stem cells enhanced PC12 cell viability and dopamine secretion in a cell dose-dependent manner. MitoLight staining was used to confirm that PC12 cells co-cultured with bone marrow-derived mesenchymal stem cells demonstrate reduced levels of cell apoptosis. Immunocytochemistry and western blot analysis found the quantity of α-synuclein accumulation was significantly reduced in PC12 cell and bone marrow-derived mesenchymal stem cell co-cultures. These results indicate that bone marrow-derived mesenchymal stem cells can attenuate 6-hydroxydopamine-induced cytotoxicity by reducing abnormal α-synuclein accumulation in PC12 cells.

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

    Science.gov (United States)

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

    2011-01-01

    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 18Gy-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.

  14. Cloning of two adenosine receptor subtypes from mouse bone marrow-derived mast cells.

    Science.gov (United States)

    Marquardt, D L; Walker, L L; Heinemann, S

    1994-05-01

    Adenosine potentiates the stimulated release of mast cell mediators. Pharmacologic studies suggest the presence of two adenosine receptors, one positively coupled to adenylate cyclase and the other coupled to phospholipase C activation. To identify mast cell adenosine receptor subtypes, cDNAs for the A1 and A2a adenosine receptors were obtained by screening a mouse brain cDNA library with the use of PCR-derived probes. Mouse bone marrow-derived mast cell cDNA libraries were constructed and screened with the use of A1 and A2a cDNA probes, which revealed the presence of A2a, but not A1, receptor clones. A putative A2b receptor was identified by using low stringency mast cell library screening. Northern blotting of mast cell poly(A)+ RNA with the use of receptor subtype probes labeled single mRNA bands of 2.4 kb and 1.8 kb for the A2a and A2b receptors, respectively. In situ cells. An A2a receptor-specific agonist failed to enhance mast cell mediator release, which suggests that the secretory process is modulated through the A2b and/or another receptor subtype. By using RNase protection assays, we found that mast cells that had been cultured in the presence of N-ethylcarboxamidoadenosine for 24 h exhibited a decrease in both A2a and A2b receptor RNA levels. Cells that had been cultured for 1 to 2 days in the presence of dexamethasone demonstrated increased amounts of A2a receptor mRNA, but no identifiable change in A2b receptor mRNA. Mast cells possess at least two adenosine receptor subtypes that may be differentially regulated.

  15. BST2 Mediates Osteoblast Differentiation via the BMP2 Signaling Pathway in Human Alveolar-Derived Bone Marrow Stromal Cells.

    Science.gov (United States)

    Yoo, Su-Hyang; Kim, Jae Goo; Kim, Beom-Su; Lee, Jun; Pi, Sung-Hee; Lim, Hyun-Dae; Shin, Hong-In; Cho, Eui-Sic; You, Hyung-Keun

    2016-01-01

    The molecular mechanisms controlling the differentiation of bone marrow stromal stem cells into osteoblasts remain largely unknown. In this study, we investigated whether bone marrow stromal antigen 2 (BST2) influences differentiation toward the osteoblasts lineage. BST2 mRNA expression in human alveolar-derived bone marrow stromal cells (hAD-BMSCs) increased during differentiation into osteoblasts. hAD-BMSCs differentiation into osteoblasts and the mRNA expression of the bone-specific markers alkaline phosphatase, collagen type α 1, bone sialoprotein, osteocalcin, and osterix were reduced by BST2 knockdown using siRNA. Furthermore, BST2 knockdown in hAD-BMSCs resulted in decreased RUNX2 mRNA and protein expression. We hypothesized that BST2 is involved in differentiation of into osteoblasts via the BMP2 signaling pathway. Accordingly, we evaluated the mRNA expression levels of BMP2, BMP receptors (BMPR1 and 2), and the downstream signaling molecules SMAD1, SMAD4, and p-SMAD1/5/8 in BST2 knockdown cells. BMP2 expression following the induction of differentiation was significantly lower in BST2 knockdown cells than in cells treated with a non-targeting control siRNA. Similar results were found for the knockdown of the BMP2 receptor- BMPR1A. We also identified significantly lower expression of SMAD1, SMAD4, and p-SMAD1/5/8 in the BST2 knockdown cells than control cells. Our data provide the first evidence that BST2 is involved in the osteogenic differentiation of bone marrow stromal cells via the regulation of the BMP2 signaling pathway.

  16. Graft-derived anti-HPA-2b production after allogeneic bone-marrow transplantation

    DEFF Research Database (Denmark)

    Taaning, E; Jacobsen, N; Morling, N

    1994-01-01

    We report on a male who received a bone-marrow allograft from his HLA identical sister for acute myelogenous leukaemia. After transplantation, the patient suffered from refractoriness to the transfusions of HLA-matched platelets and a strong platelet-specific antibody, anti-HPA-2b, of IgG1 subcla...

  17. Culture of equine bone marrow mononuclear fraction and adipose tissue-derived stromal vascular fraction cells in different media

    Directory of Open Access Journals (Sweden)

    Gesiane Ribeiro

    2013-12-01

    Full Text Available The objective of this study was to evaluate the culture of equine bone marrow mononuclear fraction and adipose tissue - derived stromal vascular fraction cells in two different cell culture media. Five adult horses were submitted to bone marrow aspiration from the sternum, and then from the adipose tissue of the gluteal region near the base of the tail. Mononuclear fraction and stromal vascular fraction were isolated from the samples and cultivated in DMEM medium supplemented with 10% fetal bovine serum or in AIM-V medium. The cultures were observed once a week with an inverted microscope, to perform a qualitative analysis of the morphology of the cells as well as the general appearance of the cell culture. Colony-forming units (CFU were counted on days 5, 15 and 25 of cell culture. During the first week of culture, differences were observed between the samples from the same source maintained in different culture media. The number of colonies was significantly higher in samples of bone marrow in relation to samples of adipose tissue.

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

    Energy Technology Data Exchange (ETDEWEB)

    Kawahara, Takeshi, E-mail: tkawafb@shinshu-u.ac.jp [Integrated Department of Sciences of Functional Foods, Graduate School of Agriculture, Shinshu University, Nagano (Japan)

    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.

  19. Establishment of induced pluripotent stem cells from aged mice using bone marrow-derived myeloid cells

    Institute of Scientific and Technical Information of China (English)

    Zhao Cheng; Sachiko Ito; Naomi Nishio; Hengyi Xiao; Rong Zhang; Haruhiko Suzuki; Yayoi Okawa; Toyoaki Murohara; Ken-ichi Isobe

    2011-01-01

    If induced pluripotent stem (iPS) cells are to be used to treat damaged tissues or repair organs in elderly patients, it will be necessaryto establish iPS cells from their tissues. To determine the feasibility of using this technology with elderly patients, we asked if itwas indeed possible to establish iPS cells from the bone marrow (BM) of aged mice. BM cells from aged C57BL/6 mice carrying thegreen fluorescence protein (GFP) gene were cultured with granulocyte macrophage-colony stimulating factor (GM-CSF) for 4 days.Four factors (Oct3/4, Sox2, Klf4 and c-Myc) were introduced into the BM-derived myeloid (BM-M) cells. The efficiency of generating iPS cells from aged BM cultured in GM-CSF was low. However, we succeeded in obtaining BM-M-iPS cells from aged C57BL/6 mice,which carried GFP. Our BM-M-iPS cells expressed SSEA-1 and Pou5f1 and were positive for alkaline phosphatase staining. The iPScells did make teratoma with three germ layers following injection into syngeneic C57BL/6 mice, and can be differentiated to threegerm layers in vitro. By co-culturing with OP9, the BM-M-iPS cells can be differentiated to the myeloid lineage. The differentiated BM-M-iPS cells proliferated well in the presence of GM-CSF, and lost expression of Nanog and Pou5f1, at least in part, due to methylation of their promoters. On the contrary, Tnf and Il1b gene expression was upregulated and their promoters were hypornethylated.

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

    Science.gov (United States)

    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.

  1. Comparative capability of menstrual blood versus bone marrow derived stem cells in neural differentiation.

    Science.gov (United States)

    Azedi, Fereshteh; Kazemnejad, Somaieh; Zarnani, Amir Hassan; Soleimani, Masoud; Shojaei, Amir; Arasteh, Shaghayegh

    2017-02-01

    In order to characterize the potency of menstrual blood stem cells (MenSCs) for future cell therapy of neurological disorders instead of bone marrow stem cells (BMSCs) as a well-known and conventional source of adult stem cells, we examined the in vitro differentiation potential of these stem cells into neural-like cells. The differentiation potential of MenSCs to neural cells in comparison with BMSCs was assessed under two step neural differentiation including conversion to neurosphere-like cells and final differentiation. The expression levels of Nestin, Microtubule-associated protein 2, gamma-aminobutyric acid type B receptor subunit 1 and 2, and Tubulin, beta 3 class III mRNA and/or protein were up-regulated during development of MenSCs into neurosphere-like cells (NSCs) and neural-like cells. The up-regulation level of these markers in differentiated neural-like cells from MenSCs was comparable with differentiated cells from BMSCs. Moreover, both differentiated MenSCs and BMSCs expressed high levels of potassium, calcium and sodium channel genes developing functional channels with electrophysiological recording. For the first time, we demonstrated that MenSCs are a unique cell population with differentiation ability into neural-like cells comparable to BMSCs. In addition, we have introduced an approach to generate NSCs from MenSCs and BMSCs and their further differentiation into neural-like cells in vitro. Our results hold a promise to future stem cell therapy of neurological disorders using NSCs derived from menstrual blood, an accessible source in every woman.

  2. The signalling imprints of nanoparticle uptake by bone marrow derived dendritic cells.

    Science.gov (United States)

    Karlson, Tanya De L; Kong, Ying Ying; Hardy, Charles L; Xiang, Sue Dong; Plebanski, Magdalena

    2013-05-01

    Nanoparticles (NP) possess remarkable adjuvant and carrier capacity, therefore are used in the development of various vaccine formulations. Our previous studies demonstrated that inert non-toxic 40-50 nm polystyrene NP (PS-NP) can promote strong CD8 T cell and antibody responses to the antigen, in the absence of observable inflammatory responses. Furthermore, instillation of PS-NP inhibited the development of allergic airway inflammation by induction of an immunological imprint via modulation of dendritic cell (DC) function without inducing oxidative stress in the lungs in mice. This is in contrast to many studies which show that a variety of ambient and man-made NP promote lung immunopathology, raising concerns generally about the safe use of NPs in biomedicine. Most NPs are capable of inducing inflammatory pathways in DC largely mediated by signalling via the extracellular signal-regulated kinase 1/2 (ERK). Herein, we investigate whether PS-NPs also activate ERK in DC in vitro. Our data show that PS-NP do not induce ERK activation in two different types of bone marrow derived (BM) DC cultures (expanded with GM-CSF or with GM-CSF together with IL-4). The absence of such signalling was not due to lack of PS-NP uptake by BM-DC as confirmed by confocal microscopy and flow cytometry. The process of NP uptake by DC usually initiates ERK signalling, suggesting an unusual uptake pathway may be engaged by PS-NPs. Indeed, data herein showns that uptake of PS-NP by BM-DC was substantially inhibited by phorbol myristate acetate (PMA) but not cytochalasin D (CCD), suggesting an uptake pathway utilising caveole for PS-NP. Together these data show that BM-DC take up PS-NP via a caveole-dependent pathway which does not trigger ERK signalling which may explain their efficient uptake by DC, without the concomitant activation of conventional inflammatory pathways.

  3. Comparative analysis of canine monocyte- and bone-marrow-derived dendritic cells.

    Science.gov (United States)

    Ricklin Gutzwiller, Meret Elisabeth; Moulin, Hervé Raphaël; Zurbriggen, Andreas; Roosje, Petra; Summerfield, Artur

    2010-01-01

    Dendritic cells (DC) represent a heterogeneous cell family of major importance for innate immune responses against pathogens and antigen presentation during infection, cancer, allergy and autoimmunity. The aim of the present study was to characterize canine DC generated in vitro with respect to their phenotype, responsiveness to toll-like receptor (TLR) ligands and T-cell stimulatory capacity. DC were derived from monocytes (MoDC) and from bone marrow hematopoietic cells cultured with either Flt3-ligand (FL-BMDC) or with GM-CSF (GM-BMDC). All three methods generated cells with typical DC morphology that expressed CD1c, CD11c and CD14, similar to macrophages. However, CD40 was only found on DC, CD206 on MPhi and BMDC, but not on monocytes and MoDC. CD1c was not found on monocytes but on all in vitro differentiated cells. FL-BMDC and GM-BMDC were partially positive for CD4 and CD8. CD45RA was expressed on a subset of FL-BMDC but not on MoDC and GM-BMDC. MoDC and FL-DC responded well to TLR ligands including poly-IC (TLR2), Pam3Cys (TLR3), LPS (TLR4) and imiquimod (TLR7) by up-regulating MHC II and CD86. The generated DC and MPhi showed a stimulatory capacity for lymphocytes, which increased upon maturation with LPS. Taken together, our results are the basis for further characterization of canine DC subsets with respect to their role in inflammation and immune responses.

  4. Age-associated changes in microRNA expression in bone marrow derived dendritic cells.

    Science.gov (United States)

    Park, Seungbum; Kang, Soowon; Min, Kyung Hoon; Woo Hwang, Kwang; Min, Hyeyoung

    2013-01-01

    MiRNAs have shown to regulate aging process at the level of cellular senescence, tissue aging, and lifespan of whole organism. Given that many miRNAs also function as important regulators of hematopoietic system as well as aging process, it is highly likely that miRNAs would be involved in the changes of myeloid function and differentiation during aging. Therefore, here we examine differential expression of miRNAs in aged myeloid lineage cells and assess if altered miRNA expression pattern would reflect the change of miRNA targets and related function. We demonstrated that the expressions of myelogenic miRNAs such as miR-155, miR-223, miR-146a, miR-146b, miR-132, miR-142-5p, and miR-142-3p were increased in aged bone marrow derived dendritic cells (BMDC) under normal and activated conditions. We also observed that the expressions of IRAK1 and TRAF6, the targets of miR-146a, and DC-SIGN, a target of miR-155 were diminished while miR-146a and miR-155 were augmented during aging. In addition, we found that the production of pro-inflammatory cytokines, which is mediated by the activation of NF-kB pathway via IRAK1 and TRAF6, was greatly reduced in aged BMDC. Taken together, our data reveal that age-associated changes occur in miRNA expression in BMDC, and this altered miRNA expression affects miRNA target expression and compromises BMDC function such as cytokine production during aging.

  5. Accelerated differentiation of bone marrow-derived dendritic cells in atopic prone mice.

    Science.gov (United States)

    Koike, Eiko; Takano, Hirohisa; Inoue, Ken-Ichiro; Yanagisawa, Rie

    2008-12-20

    NC/Nga mice are atopic prone mice that can be an animal model for human atopic dermatitis (AD). Dendritic cells (DC) as professional antigen-presenting cells (APC) are the most capable inducers of immune responses. The present study using BALB/c, C57BL/6J, and NC/Nga male mice investigated whether differentiation and function of DC were associated with atopic prone. Bone marrow-derived DC (BMDC) were differentiated by culture with granulocyte macrophage colony stimulating factor (GM-CSF). At days 0, 6, and 8 of culture with GM-CSF, the expression of MHC class II, co-stimulatory molecules (CD80, CD86), and of DC markers (CD11c, DEC205) was measured by flow cytometry. Antigen-presenting activity of BMDC and cytokine production were measured by ELISA. The cell numbers and the expression of MHC class II, co-stimulatory molecules, and of DC markers on BMDC from NC/Nga mice were significantly larger than those from BALB/c and C57BL/6J mice. Antigen-presenting activity of BMDC was significantly greater in NC/Nga and C57BL/6J mice than in BALB/c mice. BMDC-stimulated IFN-gamma production from T-cells was significantly lower in NC/Nga or BALB/c mice than in C57BL/6J mice, whereas IL-4 production was significantly greater in NC/Nga and C57BL/6J mice than in BALB/c mice. Taken together, GM-CSF-stimulated differentiation of BMDC was more accelerated in atopic prone NC/Nga mice than in the other strains of mice. The enhancement of differentiation and function of DC caused by genetic background may be related, at least partly, to the induction or aggravation of allergic/atopic diseases.

  6. Carbon black nanoparticles promote the maturation and function of mouse bone marrow-derived dendritic cells.

    Science.gov (United States)

    Koike, Eiko; Takano, Hirohisa; Inoue, Ken-Ichiro; Yanagisawa, Rie; Kobayashi, Takahiro

    2008-09-01

    Particulate matter including carbon black (CB) nanoparticles can enhance antigen-related inflammation and immunoglobulin production in vivo. Dendritic cells (DC) as antigen-presenting cells (APC) are the most capable inducers of immune responses. The present study was designed to determine whether CB nanoparticles affect the maturation/activation and function of DC in vitro. DC were differentiated from bone marrow (BM) cells of BALB/c mice by culture with granulocyte macrophage colony stimulating factor (GM-CSF). At day 8 of culture, BM-derived DC (BMDC) were exposed to CB nanoparticles with a diameter of 14nm or 56nm for 24h. The expression of major histocompatibility complex (MHC) class II, DEC205, CD80, and CD86 (maturation/activation markers of BMDC) was measured by flow cytometry. BMDC function was evaluated by an allogeneic mixed lymphocyte reaction (MLR) assay. CB nanoparticles significantly increased the expression of DEC205 and CD86 in BMDC and tended to increase MHC class II and CD80 expression; however, a size-dependent effect was not observed. On the other hand, BMDC-mediated MLR was significantly enhanced by the CB nanoparticles and the enhancement was greater by 14nm CB nanoparticles than by 56nm CB nanoparticles. Taken together, CB nanoparticles can promote the maturation/activation and function of BMDC, which could be related to their effects on allergic diseases and/or responses. In addition, BMDC-mediated MLR might be useful assay for in vitro screening for adjuvant activity of environmental toxicants.

  7. Caloric restriction and the adipokine leptin alter the SDF-1 signaling axis in bone marrow and in bone marrow derived mesenchymal stem cells.

    Science.gov (United States)

    Periyasamy-Thandavan, Sudharsan; Herberg, Samuel; Arounleut, Phonepasong; Upadhyay, Sunil; Dukes, Amy; Davis, Colleen; Johnson, Maribeth; McGee-Lawrence, Meghan; Hamrick, Mark W; Isales, Carlos M; Hill, William D

    2015-07-15

    Growing evidence suggests that the chemokine stromal cell-derived factor-1 (SDF-1) is essential in regulating bone marrow (BM) derived mesenchymal stromal/stem cell (BMSC) survival, and differentiation to either a pro-osteogenic or pro-adipogenic fate. This study investigates the effects of caloric restriction (CR) and leptin on the SDF-1/CXCR4 axis in bone and BM tissues in the context of age-associated bone loss. For in vivo studies, we collected bone, BM cells and BM interstitial fluid from 12 and 20 month-old C57Bl6 mice fed ad-libitum (AL), and 20-month-old mice on long-term CR with, or without, intraperitoneal injection of leptin for 10 days (10 mg/kg). To mimic conditions of CR in vitro, 18 month murine BMSCs were treated with (1) control (Ctrl): normal proliferation medium, (2) nutrient restriction (NR): low glucose, low serum medium, or (3) NR + leptin: NR medium + 100 ng/ml leptin for 6-48 h. In BMSCs both protein and mRNA expression of SDF-1 and CXCR4 were increased by CR and CR + leptin. In contrast, the alternate SDF-1 receptor CXCR7 was decreased, suggesting a nutrient signaling mediated change in SDF-1 axis signaling in BMSCs. However, in bone SDF-1, CXCR4 and 7 gene expression increase with age and this is reversed with CR, while addition of leptin returns this to the "aged" level. Histologically bone formation was lower in the calorically restricted mice and BM adipogenesis increased, both effects were reversed with the 10 day leptin treatment. This suggests that in bone CR and leptin alter the nutrient signaling pathways in different ways to affect the local action of the osteogenic cytokine SDF-1. Studies focusing on the molecular interaction between nutrient signaling by CR, leptin and SDF-1 axis may help to address age-related musculoskeletal changes.

  8. Overexpression of microRNA-124 promotes the neuronal differentiation of bone marrow-derived mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Defeng Zou; Yi Chen; Yaxin Han; Chen Lv; Guanjun Tu

    2014-01-01

    microRNAs (miRNAs) play an important regulatory role in the self-renewal and differentiation of stem cells. In this study, we examined the effects of miRNA-124 (miR-124) overexpression in bone marrow-derived mesenchymal stem cells. In particular, we focused on the effect of overexpression on the differentiation of bone marrow-derived mesenchymal stem cells into neurons. First, we used GeneChip technology to analyze the expression of miRNAs inbone marrow-derived mesen-chymal stem cells, neural stem cells and neurons. miR-124 expression was substantially reduced inbone marrow-derived mesenchymal stem cells compared with the other cell types. We con-structed a lentiviral vector overexpressing miR-124 and transfected it intobone marrow-derived mesenchymal stem cells. Intracellular expression levels of the neuronal early markersβ-III tu-bulin and microtubule-associated protein-2 were signiifcantly increased, and apoptosis induced by oxygen and glucose deprivation was reduced in transfected cells. After miR-124-transfected bone marrow-derived mesenchymal stem cells were transplanted into the injured rat spinal cord, a large number of cells positive for the neuronal marker neurofilament-200 were observed in the transplanted region. The Basso-Beattie-Bresnahan locomotion scores showed that the motor function of the hind limb of rats with spinal cord injury was substantially improved. These re-sults suggest that miR-124 plays an important role in the differentiation ofbone marrow-derived mesenchymal stem cells into neurons. Our ifndings should facilitate the development of novel strategies for enhancing the therapeutic efifcacy ofbone marrow-derived mesenchymal stem cell transplantation for spinal cord injury.

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

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    Steffen K Meurer

    Full Text Available 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.

  10. Brain-derived neurotrophic factor from bone marrow-derived cells promotes post-injury repair of peripheral nerve.

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    Yoshinori Takemura

    Full Text Available Brain-derived neurotrophic factor (BDNF stimulates peripheral nerve regeneration. However, the origin of BNDF and its precise effect on nerve repair have not been clarified. In this study, we examined the role of BDNF from bone marrow-derived cells (BMDCs in post-injury nerve repair. Control and heterozygote BDNF knockout mice (BDNF+/- received a left sciatic nerve crush using a cerebral blood clip. Especially, for the evaluation of BDNF from BMDCs, studies with bone marrow transplantation (BMT were performed before the injury. We evaluated nerve function using a rotarod test, sciatic function index (SFI, and motor nerve conduction velocity (MNCV simultaneously with histological nerve analyses by immunohistochemistry before and after the nerve injury until 8 weeks. BDNF production was examined by immunohistochemistry and mRNA analyses. After the nerve crush, the controls showed severe nerve dysfunction evaluated at 1 week. However, nerve function was gradually restored and reached normal levels by 8 weeks. By immunohistochemistry, BDNF expression was very faint before injury, but was dramatically increased after injury at 1 week in the distal segment from the crush site. BDNF expression was mainly co-localized with CD45 in BMDCs, which was further confirmed by the appearance of GFP-positive cells in the BMT study. Variant analysis of BDNF mRNA also confirmed this finding. BDNF+/- mice showed a loss of function with delayed histological recovery and BDNF+/+→BDNF+/- BMT mice showed complete recovery both functionally and histologically. These results suggested that the attenuated recovery of the BDNF+/- mice was rescued by the transplantation of BMCs and that BDNF from BMDCs has an essential role in nerve repair.

  11. The Bone Marrow-Derived Stromal Cells: Commitment and Regulation of Adipogenesis

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    Tencerova, Michaela; Kassem, Moustapha

    2016-01-01

    Bone marrow (BM) microenvironment represents an important compartment of bone that regulates bone homeostasis and the balance between bone formation and bone resorption depending on the physiological needs of the organism. Abnormalities of BM microenvironmental dynamics can lead to metabolic bone diseases. BM stromal cells (also known as skeletal or mesenchymal stem cells) [bone marrow stromal stem cell (BMSC)] are multipotent stem cells located within BM stroma and give rise to osteoblasts and adipocytes. However, cellular and molecular mechanisms of BMSC lineage commitment to adipocytic lineage and regulation of BM adipocyte formation are not fully understood. In this review, we will discuss recent findings pertaining to identification and characterization of adipocyte progenitor cells in BM and the regulation of differentiation into mature adipocytes. We have also emphasized the clinical relevance of these findings. PMID:27708616

  12. Paracrine Mechanisms of Intravenous Bone Marrow-Derived Mononuclear Stem Cells in Chronic Ischemic Stroke

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    Ashu Bhasin

    2016-10-01

    Full Text Available Background: The emerging role of stem cell technology and transplantation has helped scientists to study their potential role in neural repair and regeneration. The fate of stem cells is determined by their niche, consisting of surrounding cells and the secreted trophic growth factors. This interim report evaluates the safety, feasibility and efficacy (if any of bone marrow-derived mononuclear stem cells (BM-MNC in chronic ischemic stroke by studying the release of serum vascular endothelial growth factor (VEGF and brain-derived neurotrophic growth factor (BDNF. Methods: Twenty stroke patients and 20 age-matched healthy controls were recruited with the following inclusion criteria: 3 months to 1.5 years from the index event, Medical Research Council (MRC grade of hand muscles of at least 2, Brunnstrom stage 2-5, conscious, and comprehendible. They were randomized to one group receiving autologous BM-MNC (mean 60-70 million and to another group receiving saline infusion (placebo. All patients were administered a neuromotor rehabilitation regime for 8 weeks. Clinical assessments [Fugl Meyer scale (FM, modified Barthel index (mBI, MRC grade, Ashworth tone scale] were carried out and serum VEGF and BDNF levels were assessed at baseline and at 8 weeks. Results: No serious adverse events were observed during the study. There was no statistically significant clinical improvement between the groups (FM: 95% CI 15.2-5.35, p = 0.25; mBI: 95% CI 14.3-4.5, p = 0.31. VEGF and BDNF expression was found to be greater in group 1 compared to group 2 (VEGF: 442.1 vs. 400.3 pg/ml, p = 0.67; BDNF: 21.3 vs. 19.5 ng/ml without any statistically significant difference. Conclusion: Autologous mononuclear stem cell infusion is safe and tolerable by chronic ischemic stroke patients. The released growth factors (VEGF and BDNF in the microenvironment could be due to the paracrine hypothesis of stem cell niche and neurorehabilitation regime.

  13. Cartilage Derived from Bone Marrow Mesenchymal Stem Cells Expresses Lubricin In Vitro and In Vivo.

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    Yusuke Nakagawa

    Full Text Available Lubricin expression in the superficial cartilage will be a crucial factor in the success of cartilage regeneration. Mesenchymal stem cells (MSCs are an attractive cell source and the use of aggregates of MSCs has some advantages in terms of chondrogenic potential and efficiency of cell adhesion. Lubricin expression in transplanted MSCs has not been fully elucidated so far. Our goals were to determine (1 whether cartilage pellets of human MSCs expressed lubricin in vitro chondrogenesis, (2 whether aggregates of human MSCs promoted lubricin expression, and (3 whether aggregates of MSCs expressed lubricin in the superficial cartilage after transplantation into osteochondral defects in rats.For in vitro analysis, human bone marrow (BM MSCs were differentiated into cartilage by pellet culture, and also aggregated using the hanging drop technique. For an animal study, aggregates of BM MSCs derived from GFP transgenic rats were transplanted to the osteochondral defect in the trochlear groove of wild type rat knee joints. Lubricin expression was mainly evaluated in differentiated and regenerated cartilages.In in vitro analysis, lubricin was detected in the superficial zone of the pellets and conditioned medium. mRNA expression of Proteoglycan4 (Prg4, which encodes lubricin, in pellets was significantly higher than that of undifferentiated MSCs. Aggregates showed different morphological features between the superficial and deep zone, and the Prg4 mRNA expression increased after aggregate formation. Lubricin was also found in the aggregate. In a rat study, articular cartilage regeneration was significantly better in the MSC group than in the control group as shown by macroscopical and histological analysis. The transmission electron microscope showed that morphology of the superficial cartilage in the MSC group was closer to that of the intact cartilage than in the control group. GFP positive cells remained in the repaired tissue and expressed lubricin in

  14. CXCR2 modulates bone marrow vascular repair and haematopoietic recovery post-transplant.

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    Hale, Sarah J M; Hale, Ashley B H; Zhang, Youyi; Sweeney, Dominic; Fisher, Nita; van der Garde, Mark; Grabowska, Rita; Pepperell, Emma; Channon, Keith; Martin-Rendon, Enca; Watt, Suzanne M

    2015-05-01

    Murine models of bone marrow transplantation show that pre-conditioning regimens affect the integrity of the bone marrow endothelium and that the repair of this vascular niche is an essential pre-requisite for successful haematopoietic stem and progenitor cell engraftment. Little is known about the angiogenic pathways that play a role in the repair of the human bone marrow vascular niche. We therefore established an in vitro humanized model, composed of bone marrow stromal and endothelial cells and have identified several pro-angiogenic factors, VEGFA, ANGPT1, CXCL8 and CXCL16, produced by the stromal component of this niche. We demonstrate for the first time that addition of CXCL8 or inhibition of its receptor, CXCR2, modulates blood vessel formation in our bone marrow endothelial niche model. Compared to wild type, Cxcr2(-/-) mice displayed a reduction in bone marrow cellularity and delayed platelet and leucocyte recovery following myeloablation and bone marrow transplantation. The delay in bone marrow recovery correlated with impaired bone marrow vascular repair. Taken together, our data demonstrate that CXCR2 regulates bone marrow blood vessel repair/regeneration and haematopoietic recovery, and clinically may be a therapeutic target for improving bone marrow transplantation.

  15. Identification of microRNAs regulating the developmental pathways of bone marrow derived mast cells.

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    Yang Xiang

    Full Text Available BACKGROUND: MicroRNAs (miRNAs play important roles in leukocyte differentiation, although those utilised for specific programs and key functions remain incompletely characterised. As a global approach to gain insights into the potential regulatory role of miRNA in mast cell differentiation we characterised expression in BM cultures from the initiation of differentiation. In cultures enriched in differentiating mast cells we characterised miRNA expression and identified miRNA targeting the mRNA of putative factors involved in differentiation pathways and cellular identity. Detailed pathway analysis identified a unique miRNA network that is intimately linked to the mast cell differentiation program. METHODOLOGY/PRINCIPAL FINDINGS: We identified 86 unique miRNAs with expression patterns that were up- or down- regulated at 5-fold or more during bone marrow derived mast cells (BMMC development. By employing TargetScan and MeSH databases, we identified 524 transcripts involved in 30 canonical pathways as potentially regulated by these specific 86 miRNAs. Furthermore, by applying miRanda and IPA analyses, we predict that 7 specific miRNAs of this group are directly associated with the expression of c-Kit and FcεRIα and likewise, that 18 miRNAs promote expression of Mitf, GATA1 and c/EBPα three core transcription factors that direct mast cell differentiation. Furthermore, we have identified 11 miRNAs that may regulate the expression of STATs-3, -5a/b, GATA2 and GATA3 during differentiation, along with 13 miRNAs that target transcripts encoding Ndst2, mMCP4 and mMCP6 and thus may regulate biosynthesis of mast cell secretory mediators. CONCLUSIONS/SIGNIFICANCE: This investigation characterises changes in miRNA expression in whole BM cultures during the differentiation of mast cells and predicts functional links between miRNAs and their target mRNAs for the regulation of development. This information provides an important resource for further

  16. Bone marrow-derived mesenchymal stem cells protect against experimental liver fibrosis in rats

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    Dong-Chang Zhao; Jun-Xia Lei; Rui Chen; Wei-Hua Yu; Xiu-Ming Zhang; Shu-Nong Li; Peng Xiang

    2005-01-01

    AIM: Recent reports have shown the capacity of mesenchymal stem cells (MSCs) to differentiate into hepatocytes in vitro and in vivo. MSCs administration could repair injured liver, lung, or heart through reducing inflammation, collagen deposition, and remodeling. These results provide a clue to treatment of liver fibrosis. The aim of this study was to investigate the effect of infusion of bone marrow (BM)-derived MSCs on the experimental liver fibrosis in rats.METHODS: MSCs isolated from BM in male Fischer 344 rats were infused to female Wistar rats induced with carbon tetrachloride (CCl4) or dimethylnitrosamine (DMN).There were two random groups on the 42nd d of CCl4:CCl4/MSCs, to infuse a dose of MSCs alone; CCl4/saline,to infuse the same volume of saline as control. There were another three random groups after exposure to DMN: DMN10/MSCs, to infuse the same dose of MSCs on d 10; DMN10/saline, to infuse the same volume of saline on d 10; DMN20/MSCs, to infuse the same dose of MSCson d 20. The morphological and behavioral changes ofrats were monitored everyday. After 4-6 wk of MSCs administration, all rats were killed and fibrosis index were assessed by histopathology and radioimmunoassay. Smooth muscle alpha-actin (alpha-SMA) of liver were tested by immunohistochemistry and quantified by IBAS 2.5 software. Male rats sex determination region on the Y chromosome (sry) gene were explored by PCR.RESULTS: Compared to controls, infusion of MSCsreduced the mortality rates of incidence in CCl4-induced model (10% vs 20%) and in DMN-induced model (2040% vs 90%).The amount of collagen deposition and alpha-SMA staining was about 40-50% lower in liver of rats with MSCs than that of rats without MSCs. The similar results were observed in fibrosis index. And the effect of the inhibition of fibrogenesis was greater in DMN10/MSCs than in DMN20/MSCs. The sry gene was positive in the liver of rats with MSCs treatment by PCR.CONCLUSION: MSCs treatment can protect against

  17. Neural differentiation potential of human bone marrow-derived mesenchymal stromal cells: misleading marker gene expression

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    Montzka Katrin

    2009-03-01

    Full Text Available Abstract Background In contrast to pluripotent embryonic stem cells, adult stem cells have been considered to be multipotent, being somewhat more restricted in their differentiation capacity and only giving rise to cell types related to their tissue of origin. Several studies, however, have reported that bone marrow-derived mesenchymal stromal cells (MSCs are capable of transdifferentiating to neural cell types, effectively crossing normal lineage restriction boundaries. Such reports have been based on the detection of neural-related proteins by the differentiated MSCs. In order to assess the potential of human adult MSCs to undergo true differentiation to a neural lineage and to determine the degree of homogeneity between donor samples, we have used RT-PCR and immunocytochemistry to investigate the basal expression of a range of neural related mRNAs and proteins in populations of non-differentiated MSCs obtained from 4 donors. Results The expression analysis revealed that several of the commonly used marker genes from other studies like nestin, Enolase2 and microtubule associated protein 1b (MAP1b are already expressed by undifferentiated human MSCs. Furthermore, mRNA for some of the neural-related transcription factors, e.g. Engrailed-1 and Nurr1 were also strongly expressed. However, several other neural-related mRNAs (e.g. DRD2, enolase2, NFL and MBP could be identified, but not in all donor samples. Similarly, synaptic vesicle-related mRNA, STX1A could only be detected in 2 of the 4 undifferentiated donor hMSC samples. More significantly, each donor sample revealed a unique expression pattern, demonstrating a significant variation of marker expression. Conclusion The present study highlights the existence of an inter-donor variability of expression of neural-related markers in human MSC samples that has not previously been described. This donor-related heterogeneity might influence the reproducibility of transdifferentiation protocols as

  18. Differentiation of bone marrow-derived mesenchymal stem cells from diabetic patients into insulin-producing cells in vitro

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    SUN Yu; LI Hui; WANG Ke-xin; CHEN Li; HOU Xin-guo; HOU Wei-kai; DONG Jian-jun; SUN Lei; TANG Kuan-xiao; WANG Bin; SONG Jun

    2007-01-01

    Bckground Stem cells, which have the ability to differentiate into insulin-producing cells (IPCs), would provide a potentially unlimited source of islet cells for transplantation and alleviate the major limitations of availability and allogeneic rejection. Therefore, the utilization of stem cells is becoming the most promising therapy for diabetes mellitus (DM). Here,we studied the differentiation capacity of the diabetic patient's bone marrow-derived mesenchymal stem cells (MSCs) and tested the feasibility of using MSCs for β-cell replacement.Methods Bone marrow-derived MSCs were obtained from 10 DM patients (5 type 1 DM and 5 type 2 DM) and induced to IPCs under a three-stage protocol. Representative cell surface antigen expression profiles of MSCs were analysed by flow cytometric analysis. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect multiple genes related to pancreatic β-cell development and function. The identity of the IPCs was illustrated by the analysis of morphology, ditizone staining and immunocytochemistry. Release of insulin by these cells was confirmed by immunoradioassay.Results Flow cytometric analysis of MSCs at passage 3 showed that these cells expressed high levels of CD29 (98.28%), CD44 (99.56%) and CD106 (98.34%). Typical islet-like cell clusters were observed at the end of the protocol (18 days). Ditizone staining and immunohistochemistry for insulin were both positive. These differentiated cells at stage 2 (10 days) expressed nestin, pancreatic duodenal homeobox-1 (PDX-1), Neurogenin3, Pax4, insulin, glucagon, but at stage 3 (18 days) we observed the high expression of PDX-1, insulin, glucagon. Insulin was secreted by these cells in response to different concentrations of glucose stimulation in a regulated manner (P<0.05).Conclusions Bone marrow-derived MSCs from DM patients can differentiate into functional IPCs under certain conditions in vitro. Using diabetic patient's own bone marrow-derived MSCs as

  19. The bone marrow microenvironment enhances multiple myeloma progression by exosome-mediated activation of myeloid-derived suppressor cells.

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    Wang, Jinheng; De Veirman, Kim; De Beule, Nathan; Maes, Ken; De Bruyne, Elke; Van Valckenborgh, Els; Vanderkerken, Karin; Menu, Eline

    2015-12-22

    Exosomes, extracellular nanovesicles secreted by various cell types, modulate the bone marrow (BM) microenvironment by regulating angiogenesis, cytokine release, immune response, inflammation, and metastasis. Interactions between bone marrow stromal cells (BMSCs) and multiple myeloma (MM) cells play crucial roles in MM development. We previously reported that BMSC-derived exosomes directly promote MM cell growth, whereas the other possible mechanisms for supporting MM progression by these exosomes are still not clear. Here, we investigated the effect of BMSC-derived exosomes on the MM BM cells with specific emphasis on myeloid-derived suppressor cells (MDSCs). BMSC-derived exosomes were able to be taken up by MM MDSCs and induced their expansion in vitro. Moreover, these exosomes directly induced the survival of MDSCs through activating STAT3 and STAT1 pathways and increasing the anti-apoptotic proteins Bcl-xL and Mcl-1. Inhibition of these pathways blocked the enhancement of MDSC survival. Furthermore, these exosomes increased the nitric oxide release from MM MDSCs and enhanced their suppressive activity on T cells. Taken together, our results demonstrate that BMSC-derived exosomes activate MDSCs in the BM through STAT3 and STAT1 pathways, leading to increased immunosuppression which favors MM progression.

  20. Mobilization of endogenous bone marrow derived endothelial progenitor cells and therapeutic potential of parathyroid hormone after ischemic stroke in mice.

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    Li-Li Wang

    Full Text Available Stroke is a major neurovascular disorder threatening human life and health. Very limited clinical treatments are currently available for stroke patients. Stem cell transplantation has shown promising potential as a regenerative treatment after ischemic stroke. The present investigation explores a new concept of mobilizing endogenous stem cells/progenitor cells from the bone marrow using a parathyroid hormone (PTH therapy after ischemic stroke in adult mice. PTH 1-34 (80 µg/kg, i.p. was administered 1 hour after focal ischemia and then daily for 6 consecutive days. After 6 days of PTH treatment, there was a significant increase in bone marrow derived CD-34/Fetal liver kinase-1 (Flk-1 positive endothelial progenitor cells (EPCs in the peripheral blood. PTH treatment significantly increased the expression of trophic/regenerative factors including VEGF, SDF-1, BDNF and Tie-1 in the brain peri-infarct region. Angiogenesis, assessed by co-labeled Glut-1 and BrdU vessels, was significantly increased in PTH-treated ischemic brain compared to vehicle controls. PTH treatment also promoted neuroblast migration from the subventricular zone (SVZ and increased the number of newly formed neurons in the peri-infarct cortex. PTH-treated mice showed significantly better sensorimotor functional recovery compared to stroke controls. Our data suggests that PTH therapy improves endogenous repair mechanisms after ischemic stroke with functional benefits. Mobilizing endogenous bone marrow-derived stem cells/progenitor cells using PTH and other mobilizers appears an effective and feasible regenerative treatment after ischemic stroke.

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

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    Clifford Lin

    Full Text Available 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.

  2. Bone marrow-derived dendritic cells pulsed with tumor lysates induce anti-tumor immunity against gastric cancer ex vivo

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    AIM: To investigate whether bone marrow-derived denritic cells pulsed with tumor lysates induce immunity against gastric cancer ex vivo. METHODS: c-kit+ hematopoietic progenitor cells were magnetically isolated with a MiniMACS separator from BALB/c mice bone marrow cells. These cells were cultured with cytokines GM-CSF, IL-4, and TNFα to induce their maturation. They were analysed by morphological observation, phenotype analysis, and mixed lymphocyte reaction (MLR). Bone marrow-derived DCs (BM-DCs) were pulsed with tumor cell lysate obtained by rapid freezing and thawing at a 1:3 DC:tumor cell ratio. Finally, cytotoxic T lymphocyte (CTL) activity and interferon gamma (IFNy) secretion was evaluated ex vivo.RESULTS: c-kit+ hematopoietic progenitor cells from mice bone marrow cells cultured with cytokines for 8 d showed the character of typical mature DCs. Norphologically, observed by light microscope, these cells were large with oval or irregularly shaped nuclei and with many small dendrites. Phenotypically, FACS analysis showed that they expressed.high levels of Ia, DEC-205, CD11b, CD80 and CD86 antigen, moderate levels of CD40, and negative for F4/80. Functionally, these ceils gained the capacity to stimulate allogeneic T cells in MLR assay. However, immature DCs cultured with cytokines for 5 d did not have typical DCs phenotypic markers and could not stimulate allogeneic T cells. Ex vivo primed T cells with SGC-7901 tumor cell lysate-pulsed (TP) DCs were able to induce effective CTL activity against SGC-7901 tumor cells (E:T = 100:1, 69.55% ± 6.05% specific lysis), but not B16 tumor cells, and produced higher levels of IFNγ, when stimulated with SGC-7901 tumor cells but not when stimulated with B16 tumor cells (1575.31 ± 60.25 pg/mL in SGC-7901 group vs 164.11 ± 18.52 pg/mL in B16 group, P < 0.01).CONCLUSION: BM-derived DCs pulsed with tumor lysates can induce anti-tumor immunity specific to gastric cancer ex vivo.

  3. Engineered bone marrow-derived cell sheets restore structure and function of radiation-injured rat urinary bladders.

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    Imamura, Tetsuya; Ogawa, Teruyuki; Minagawa, Tomonori; Yokoyama, Hitoshi; Nakazawa, Masaki; Nishizawa, Osamu; Ishizuka, Osamu

    2015-05-01

    Previously, we reported that implantation of isolated single bone marrow-derived cells into radiation-injured urinary bladders could restore structure and function. However, injections of isolated single cells had some limitations. Thus, in this study, we produced bone marrow-derived cell sheets in temperature-responsive culture dishes that release the monolayer sheets intact. We then determined whether the produced cell sheets could restore function to irradiated urinary bladders. Twenty female 10-week-old Sprague-Dawley (SD) rats were irradiated with 2 gray once a week for 5 weeks. Bone marrow cells harvested from two male 17-week-old green fluorescence protein-transfected SD rats were placed in primary culture for 7 days. Bone marrow cell-derived outgrowths were harvested by enzymatic digestion and transferred into the atelocollagen-coated temperature-responsive culture dishes for 2 days. To harvest the secondarily cultured cells as monolayer sheets, a support membrane was put in each culture dish, and then the temperature was reduced to 20°C. Each released cell sheet was then patched onto the irradiated anterior bladder wall (n=10). As controls, cell-free sheets were similarly patched (n=10). After 4 weeks, transplanted cells were detected on the bladder walls. The cell sheet-transplanted bladders had smooth muscle layers and acetylcholinesterase-positive nerve fibers in proportions that were significantly larger than those of the control bladders. In addition, the cell sheet-transplanted bladders had reduced prolyl 4-hydroxylase beta (P4HB)-positive regions of collagen synthesis and apoptosis within the smooth muscle layers. In cystometric investigations, threshold pressures, voiding interval, micturition volume, and bladder capacity in the cell sheet-transplantation group were significantly higher than those in the control group. Residual volume of the cell sheet-transplantation group was significantly lower compared with the control. There were 24 growth

  4. Comparative effects on type 2 diabetes of mesenchymal stem cells derived from bone marrow and adipose tissue

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    Li ZANG

    2016-08-01

    Full Text Available Objective  To compare the effects on type 2 diabetes of mesenchymal stem cells (MSCs derived from bone marrow and adipose tissue. Methods  Thirty type 2 diabetic rat models were established by an eight weeks high-fat diet (HFD with a low dose streptozotocin (STZ, 25mg/kg, and randomly assigned into three groups (10 each: diabetes group (T2DM, bone marrow MSCs transplantation group (BMSC and adipose tissue MSCs transplantation group (ADSC. Ten normal rats were set as control. MSCs were isolated from bone marrow or inguinal adipose tissue of normal rats. One week after STZ injection, 3×10 6 MSCs suspended in 1ml PBS were infused into rats via tail vein. The blood glucose was measured every day after MSCs transplantation, the intraperitoneal glucose tolerance test (IPGTT and intraperitoneal insulin tolerance test (IPITT were performed the 7th day after transplantation to evaluate the effects of MSCs on diabetic rats. Pancreatic tissues were collected for insulin/glucagon immunofluorescence staining. Results  After MSCs transplantation, the blood glucose decreased gradually and continuously in type 2 diabetic rats, with glucose tolerance and insulin sensitivity improved greatly. The improved insulin sensitivity was further confirmed by a decreased HOMA-IR (homeostasis model of assessment for insulin resistance index and increased pancreas islet β-cells (P<0.05. However, no significant differences were observed between BMSC and ADSC group. Conclusion  Both BMSC and ADSC have the same effect on type 2 diabetic rats, so the ADSC will be the ideal stem cells for treatment of type 2 diabetes. DOI: 10.11855/j.issn.0577-7402.2016.07.03

  5. Orp8 deficiency in bone marrow-derived cells reduces atherosclerotic lesion progression in LDL receptor knockout mice.

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    Erik van Kampen

    Full Text Available INTRODUCTION: Oxysterol binding protein Related Proteins (ORPs mediate intracellular lipid transport and homeostatic regulation. ORP8 downregulates ABCA1 expression in macrophages and cellular cholesterol efflux to apolipoprotein A-I. In line, ORP8 knockout mice display increased amounts of HDL cholesterol in blood. However, the role of macrophage ORP8 in atherosclerotic lesion development is unknown. METHODS AND RESULTS: LDL receptor knockout (KO mice were transplanted with bone marrow (BM from ORP8 KO mice and C57Bl/6 wild type mice. Subsequently, the animals were challenged with a high fat/high cholesterol Western-type diet to induce atherosclerosis. After 9 weeks of Western-Type diet feeding, serum levels of VLDL cholesterol were increased by 50% in ORP8 KO BM recipients compared to the wild-type recipients. However, no differences were observed in HDL cholesterol. Despite the increase in VLDL cholesterol, lesions in mice transplanted with ORP8 KO bone marrow were 20% smaller compared to WT transplanted controls. In addition, ORP8 KO transplanted mice displayed a modest increase in the percentage of macrophages in the lesion as compared to the wild-type transplanted group. ORP8 deficient macrophages displayed decreased production of pro-inflammatory factors IL-6 and TNFα, decreased expression of differentiation markers and showed a reduced capacity to form foam cells in the peritoneal cavity. CONCLUSIONS: Deletion of ORP8 in bone marrow-derived cells, including macrophages, reduces lesion progression after 9 weeks of WTD challenge, despite increased amounts of circulating pro-atherogenic VLDL. Reduced macrophage foam cell formation and lower macrophage inflammatory potential are plausible mechanisms contributing to the observed reduction in atherosclerosis.

  6. Myogenic reprogramming of bone marrow derived cells in a W⁴¹Dmd(mdx deficient mouse model.

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    Stuart Walsh

    Full Text Available Lack of expression of dystrophin leads to degeneration of muscle fibers and infiltration of connective and adipose tissue. Cell transplantation therapy has been proposed as a treatment for intractable muscle degenerative disorders. Several reports have demonstrated the ability of bone-marrow derived cells (BMDC to contribute to non-haematopoietic tissues including epithelium, heart, liver, skeletal muscle and brain following transplantation by means of fusion and reprogramming. A key issue is the extent to which fusion and reprogramming can occur in vivo, particularly under conditions of myogenic deterioration.To investigate the therapeutic potential of bone marrow transplantation in monogenetic myopathy, green fluorescent protein-positive (GFP+ bone marrow cells were transplanted into non-irradiated c-kit receptor-deficient (W⁴¹ mdx mice. This model allows BMDC reconstitution in the absence of irradiation induced myeloablation. We provide the first report of BMDC fusion in a W⁴¹Dmd(mdx deficient mouse model.In the absence of irradiation induced injury, few GFP+ cardiomyocytes and muscle fibres were detected 24 weeks post BMT. It was expected that the frequency of fusion in the hearts of W⁴¹Dmd(mdx mice would be similar to frequencies observed in infarcted mice. Although, it is clear from this study that individual cardiomyocytes with monogenetic deficiencies can be rescued by fusion, it is as clear that in the absence of irradiation, the formation of stable and reprogrammed fusion hybrids occurs, with the current techniques, at very low levels in non-irradiated recipients.

  7. Bone marrow-derived cells may not be the original cells for carcinogen-induced mouse gastrointestinal carcinomas.

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    Chen Yang

    Full Text Available AIM: It has been reported that bone marrow-derived cells (BMDC can be original cells of mouse gastric cancers induced by Helicobacter felis (H. felis infection. However, it is unknown whether BMDCs are also the original cells of mouse gastrointestinal cancers induced by gastric carcinogens N-nitroso-N-methylurea (NMU and H. felis infection. METHODS: C57BL/6 recipient mice were initially irradiated with 10Gy X-ray, reconstituted with bone marrow cells from the C57BL/6-Tg (CAG-EGFP donor mice to label BMDCs with green fluorescence protein (GFP. After 4 weeks of recovery, the bone marrow-transplanted mice were given NMU in drinking water (240 ppm and subsequently infected with H. felis by gavage. Eighty weeks later, all mice were euthanized for pathological examination. The BMDCs expressing GFP were detected in tissues using direct GFP fluorescence confocal microscopy analysis and immunohistochemistry staining (IHC assays. RESULTS: Neoplastic lesions were induced by NMU treatment and/or H. felis infection at the antrum of the glandular stomach and small intestine. In the direct GFP fluorescence confocal assay, GFP(+ epithelial cell cluster or glands were not observed in these gastrointestinal tumors, however, most GFP(+ BMDCs sporadically located in the tumor stromal tissues. Some of these GFP(+ stromal BMDCs co-expressed the hematopoietic marker CD45 or myofibroblasts markers αSMA and SRF. In the indirect GFP IHC assay, similar results were observed among 11 gastric intraepithelial neoplasia lesions and 2 small intestine tumors. CONCLUSION: These results demonstrated that BMDCs might not be the source of gastrointestinal tumor cells induced by NMU and/or H. felis infection.

  8. The influence of surface mineral and osteopontin on the formation and function of murine bone marrow-derived osteoclasts.

    Science.gov (United States)

    Rajachar, Rupak M; Truong, Anh Q; Giachelli, Cecilia M

    2008-10-01

    The phosphorylated glycoprotein osteopontin (OPN) is involved in the regulation of biomineralization under normal and pathological conditions. Its actions include inhibiting apatite crystal growth and promoting the formation and function of mineral resorbing cells, including osteoclasts (OCL). The purpose of this study was to develop stable apatitic mineral surfaces and determine their influence on OCL formation and mineral resorption from bone marrow macrophages derived from OPN wild-type (OPN+/+) and OPN deficient (OPN-/-) mice. We demonstrated that these mineral coatings were stable and supported bone marrow-derived macrophage differentiation to OCL under our culture conditions. Macrophages harvested from OPN-/- mice had a greater capacity to form OCL than macrophages from OPN+/+ mice when allowed to differentiate on tissue culture plastic. In contrast, when allowed to differentiate on a mineral surface, no difference in OCL formation was observed. Interestingly, OPN+/+ OCL were more efficient at mineral dissolution than OPN-/- OCL, and this difference was observed regardless of differentiating surface. Our results suggest that mineralized substrates as well as ability to synthesize OPN both control OCL function in our model system. The exact nature of these effects may be dependent on variables related to mineral substrate presentation.

  9. Adipose derived stem cell transplantation is better than bone marrow mesenchymal stem cell transplantation in treating hindlimb ischemia in mice

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    Ngoc Bich Vu

    2016-09-01

    Full Text Available Introduction: Bone marrow derived MSCs (BM-MSCs and adipose derived MSCs (AD-MSCs are among the types of stem cells most commonly studied. Our study aims to compare the therapeutic efficacy of allograft AD-MSCs versus BM-MSCs in a mouse model of hindlimb ischemia. Methods: AD-MSCs were isolated from belly fat and BM-MSCs were isolated from femur bone marrow. They were used to treat mice with acute hindlimb ischemia. Treatment efficacy was compared among 4 groups: injected with BM-MSCs, injected with AD-MSCs, non-treated and injected with phosphate buffered saline. Mice in the groups were evaluated for the following: necrosis grade of leg, leg edema, blood flow, muscle cell restructure and new blood vessel formation. Results: Results showed that AD-MSC transplantation significantly recovered acute limb ischemia, with 76.5% of mice fully recovered, while the ratio was only 48.5% in BM-MSC transplanted group, and 0% in the non-treated and PBS groups. Evaluation of leg edema, blood flow, muscle cell restructure and new blood vessel formation also supported the observation that AD-MSC transplantation was superior over BM-MSC transplantation. Conclusion: Therefore, AD-MSCs may serve as the more suitable MSC for hindlimb ischemia treatment and angiogenesis therapy. [Biomed Res Ther 2016; 3(9.000: 844-856

  10. Paracrine effects of bone marrow-derived endothelial progenitor cells: cyclooxygenase-2/prostacyclin pathway in pulmonary arterial hypertension.

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    Dong-Mei Jiang

    Full Text Available BACKGROUND: Endothelial dysfunction is the pathophysiological characteristic of pulmonary arterial hypertension (PAH. Some paracrine factors secreted by bone marrow-derived endothelial progenitor cells (BMEPCs have the potential to strengthen endothelial integrity and function. This study investigated whether BMEPCs have the therapeutic potential to improve monocrotaline (MCT-induced PAH via producing vasoprotective substances in a paracrine fashion. METHODS AND RESULTS: Bone marrow-derived mononuclear cells were cultured for 7 days to yield BMEPCs. 24 hours or 3 weeks after exposure to BMEPCs in vitro or in vivo, the vascular reactivity, cyclooxygenase-2 (COX-2 expression, prostacyclin (PGI2 and cAMP release in isolated pulmonary arteries were examined respectively. Treatment with BMEPCs could improve the relaxation of pulmonary arteries in MCT-induced PAH and BMEPCs were grafted into the pulmonary bed. The COX-2/prostacyclin synthase (PGIS and its progenies PGI2/cAMP were found to be significantly increased in BMEPCs treated pulmonary arteries, and this action was reversed by a selective COX-2 inhibitor, NS398. Moreover, the same effect was also observed in conditioned medium obtained from BMEPCs culture. CONCLUSIONS: Implantation of BMEPCs effectively ameliorates MCT-induced PAH. Factors secreted in a paracrine fashion from BMEPCs promote vasoprotection by increasing the release of PGI2 and level of cAMP.

  11. Levamisole promotes murine bone marrow derived dendritic cell activation and drives Th1 immune response in vitro and in vivo.

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    Fu, Yubing; Wang, Ting; Xiu, Lei; Shi, Xiaojie; Bian, Ziyao; Zhang, Yongli; Ruhan, A; Wang, Xiao

    2016-02-01

    Our lab previously found that levamisole (LMS) as an adjuvant enhanced the efficacy of vaccine against infectious pathogens. However, the cellular and molecular mechanisms remain to be defined. In this study, we showed that BALB/c bone marrow-derived DC stimulated with LMS resulted in enhanced cell-surface expression of CD80, CD86, CD40 and MHC class II, as well as enhanced production of IL-12p70, TNF-α and IL-1β. Interestingly, the LMS activated DCs were able to stimulate CD4(+) T cell proliferation and facilitated Th1 differentiation by increasing the secretion of IFN-γ in an allogeneic mixed leukocyte reaction. Furthermore, to confirm the in vitro data, we investigated the effect of LMS on antigen-specific antibody and cytokine production in BALB/c mice. Immunization with LMS plus OVA showed that anti-OVA IgG2a and IFN-γ were increased significantly compared with OVA alone in BALB/c mice. In conclusion, our results suggested that murine bone marrow-derived DC, played a crucial role in the effect of LMS on the induction of Th1 responses, which probably was due to its ability to promote DC maturation and secrete proinflammatory cytokines.

  12. A novel role for platelet secretion in angiogenesis: mediating bone marrow-derived cell mobilization and homing.

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    Feng, Weiyi; Madajka, Maria; Kerr, Bethany A; Mahabeleshwar, Ganapati H; Whiteheart, Sidney W; Byzova, Tatiana V

    2011-04-07

    Angiogenesis alleviates hypoxic stress in ischemic tissues or during tumor progression. In addition to endothelial cell proliferation and migration, the angiogenic process requires bone marrow-derived cell (BMDC) recruitment to sites of neovascularization. However, the mechanism of communication between hypoxic tissues and the BM remains unknown. Using 2 models of hypoxia-induced angiogenesis (ischemic hindlimb surgery and subcutaneous tumor growth), we show that platelet infusion promotes BMDC mobilization into the circulation, BMDC recruitment into growing neovasculature, tumor vascularization, and blood flow restoration in ischemic limbs, whereas platelet depletion inhibits these effects. Thus, platelets are required for BMDC recruitment into ischemia-induced vasculature. Secretion of platelet α-granules, but neither dense granules nor platelet aggregation is crucial for BMDC homing and subsequent angiogenesis, as determined using VAMP-8(-/-), Pearl, and integrin Beta 3(-/-) platelets. Finally, platelets sequester tumor-derived promoters of angiogenesis and BMDC mobilization, which are counterbalanced by the antiangiogenic factor thrombospondin-1. A lack of thrombospondin-1 in platelets leads to an imbalance in proangiogenic and antiangiogenic factors and accelerates tumor growth and vascularization. Our data demonstrate that platelets stimulate BMDC homing in a VAMP-8-dependent manner, revealing a previously unknown role for platelets as key mediators between hypoxic tissues and the bone marrow during angiogenesis.

  13. Bone marrow derived cell-seeded extracellular matrix: A novel biomaterial in the field of wound management

    Directory of Open Access Journals (Sweden)

    V. Remya

    2014-11-01

    Full Text Available Aim: Extensive or irreversible damage to the skin often requires additional skin substitutes for reconstruction. Biomaterials have become critical components in the development of effective new medical therapies for wound care. Materials and Methods: In the present study, a cell matrix construct (bone marrow-derived cells (BMdc seeded extracellular matrix [ECM] was used as a biological substitute for the repair of full-thickness skin wound. ECM was developed by decellularizing fish swim bladder (FSB. Goat bone marrow-derived cells (G-BMdc were seeded over this decellularized matrix. Efficacy of this cell matrix construct in wound repair was tested by implanting it over 20 mm2 × 20 mm2 size fullthickness skin wound created over the dorsum of rat. The study was conducted in 16 clinically healthy adult rats of either sex. The animals were randomly divided into 2 equal groups of 8 animals each. In Group I, animal’s wounds were repaired with a cellular FSB matrix. In Group II, wounds were repaired with G-BMdc seeded a cellular FSB matrix. Immune response and efficacy of healing were analyzed. Results: Quality of healing and immuno tolerance to the biological substitute was significantly better in Group II than Group I. Conclusion: Seeding with BMdc increases the wound healing potency and modulates the immune response to a significantly negligible level. The BMdc seeded acellular FSB matrix was found to be a novel biomaterial for wound management.

  14. Cryptococcus neoformans activates bone marrow-derived conventional dendritic cells rather than plasmacytoid dendritic cells and down-regulates macrophages.

    Science.gov (United States)

    Siegemund, Sabine; Alber, Gottfried

    2008-04-01

    Induction of IL-12 and IL-23 is essential for protective immunity against Cryptococcusneoformans. The contribution of dendritic cells vs. macrophages to IL-12/23 production in response to C. neoformans infection is unclear. Activation of conventional bone marrow-derived dendritic cells (BMDC), plasmacytoid BMDC, and bone marrow-derived macrophages (BMMPhi) was assessed by analyzing cytokine responses and the expression of MHC-II, CD86, and CD80 in each cell type. Cryptococcus neoformans induced the release of IL-12/23p40 by BMDC, but not by BMMPhi, in a TLR2- and TLR4-independent but MyD88-dependent manner. Conventional BMDC rather than plasmacytoid BMDC up-regulated MHC-II and CD86, while BMMPhi down-regulated MHC-II and CD86 in response to C. neoformans. The up-regulation of MHC-II and CD86 on BMDC required MyD88. Our data point to conventional DC as critical IL-12/23-producing antigen-presenting cells during cryptococcosis.

  15. Adenovirus-mediated human brain-derived neurotrophic factor gene-modified bone marrow mesenchymal stem cell transplantation for spinal cord injury

    Institute of Scientific and Technical Information of China (English)

    Changsheng Wang; Jianhua Lin; Chaoyang Wu; Rongsheng Chen

    2011-01-01

    Rat bone marrow mesenchymal stem cells expressing brain-derived neurotrophic factor were successfully obtained using a gene transfection method, then intravenously transplanted into rats with spinal cord injury. At 1, 3, and 5 weeks after transplantation, the expression of ??brain-derived neurotrophic factor and neurofilament-200 was upregulated in the injured spinal cord, spinal cord injury was alleviated, and Basso-Beattie-Bresnahan scores of hindlimb motor function were significantly increased. This evidence suggested that intravenous transplantation of adenovirus- mediated brain-derived neurotrophic factor gene-modified rat bone marrow mesenchymal stem cells could play a dual role, simultaneously providing neural stem cells and neurotrophic factors.

  16. Bone marrow-derived hematopoietic stem and progenitor cells infiltrate allogeneic and syngeneic transplants.

    Science.gov (United States)

    Fan, Z; Enjoji, K; Tigges, J C; Toxavidis, V; Tchipashivili, V; Gong, W; Strom, T B; Koulmanda, M

    2014-12-01

    Lineage (CD3e, CD11b, GR1, B220 and Ly-76) negative hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) infiltrate islet allografts within 24 h posttransplantation. In fact, lineage(negative) Sca-1(+) cKit(+) ("LSK") cells, a classic signature for HSCs, were also detected among these graft infiltrating cells. Lineage negative graft infiltrating cells are functionally multi-potential as determined by a standard competitive bone marrow transplant (BMT) assay. By 3 months post-BMT, both CD45.1 congenic, lineage negative HSCs/HPCs and classic "LSK" HSCs purified from islet allograft infiltrating cells, differentiate and repopulate multiple mature blood cell phenotypes in peripheral blood, lymph nodes, spleen, bone marrow and thymus of CD45.2 hosts. Interestingly, "LSK" HSCs also rapidly infiltrate syngeneic islet transplants as well as allogeneic cardiac transplants and sham surgery sites. It seems likely that an inflammatory response, not an adaptive immune response to allo-antigen, is responsible for the rapid infiltration of islet and cardiac transplants by biologically active HSCs/HPCs. The pattern of hematopoietic differentiation obtained from graft infiltrating HSCs/HPCs, cells that are recovered from inflammatory sites, as noted in the competitive BMT assay, is not precisely the same as that of intramedullary HSCs. This does not refute the obvious multi-lineage potential of graft infiltrating HSCs/HPCs.

  17. Immune Suppressive Effects of Tonsil-Derived Mesenchymal Stem Cells on Mouse Bone-Marrow-Derived Dendritic Cells

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    Minhwa Park

    2015-01-01

    Full Text Available Mesenchymal stem cells (MSCs are considered valuable sources for cell therapy because of their immune regulatory function. Here, we investigated the effects of tonsil-derived MSCs (T-MSCs on the differentiation, maturation, and function of dendritic cells (DCs. We examined the effect of T-MSCs on differentiation and maturation of bone-marrow- (BM- derived monocytes into DCs and we found suppressive effect of T-MSCs on DCs via direct contact as well as soluble mediators. Moreover, T cell proliferation, normally increased in the presence of DCs, was inhibited by T-MSCs. Differentiation of CD4+ T cell subsets by the DC-T cell interaction also was inhibited by T-MSCs. The soluble mediators suppressed by T-MSCs were granulocyte-macrophage colony-stimulating factor (GM-CSF, RANTES, interleukin-6 (IL-6, and monocyte chemoattractant protein-1 (MCP-1. Taken together, T-MSCs exert immune modulatory function via suppression of the differentiation, maturation, and function of BM-derived DCs. Our data suggests that T-MSCs could be used as a novel source of stem cell therapy as immune modulators.

  18. Brain-derived neurotrophic factor (BDNF) gene delivery into the CNS using bone marrow cells as vehicles in mice.

    Science.gov (United States)

    Makar, T K; Trisler, D; Eglitis, M A; Mouradian, M M; Dhib-Jalbut, S

    2004-02-19

    Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, is protective in animal models of neurodegenerative diseases. However, BDNF has a short half-life and its efficacy in the CNS when delivered peripherally is limited due to the blood-brain barrier. In the present study, bone marrow cells were used as vehicles to deliver the BDNF gene into the CNS. Marrow cells obtained from 6 to 8 week-old SJL/J mice were transduced with BDNF expressing pro-virus. RT-PCR analysis revealed that BDNF mRNA was expressed in transduced but not in non-transduced marrow cells. Additionally, virus transduced marrow cells expressed the BDNF protein (296+/-1.2 unit/ml). BDNF-transduced marrow cells were then transplanted into irradiated mice through the tail vein. Three months post-transplantation, significant increases in BDNF as well as glutamic acid decarboxylase (GAD(67)) mRNA were detected in the brains of BDNF transplanted mice compared to untransplanted animals, indicating biological activity of the BDNF transgene. Thus, bone marrow cells can be used as vehicles to deliver the BDNF gene into the brain with implications for the treatment of neurological diseases.

  19. Systemic Injection of RPE65-Programmed Bone Marrow-Derived Cells Prevents Progression of Chronic Retinal Degeneration.

    Science.gov (United States)

    Qi, Xiaoping; Pay, S Louise; Yan, Yuanqing; Thomas, James; Lewin, Alfred S; Chang, Lung-Ji; Grant, Maria B; Boulton, Michael E

    2017-04-05

    Bone marrow stem and progenitor cells can differentiate into a range of non-hematopoietic cell types, including retinal pigment epithelium (RPE)-like cells. In this study, we programmed bone marrow-derived cells (BMDCs) ex vivo by inserting a stable RPE65 transgene using a lentiviral vector. We tested the efficacy of systemically administered RPE65-programmed BMDCs to prevent visual loss in the superoxide dismutase 2 knockdown (Sod2 KD) mouse model of age-related macular degeneration. Here, we present evidence that these RPE65-programmed BMDCs are recruited to the subretinal space, where they repopulate the RPE layer, preserve the photoreceptor layer, retain the thickness of the neural retina, reduce lipofuscin granule formation, and suppress microgliosis. Importantly, electroretinography and optokinetic response tests confirmed that visual function was significantly improved. Mice treated with non-modified BMDCs or BMDCs pre-programmed with LacZ did not exhibit significant improvement in visual deficit. RPE65-BMDC administration was most effective in early disease, when visual function and retinal morphology returned to near normal, and less effective in late-stage disease. This experimental paradigm offers a minimally invasive cellular therapy that can be given systemically overcoming the need for invasive ocular surgery and offering the potential to arrest progression in early AMD and other RPE-based diseases.

  20. Comparison of molecular profiles of human mesenchymal stem cells derived from bone marrow, umbilical cord blood, placenta and adipose tissue.

    Science.gov (United States)

    Heo, June Seok; Choi, Youjeong; Kim, Han-Soo; Kim, Hyun Ok

    2016-01-01

    Mesenchymal stem cells (MSCs) are clinically useful due to their capacity for self-renewal, their immunomodulatory properties and tissue regenerative potential. These cells can be isolated from various tissues and exhibit different potential for clinical applications according to their origin, and thus comparative studies on MSCs from different tissues are essential. In this study, we investigated the immunophenotype, proliferative potential, multilineage differentiation and immunomodulatory capacity of MSCs derived from different tissue sources, namely bone marrow, adipose tissue, the placenta and umbilical cord blood. The gene expression profiles of stemness-related genes [octamer-binding transcription factor 4 (OCT4), sex determining region Y-box (SOX)2, MYC, Krüppel-like factor 4 (KLF4), NANOG, LIN28 and REX1] and lineage‑related and differentiation stage-related genes [B4GALNT1 (GM2/GS2 synthase), inhibin, beta A (INHBA), distal-less homeobox 5 (DLX5), runt-related transcription factor 2 (RUNX2), proliferator‑activated receptor gamma (PPARG), CCAAT/enhancer-binding protein alpha (C/EBPA), bone morphogenetic protein 7 (BMP7) and SOX9] were compared using RT-PCR. No significant differences in growth rate, colony-forming efficiency and immunophenotype were observed. Our results demonstrated that MSCs derived from bone marrow and adipose tissue shared not only in vitro tri-lineage differentiation potential, but also gene expression profiles. While there was considerable inter-donor variation in DLX5 expression between MSCs derived from different tissues, its expression appears to be associated with the osteogenic potential of MSCs. Bone marrow-derived MSCs (BM-MSCs) significantly inhibited allogeneic T cell proliferation possibly via the high levels of the immunosuppressive cytokines, IL10 and TGFB1. Although MSCs derived from different tissues and fibroblasts share many characteristics, some of the marker genes, such as B4GALNT1 and DLX5 may be useful for

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

    Science.gov (United States)

    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.

  2. Notch signaling stimulates osteogenic differentiation of human bone marrow-derived mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    LU Zhuozhuang; WU Zuze(WU Chutse); ZHANG Qunwei; WANG Hua; JIA Xiangxu; DUAN Haifeng; WANG Lisheng

    2004-01-01

    Notch signaling is one of the most important pathways mediating cell determination and differentiation. In this study, the roles of Notch signal in the regulation of osteogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs) were investigated. The expression of Notch1, Jagged1 and DTX1 detected by reverse transcription polymerase chain reaction (RT-PCR) suggested that Notch signal might exhibit a physiological regulatory role in the differentiation of MSCs. Constitutive expression of the intracellular domain of Notch1 (ICN), the active form of Notch1 protein, can activate Notch signal in cells without ligands' binding. hMSCs were isolated, expanded, and infected with retrovirus carrying green fluorescent protein (GFP) gene or ICN. Overexpression of ICN in hMSCs resulted in enhanced osteogenic differentiation induced by dexamethasone (Dex), which was characterized by an increase of cellular alkaline phosphatase (ALP) activity and calcium deposition. These results indicate that Notch stimulates differentiation of MSCs into osteoblasts.

  3. Systematic analysis of reportedly distinct populations of multipotent bone marrow-derived stem cells reveals a lack of distinction.

    Science.gov (United States)

    Lodie, Tracey A; Blickarz, Courtney E; Devarakonda, Tara J; He, Chufa; Dash, Ajeeta B; Clarke, Jennifer; Gleneck, Kristen; Shihabuddin, Lamya; Tubo, Ross

    2002-10-01

    Adult human bone marrow-derived stem cells, having the ability to differentiate into cells of multiple lineages, have been isolated and propagated by varied protocols, including positive (CD105(+))/negative (CD45(-)GlyA(-)) selection with immunomagnetic beads, or direct plating into selective culture media. Each substratum-adherent cell population was subjected to a systematic analysis of their cell surface markers and differentiation potential. In the initial stages of culture, each cell population proliferated slowly, reaching confluence in 10-14 days. Adherent cells proliferated at similar rates whether cultured in serum-free medium supplemented with basic fibroblast growth factor, medium containing 2% fetal bovine serum (FBS) supplemented with epidermal growth factor and platelet-derived growth factor, or medium containing 10% FBS alone. Cell surface marker analysis revealed that more than 95% of the cells were positive for CD105/endoglin, a putative mesenchymal stem cell marker, and negative for CD34, CD31, and CD133, markers of hematopoietic, endothelial, and neural stem cells, respectively, regardless of cell isolation and propagation method. CD44 expression was variable, apparently dependent on serum concentration. Functional similarity of the stem cell populations was also observed, with each different cell population expressing the cell type-specific markers beta-tubulin, type II collagen, and desmin, and demonstrating endothelial tube formation when cultured under conditions favoring neural, cartilage, muscle, and endothelial cell differentiation, respectively. On the basis of these data, adult human bone marrow-derived stem cells cultured in adherent monolayer are virtually indistinguishable, both physically and functionally, regardless of the method of isolation or proliferative expansion.

  4. Novel analysis of maturation of murine bone-marrow-derived dendritic cells induced by Ginkgo Seed Polysaccharides.

    Science.gov (United States)

    Chen, Yinghan; Meng, Yiming; Cao, Yan; Wen, Hua; Luo, Hong; Gao, Xinghua; Shan, Fengping

    2015-01-01

    Our understanding of the mechanisms of effect of Ginkgo Seed Polysaccharides (GSPs) on the immune system remains unclear. The aim of this work was to investigate the effect of GSPs on the maturation and function of bone-marrow-derived dendritic cells (BMDCs). The results demonstrate that GSP could exert positive immune modulation on the maturation and functions of BMDCs. This effect was evidenced by decreased changes of phagosome number inside BMDCs, decreased activity of acidic phosphatase (ACP), decreased phagocytosis of BMDCs, and increased changes of key membrane molecules on BMDCs. Upregulated production of cytokines IL-12 and TNF-α also was confirmed. Therefore, it can be concluded that GSPs can efficiently induce the maturation of BMDCs. Our exploration provides direct data and a rationale for potential application of GSPs as an immune enhancer in improving immunity and as a potent adjuvant in the design of DC-based vaccines.

  5. Effect of NK4 Transduction in Bone Marrow-Derived Mesenchymal Stem Cells on Biological Characteristics of Pancreatic Cancer Cells

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    Yun-Peng Sun

    2014-03-01

    Full Text Available Pancreatic cancer usually has a poor prognosis, and no gene therapy has yet been developed that is effective to treat it. Since a unique characteristic of bone marrow-derived mesenchymal stem cells (MSCs is that they migrate to tumor tissues, we wanted to determine whether MSCs could serve as a vehicle of gene therapy for targeting pancreatic cancer. First, we successfully extracted MSCs from SD rats. Next, MSCs were efficiently transduced with NK4, an antagonist of hepatocyte growth factor (HGF which comprising the N-terminal and the subsequent four kringle domains of HGF, by an adenoviral vector. Then, we confirmed that rat MSCs preferentially migrate to pancreatic cancer cells. Last, MSCs expressing NK4 (NK4-MSCs strongly inhibited proliferation and migration of the pancreatic cancer cell line SW1990 after co-culture. These results indicate that MSCs can serve as a vehicle of gene therapy for targeting pancreatic cancer.

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

    Science.gov (United States)

    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.

  7. Quantification of nucleated cells, CD34-positive cells and CFU-GM colonies in single bone marrow samples and bone marrow harvests derived from healthy children.

    Science.gov (United States)

    Schündeln, Michael M; Walde, Gabriele; Basu, Oliver; Havers, Werner; Kremens, Bernhard

    2014-05-01

    Little is known regarding bone marrow (BM) cellularity, CD34+ fraction, and CFU-GM colony formation in relation to age and whether healthy children require a reference range distinct from healthy adults. We therefore analyzed a series of single BM aspirates from 45 healthy children who were evaluated as potential BM donors. Thirty-three of these children subsequently donated BM. We quantified the nucleated cell count, fraction of CD34+ cells, and number of CFU-GM colonies in single aspirates and BM harvests. Single aspirates displayed a mean nucleated cell count of 31.3 × 10(6) cells/mL, a mean fraction of 1.17% CD34+ cells, and a mean colony forming potential of 66.6 CFU-GM/10(5) cells. Harvests yielded the same number of nucleated cells but increased numbers of CD34+ cells and CFU-GM compared with single aspirates. The mean nucleated cell count in BM harvests was 31.1 × 10(6) /mL with a mean fraction of 1.95% CD34+ cells and a mean of 112.4 CFU-GM colonies/10(5) cells. The concentration of nucleated cells was elevated compared with reported adult counts, while CD34+ percentage and CFU-GM counts were similar. In this series of healthy children, the fraction of CD34+ cells, CFU-GM colonies, and nucleated cells decreased with age. We did not identify gender specific differences. To our knowledge, this represents the first comprehensive study of CD34+ cell fraction, CFU-GM counts, and nucleated cell numbers in the BM of healthy children. The findings provide valuable information for practical use for BM transplantation and contribute to the understanding of hematopoiesis from birth to adulthood.

  8. Human bone marrow-derived mesenchymal stem cells transplanted into damaged rabbit heart to improve heart function

    Institute of Scientific and Technical Information of China (English)

    WANG Jian-an; FAN You-qi; LI Chang-ling; HE Hong; SUN Yong; LV Bin-jian

    2005-01-01

    Objective: The present study was designed to test whether transplantation of human bone marrow-derived mesenchymal stem cells (hMSCs) in New Zealand rabbits with myocardial infarction can improve heart function; and whether engrafted donor cells can survive and transdifferentiated into cardiomyocytes. Methods: Twenty milliliters bone marrow was obtained from healthy men by bone biopsy. A gradient centrifugation method was used to separate bone marrow cells (BMCs) and red blood cells.BMCs were incubated for 48 h and then washed with phosphate-buffered saline (PBS). The culture medium was changed twice a week for 28 d. Finally, hematopoietic cells were washed away to leave only MSCs. Human MSCs (hMSCs) were premarked by BrdU 72 h before the transplantation. Thirty-four New Zealand rabbits were randomly divided into myocardial infarction (MI)control group and cell treated group, which received hMSCs (MI+MSCs) through intramyocardial injection, while the control group received the same volume of PBS. Myocardial infarction was induced by ligation of the left coronary artery. Cell treated rabbits were treated with 5× 106 MSCs transplanted into the infarcted region after ligation of the coronary artery for 1 h, and the control group received the same volume of PBS. Cyclosporin A (oral solution; 10 mg/kg) was provided alone, 24 h before surgery and once a day after MI for 4 weeks. Echocardiography was measured in each group before the surgery and 4 weeks after the surgery to test heart function change. The hearts were harvested for HE staining and immunohistochemical studies after MI and cell transplantation for 4 weeks. Results: Our data showed that cardiac function was significantly improved by hMSC transplantation in rabbit infarcted hearts 4 weeks after MI (ejection fraction: 0.695±0.038 in the cell treated group (n=12) versus0.554±0.065 in the control group (n=13) (P<0.05). Surviving hMSCs were identified by BrdU positive spots in infarcted region and

  9. Analysis of maturation states of rat bone marrow-derived dendritic cells using an improved culture technique.

    Science.gov (United States)

    Grauer, Oliver; Wohlleben, Gisela; Seubert, Silvia; Weishaupt, Andreas; Kämpgen, Eckhart; Gold, Ralf

    2002-04-01

    In this study, we examined in more detail the development of rat bone marrow-derived dendritic cells (BMDC). A two-stage culture system was used to propagate BMDC from rat bone marrow precursors. BMDC developed within clusters of proliferating cells after repetitive addition of rat granulocyte/macrophage colony-stimulating factor and rat interleukin (IL)-4 at a concentration of 5 ng/ml to the cultures. Fluorescence-activated cell sorter analysis performed at an early stage of development (day 6) revealed an immature phenotype with intermediate levels of major histocompatibility complex (MHC) class II expression and low levels of the costimulator molecules CD80 and CD86. Upon further culture, a strong upregulation of MHC class II, costimulatory and adhesion molecules could be observed, whereas macrophage marker antigens were downregulated. Late-stage BMDC (day 10) showed a high expression of MHC class I and II, ICAM-1, Ox62 and CD11c, and revealed a split pattern of B7-1 and B7-2. The cell yield was about 40% of the initially plated bone marrow cells with 80% MHC class II-high and less than 20% MHC class II-low positive cells. Full maturation of rat BMDC (day 12) with an almost uniform expression of B7 was achieved by subsequent subculture and further stimulation with rat tumour necrosis factor alpha (TNF-alpha), lipopolysaccharide (LPS) or soluble CD40 ligand (CD40L). Analysis of the cell supernatant revealed a strong IL-12 production after LPS or CD40L, and to a lesser extent after TNF-alpha stimulation. Additionally, LPS-treated, but not CD40L-treated BMDC secreted TNF-alpha into the supernatant. Early-stage BMDC sufficiently triggered a T cell receptor (TCR) downregulation, but did not stimulate naive T cells in an allogeneic mixed leukocyte reaction (MLR) and revealed a low stimulatory capacity in an antigen-specific T cell assay. In contrast, late-stage BMDC and especially fully mature BMDC strongly induced TCR internalisation, elicited high T cell responses

  10. Bone marrow-derived and peritoneal macrophages have different inflammatory response to oxLDL and M1/M2 marker expression - implications for atherosclerosis research

    DEFF Research Database (Denmark)

    Bisgaard, Line S; Mogensen, Christina K; Rosendahl, Alexander;

    2016-01-01

    Macrophages are heterogeneous and can polarize into specific subsets, e.g. pro-inflammatory M1-like and re-modelling M2-like macrophages. To determine if peritoneal macrophages (PEMs) or bone marrow derived macrophages (BMDMs) resembled aortic macrophages from ApoE-/- mice, their M1/M2 phenotype,...

  11. Genetic response and morphologic characterization of chicken bone-marrow derived dendritic cells during infection with high and low pathogenic avian influenza viruses

    Science.gov (United States)

    Dendritic cells (DC) are professional antigen-presenting cells of the immune system that function to initiate primary immune responses. Progenitors of DCs are derived from haematopoietic stem cells in the bone marrow (BM) that migrate in non-lymphoid tissues to develop into immature DCs. Here, they ...

  12. Bone marrow-derived myofibroblasts contribute to the renal interstitial myofibroblast population and produce procollagen I after ischemia/reperfusion in rats

    NARCIS (Netherlands)

    Broekema, Martine; Harmsen, Martin C.; van Luyn, Marja J. A.; Koerts, Jasper A.; Petersen, Arjen H.; van Kooten, Theo G.; van Goor, Harry; Navis, Gerjan; Popa, Eliane R.

    2007-01-01

    Bone marrow-derived cells (BMDC) have been proposed to exert beneficial effects after renal ischemia/reperfusion injury (IRI) by engraftment in the tubular epithelium. However, BMDC can give rise to myofibroblasts and may contribute to fibrosis. BMDC contribution to the renal interstitial myofibrobl

  13. Influence of serum from liver-damaged rats on differentiation tendency of bone marrow-derived stem cells

    Institute of Scientific and Technical Information of China (English)

    Hai Hong; Jian-Zhi Chen; Feng Zhou; Ling Xue; Guo-Qiang Zhao

    2004-01-01

    AIM: Recent studies in both rodents and humans indicated that bone marrow (BM)-derived stem cells were able to home to the liver after they were damaged and demonstrated plasticity in becoming hepatocytes. However, the question remains as to how these stem cells are activated and led to the liver and where the signals initiating the mechanisms of activation and differentiation of stem cells originate. The aim of this study was to investigate the influence of serum from liver-damaged rats on differentiation tendency of bone marrow-derived stem cells.METHODS: Serum samples were collected from rats treated with a 2-acetylaminofluorene (2-AAF)/carbon tetrachloride (CCl4) program for varying time points and then used as stimulators of cultured BM stem cells. Expression of M2- and L-type isozymes of rat pyruvate kinase, albumin as well as integrin-β1 were then examined by reverse transcription polymerase chain reaction (RT-PCR) to estimate the differentiation state of BM stem cells.RESULTS: Expression of M2-type isozyme of pyruvate kinase (M2-PK), a marker of immature hepatocytes, was detected in each group stimulated with experimental serum, but not in controls including mature hepatocytes, BM stem cells without serum stimulation, and BM stem cells stimulated with normal control serum. As a marker expressed in the development of liver, the expression signal of integrin-β1 was also detectable in each group stimulated with experimental serum. However, expression of L-type isozyme of pyruvate kinase (L-PK) and albumin, marker molecules of mature hepatocytes, was not detected in groups stimulated with experimental serum.CONCLUSION: Under the influence of serum from rats with liver failure, BM stem cells begin to differentiate along a direction to hepatocyte lineage and to possess some features of immature hepatocytes.

  14. Effects of BMP2 and VEGF165 on the osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells.

    Science.gov (United States)

    Lin, Zhaowei; Wang, Jiang-Sheng; Lin, Lijun; Zhang, Jingwen; Liu, Yunlong; Shuai, Ming; Li, Qi

    2014-03-01

    Bone marrow-derived mesenchymal stem cells (MSCs) are dominant seed cell sources for bone regeneration. Bone morphogenetic proteins (BMPs) initiate cartilage and bone formation in a sequential cascade. Vascular endothelial growth factor (VEGF) is an essential coordinator of extracellular matrix remodeling, angiogenesis and bone formation. In the present study, the effects of the vascular endothelial growth factor 165 (VEGF165) and bone morphogenetic protein 2 (BMP2) genes on bone regeneration were investigated by the lentivirus-mediated cotransfection of the two genes into rat bone marrow-derived MSCs. The successful co-expression of the two genes in the MSCs was confirmed using quantitative polymerase chain reaction (qPCR) and western blot analysis. The results of alizarin red and alkaline phosphatase (ALP) staining at 14 days subsequent to transfection showed that the area of staining in cells transfected with BMP2 alone was higher than that in cells transfected with BMP2 and VEGF165 or untransfected control cells, while the BMP2 + VEGF165 group showed significantly more staining than the untransfected control. This indicated that BMP2 alone exhibited a stronger effect in bone regeneration than BMP2 in combination with VEGF165. Similarly, in inducing culture medium, the ALP activity of the BMP2 + VEGF165 group was notably suppressed compared with that of the BMP2 group. The overexpression of VEGF165 inhibited BMP2-induced MSC differentiation and osteogenesis in vitro. Whether or not local VEGF gene therapy is likely to affect bone regeneration in vivo requires further investigation.

  15. Analyzing the cellular contribution of bone marrow to fracture healing using bone marrow transplantation in mice.

    Science.gov (United States)

    Colnot, C; Huang, S; Helms, J

    2006-11-24

    The bone marrow is believed to play important roles during fracture healing such as providing progenitor cells for inflammation, matrix remodeling, and cartilage and bone formation. Given the complex nature of bone repair, it remains difficult to distinguish the contributions of various cell types. Here we describe a mouse model based on bone marrow transplantation and genetic labeling to track cells originating from bone marrow during fracture healing. Following lethal irradiation and engraftment of bone marrow expressing the LacZ transgene constitutively, wild type mice underwent tibial fracture. Donor bone marrow-derived cells, which originated from the hematopoietic compartment, did not participate in the chondrogenic and osteogenic lineages during fracture healing. Instead, the donor bone marrow contributed to inflammatory and bone resorbing cells. This model can be exploited in the future to investigate the role of inflammation and matrix remodeling during bone repair, independent from osteogenesis and chondrogenesis.

  16. Effects of salinomycin on human bone marrow-derived mesenchymal stem cells in vitro.

    Science.gov (United States)

    Scherzed, A; Hackenberg, S; Froelich, K; Rak, K; Technau, A; Radeloff, A; Nöth, U; Koehler, C; Hagen, R; Kleinsasser, N

    2013-04-26

    Various hypotheses on the origin of cancer stem cells (CSCs) exist, including that CSCs develop from transformed human bone marrow mesenchymal stem cells (hBMSC). Since the polyether antibiotic salinomycin selectively kills CSCs, the present study aims to elucidate the effects of salinomycin on normal hBMSC. The immunophenotype of hBMSC after salinomycin exposure was observed by flow cytometry. The multi-differentiation capacity of hBMSC was evaluated by Oil Red O and van Kossa staining. Cytotoxic effects of salinomycin were monitored by the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) assay. Furthermore, spheroid formation and migration capacity were assessed. There were no differences in the immunophenotype and multi-differentiation capacity of hBMSC induced by salinomycin treatment. Cytotoxic effects were observed at concentrations of 30 μM and above. Neither the migration capability nor the ability to form spheroids was affected. Essential functional properties of hBMSC were unaffected by salinomycin. However, dose-dependent cytotoxicity effects could be observed. Overall, low dose salinomycin showed no negative effects on hBMSC. Since mesenchymal stem cells from various sources respond differently, further in vitro studies are needed to clarify the effect of salinomycin on tissue-specific stem cells.

  17. Resorption of monetite calcium phosphate cement by mouse bone marrow derived osteoclasts.

    Science.gov (United States)

    Montazerolghaem, M; Karlsson Ott, M; Engqvist, H; Melhus, H; Rasmusson, A J

    2015-01-01

    Recently the interest for monetite based biomaterials as bone grafts has increased; since in vivo studies have demonstrated that they are degradable, osteoconductive and improve bone healing. So far osteoclastic resorption of monetite has received little attention. The current study focuses on the osteoclastic resorption of monetite cement using primary mouse bone marrow macrophages, which have the potential to differentiate into resorbing osteoclasts when treated with receptor activator NF-κB ligand (RANKL). The osteoclast viability and differentiation were analysed on monetite cement and compared to cortical bovine bone discs. After seven days live/dead stain results showed no significant difference in viability between the two materials. However, the differentiation was significantly higher on the bone discs, as shown by tartrate resistant acid phosphatase (TRAP) activity and Cathepsin K gene expression. Moreover monetite samples with differentiated osteoclasts had a 1.4 fold elevated calcium ion concentration in their culture media compared to monetite samples with undifferentiated cells. This indicates active resorption of monetite in the presence of osteoclasts. In conclusion, this study suggests that osteoclasts have a crucial role in the resorption of monetite based biomaterials. It also provides a useful model for studying in vitro resorption of acidic calcium phosphate cements by primary murine cells.

  18. Cell-autonomous sex differences in gene expression in chicken bone marrow-derived macrophages.

    Science.gov (United States)

    Garcia-Morales, Carla; Nandi, Sunil; Zhao, Debiao; Sauter, Kristin A; Vervelde, Lonneke; McBride, Derek; Sang, Helen M; Clinton, Mike; Hume, David A

    2015-03-01

    We have identified differences in gene expression in macrophages grown from the bone marrow of male and female chickens in recombinant chicken M-CSF (CSF1). Cells were profiled with or without treatment with bacterial LPS for 24 h. Approximately 600 transcripts were induced by prolonged LPS stimulation to an equal extent in the male and female macrophages. Many transcripts encoded on the Z chromosome were expressed ∼1.6-fold higher in males, reflecting a lack of dosage compensation in the homogametic sex. A smaller set of W chromosome-specific genes was expressed only in females. LPS signaling in mammals is associated with induction of type 1 IFN-responsive genes. Unexpectedly, because IFNs are encoded on the Z chromosome of chickens, unstimulated macrophages from the female birds expressed a set of known IFN-inducible genes at much higher levels than male cells under the same conditions. To confirm that these differences were not the consequence of the actions of gonadal hormones, we induced gonadal sex reversal to alter the hormonal environment of the developing chick and analyzed macrophages cultured from male, female, and female sex-reversed embryos. Gonadal sex reversal did not alter the sexually dimorphic expression of either sex-linked or IFN-responsive genes. We suggest that female birds compensate for the reduced dose of inducible IFN with a higher basal set point of IFN-responsive genes.

  19. Adipose, Bone Marrow and Synovial Joint-Derived Mesenchymal Stem Cells for Cartilage Repair

    Science.gov (United States)

    Fellows, Christopher R.; Matta, Csaba; Zakany, Roza; Khan, Ilyas M.; Mobasheri, Ali

    2016-01-01

    Current cell-based repair strategies have proven unsuccessful for treating cartilage defects and osteoarthritic lesions, consequently advances in innovative therapeutics are required and mesenchymal stem cell-based (MSC) therapies are an expanding area of investigation. MSCs are capable of differentiating into multiple cell lineages and exerting paracrine effects. Due to their easy isolation, expansion, and low immunogenicity, MSCs are an attractive option for regenerative medicine for joint repair. Recent studies have identified several MSC tissue reservoirs including in adipose tissue, bone marrow, cartilage, periosteum, and muscle. MSCs isolated from these discrete tissue niches exhibit distinct biological activities, and have enhanced regenerative potentials for different tissue types. Each MSC type has advantages and disadvantages for cartilage repair and their use in a clinical setting is a balance between expediency and effectiveness. In this review we explore the challenges associated with cartilage repair and regeneration using MSC-based cell therapies and provide an overview of phenotype, biological activities, and functional properties for each MSC population. This paper also specifically explores the therapeutic potential of each type of MSC, particularly focusing on which cells are capable of producing stratified hyaline-like articular cartilage regeneration. Finally we highlight areas for future investigation. Given that patients present with a variety of problems it is unlikely that cartilage regeneration will be a simple “one size fits all,” but more likely an array of solutions that need to be applied systematically to achieve regeneration of a biomechanically competent repair tissue. PMID:28066501

  20. Growth of Bone Marrow Derived Osteoblast-Like Cells into Coral Implant Scaffold: Preliminary Study on Malaysian Coral

    Directory of Open Access Journals (Sweden)

    K. A. AL-Salihi

    2009-01-01

    Full Text Available Problem statement: Biomaterial fabrication in Malaysia started as a consequence of the demand for cheaper implant materials. Various biomaterials have been developed utilizing local resources like Malaysian coral. Locally processed Malaysian coral need to be complemented with proper evaluation and testing including toxicology, biocompatibility, mechanical properties, physicochemical characterization and in vivo testing. The present study was carried out to assess natural coral of porites species as scaffold combined with in vitro expanded Bone Marrow Derived Osteoblast-Like cells (BM-DOL, in order to develop a tissue-engineered bone graft in a rat model. Approach: Coral was used in a block shape with a dimension of 10 mm length × 5 mm width × 5 mm thickness. BM-DOL cells were seeded into porous coral scaffold in a density of 5×106 mL-1. After 7 days of in vitro incubation in osteogenic medium, one block was processed for light (LM and Scanning Electron Microscopy (SEM observations while the other blocks were implanted subcutaneously in the back of 5 weeks-old Sprague-Dawely rats for 3 months. Coral blocks without cells were implanted as a control. The implants harvested and processed for gross inspection, histological and scanning electron microscopy observations. Results: Both LM and SEM showed attachment of well arrangement multilayer cells inside the pores of in vitro seeded coral scaffolds. Gross inspection of all in vivo coral-cell complexes implants revealed vascularized like bone tissue formation. Histological sections revealed mature bone formation occurred in the manner resemble intramembraneous bone formation. SEM observations revealed multi-layer cellular proliferation with abundant collagen covered the surface of coral implants. Control group showed resorbed coral block. Conclusion: This study demonstrated that Malaysian coral can be use as a suitable scaffold material for delivering bone marrow mesenchymal

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

    Science.gov (United States)

    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.

  2. The peripheral chimerism of bone marrow-derived stem cells after transplantation: regeneration of gastrointestinal tissues in lethally irradiated mice.

    Science.gov (United States)

    Filip, Stanislav; Mokrý, Jaroslav; Vávrová, Jiřina; Sinkorová, Zuzana; Mičuda, Stanislav; Sponer, Pavel; Filipová, Alžběta; Hrebíková, Hana; Dayanithi, Govindan

    2014-05-01

    Bone marrow-derived cells represent a heterogeneous cell population containing haematopoietic stem and progenitor cells. These cells have been identified as potential candidates for use in cell therapy for the regeneration of damaged tissues caused by trauma, degenerative diseases, ischaemia and inflammation or cancer treatment. In our study, we examined a model using whole-body irradiation and the transplantation of bone marrow (BM) or haematopoietic stem cells (HSCs) to study the repair of haematopoiesis, extramedullary haematopoiesis and the migration of green fluorescent protein (GFP(+)) transplanted cells into non-haematopoietic tissues. We investigated the repair of damage to the BM, peripheral blood, spleen and thymus and assessed the ability of this treatment to induce the entry of BM cells or GFP(+) lin(-) Sca-1(+) cells into non-haematopoietic tissues. The transplantation of BM cells or GFP(+) lin(-) Sca-1(+) cells from GFP transgenic mice successfully repopulated haematopoiesis and the haematopoietic niche in haematopoietic tissues, specifically the BM, spleen and thymus. The transplanted GFP(+) cells also entered the gastrointestinal tract (GIT) following whole-body irradiation. Our results demonstrate that whole-body irradiation does not significantly alter the integrity of tissues such as those in the small intestine and liver. Whole-body irradiation also induced myeloablation and chimerism in tissues, and induced the entry of transplanted cells into the small intestine and liver. This result demonstrates that grafted BM cells or GFP(+) lin(-) Sca-1(+) cells are not transient in the GIT. Thus, these transplanted cells could be used for the long-term treatment of various pathologies or as a one-time treatment option if myeloablation-induced chimerism alone is not sufficient to induce the entry of transplanted cells into non-haematopoietic tissues.

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

    Directory of Open Access Journals (Sweden)

    Fangming Xiu

    Full Text Available 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.

  4. Differentiation of Adipose-derived Stem Cells into Schwann Cell Phenotype in Comparison with Bone Marrow Stem Cells

    Directory of Open Access Journals (Sweden)

    Zolikha Golipoor

    2010-06-01

    Full Text Available Objective(sBone marrow is the traditional source of human multipotent mesenchymal stem cells (MSCs, but adipose tissue appears to be an alternative and more readily available source. In this study, rat adipose-derived stem cells (ADSCs were induced to differentiate into Schwann-like cells and compared with rat bone marrow stem cells (BMSCs for their Schwann-like cells differentiation potential. Materials and MethodsBMSCs and ADSCs were characterized for expression of MSCs-specific markers, osteogenic and adipogenic differentiation. They were induced to differentiate into Schwann-like cells and analyzed for expression of the Schwann specific markers. The immunocytochemical differentiation markers were S-100 and real time quantitative Real-time polymerase chain reaction (RT-PCR markers were S100, P75 and glial fibrillary acidic protein (GFAP. 3-(4, 5-Dimethylthiazol- 2-yl-2, 5-diphenyltetrazolium bromide (MTT assay and Annexin V-Fluorescein isothiocyanate (FITC/ Propidium iodide (PI double labeling method were employed to detect early stage cell apoptosis.ResultsBMSCs and ADSCs showed similarities in expression of the MSC-specific markers, osteogenic and adipogenic differentiation. Both quantitative RT-PCR and immunocytochemical analysis demonstrated that BMSCs and ADSCs had equal expression of the Schwann-specific markers following Schwann-like cells differentiation. However, gene expression of P75 was higher in BMSCs compared with ADSCs. MTT assay and flow cytometry found that of the total BMSCs and ADSCs in the culture medium, 20% to 30% of the cells died, but the remaining cell population remained strongly attached to the substrate and differentiated.ConclusionComparative analysis showed that Schwann-like cell differentiation potential of ADSCs was slightly decreased in comparison with BMSCs. Therefore, BMSCs are more favorable choice than ADSCs for tissue engineering.

  5. GMP-compliant isolation and large-scale expansion of bone marrow-derived MSC.

    Directory of Open Access Journals (Sweden)

    Natalie Fekete

    Full Text Available BACKGROUND: Mesenchymal stromal cells (MSC have gained importance in tissue repair, tissue engineering and in immunosupressive therapy during the last years. Due to the limited availability of MSC in the bone marrow, ex vivo amplification prior to clinical application is requisite to obtain therapeutic applicable cell doses. Translation of preclinical into clinical-grade large-scale MSC expansion necessitates precise definition and standardization of all procedural parameters including cell seeding density, culture medium and cultivation devices. While xenogeneic additives such as fetal calf serum are still widely used for cell culture, its use in the clinical context is associated with many risks, such as prion and viral transmission or adverse immunological reactions against xenogeneic components. METHODS AND FINDINGS: We established animal-free expansion protocols using platelet lysate as medium supplement and thereby could confirm its safety and feasibility for large-scale MSC isolation and expansion. Five different GMP-compliant standardized protocols designed for the safe, reliable, efficient and economical isolation and expansion of MSC was performed and MSC obtained were analyzed for differentiation capacity by qPCR and histochemistry. Expression of standard MSC markers as defined by the International Society for Cellular Therapy as well as expression of additional MSC markers and of various chemokine and cytokine receptors was analysed by flow cytometry. Changes of metabolic markers and cytokines in the medium were addressed using the LUMINEX platform. CONCLUSIONS: The five different systems for isolation and expansion of MSC described in this study are all suitable to produce at least 100 millions of MSC, which is commonly regarded as a single clinical dose. Final products are equal according to the minimal criteria for MSC defined by the ISCT. We showed that chemokine and integrin receptors analyzed had the same expression pattern

  6. In vitro cartilage production using an extracellular matrix-derived scaffold and bone marrow-derived mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    ZHAO Yan-hong; YANG Qiang; XIA Qun; PENG Jiang; LU Shi-bi; GUO Quan-yi; MA Xin-long

    2013-01-01

    Background Cartilage repair is a challenging research area because of the limited healing capacity of adult articular cartilage.We had previously developed a natural,human cartilage extracellular matrix (ECM)-derived scaffold for in vivo cartilage tissue engineering in nude mice.However,before these scaffolds can be used in clinical applications in vivo,the in vitro effects should be further explored.Methods We produced cartilage in vitro using a natural cartilage ECM-derived scaffold.The scaffolds were fabricated by combining a decellularization procedure with a freeze-drying technique and were characterized by scanning electron microscopy (SEM),micro-computed tomography (micro-CT),histological staining,cytotoxicity assay,biochemical and biomechanical analysis.After being chondrogenically induced,the induction results of BMSCs were analyzed by histology and Immunohisto-chemistry.The attachment and viability assessment of the cells on scaffolds were analyzed using SEM and LIVE/DEAD staining.Cell-scaffold constructs cultured in vitro for 1 week and 3 weeks were analyzed using histological and immunohistochemical methods.Results SEM and micro-CT revealed a 3-D interconnected porous structure.The majority of the cartilage ECM was found in the scaffold following the removal of cellular debris,and stained positive for safranin O and collagen Ⅱ.Viability staining indicated no cytotoxic effects of the scaffold.Biochemical analysis showed that collagen content was (708.2±44.7)μg/mg,with GAG (254.7±25.9) μg/mg.Mechanical testing showed the compression moduli (E) were (1.226±0.288) and (0.052±0.007) MPa in dry and wet conditions,respectively.Isolated canine bone marrow-derived stem cells (BMSCs) were induced down a chondrogenic pathway,labeled with PKH26,and seeded onto the scaffold.Immunofluorescent staining of the cell-scaffold constructs indicated that chondrocyte-like cells were derived from seeded BMSCs and excreted ECM.The cell-scaffold constructs contained

  7. Bone Marrow Diseases

    Science.gov (United States)

    Bone marrow is the spongy tissue inside some of your bones, such as your hip and thigh bones. It contains stem cells. The stem cells can ... the platelets that help with blood clotting. With bone marrow disease, there are problems with the stem ...

  8. Migration and Differentiation of GFP-transplanted Bone Marrow-derived Cells into Experimentally Induced Periodontal Polyp in Mice

    OpenAIRE

    MATSUDA, SAEKA; Shoumura, Masahito; Osuga, Naoto; Tsujigiwa, Hidetsugu; Nakano, Keisuke; Okafuji, Norimasa; OCHIAI, TAKANAGA; HASEGAWA, HIROMASA; Kawakami, Toshiyuki

    2016-01-01

    Perforation of floor of the dental pulp is often encountered during root canal treatment in routine clinical practice of dental caries. If perforation were large, granulation tissue would grow to form periodontal polyp. Granulation tissue consists of proliferating cells however their origin is not clear. It was shown that the cells in granulation tissue are mainly from migration of undifferentiated mesenchymal cells of the bone marrow. Hence, this study utilized GFP bone marrow transplantatio...

  9. Gene expression pattern of functional neuronal cells derived from human bone marrow mesenchymal stromal cells

    Directory of Open Access Journals (Sweden)

    Bron Dominique

    2008-04-01

    Full Text Available Abstract Background Neuronal tissue has limited potential to self-renew or repair after neurological diseases. Cellular therapies using stem cells are promising approaches for the treatment of neurological diseases. However, the clinical use of embryonic stem cells or foetal tissues is limited by ethical considerations and other scientific problems. Thus, bone marrow mesenchymal stomal cells (BM-MSC could represent an alternative source of stem cells for cell replacement therapies. Indeed, many studies have demonstrated that MSC can give rise to neuronal cells as well as many tissue-specific cell phenotypes. Methods BM-MSC were differentiated in neuron-like cells under specific induction (NPBM + cAMP + IBMX + NGF + Insulin. By day ten, differentiated cells presented an expression profile of real neurons. Functionality of these differentiated cells was evaluated by calcium influx through glutamate receptor AMPA3. Results Using microarray analysis, we compared gene expression profile of these different samples, before and after neurogenic differentiation. Among the 1943 genes differentially expressed, genes down-regulated are involved in osteogenesis, chondrogenesis, adipogenesis, myogenesis and extracellular matrix component (tuftelin, AGC1, FADS3, tropomyosin, fibronectin, ECM2, HAPLN1, vimentin. Interestingly, genes implicated in neurogenesis are increased. Most of them are involved in the synaptic transmission and long term potentialisation as cortactin, CASK, SYNCRIP, SYNTL4 and STX1. Other genes are involved in neurite outgrowth, early neuronal cell development, neuropeptide signaling/synthesis and neuronal receptor (FK506, ARHGAP6, CDKRAP2, PMCH, GFPT2, GRIA3, MCT6, BDNF, PENK, amphiregulin, neurofilament 3, Epha4, synaptotagmin. Using real time RT-PCR, we confirmed the expression of selected neuronal genes: NEGR1, GRIA3 (AMPA3, NEF3, PENK and Epha4. Functionality of these neuron-like cells was demonstrated by Ca2+ influx through glutamate

  10. Donor bone marrow-derived dendritic cells prolong corneal allograft survival and promote an intragraft immunoregulatory milieu.

    Science.gov (United States)

    O'Flynn, Lisa; Treacy, Oliver; Ryan, Aideen E; Morcos, Maurice; Cregg, Marese; Gerlach, Jared; Joshi, Lokesh; Nosov, Mikhail; Ritter, Thomas

    2013-11-01

    Investigations into cell therapies for application in organ transplantation have grown. Here, we describe the ex vivo generation of donor bone marrow-derived dendritic cells (BMDCs) and glucocorticoid-treated BMDCs with potent immunomodulatory properties for application in allogeneic transplantation. BMDCs were treated with dexamethasone (Dexa) to induce an immature, maturation-resistant phenotype. BMDC and Dexa BMDC phenotype, antigen presenting cell function, and immunomodulatory properties were fully characterized. Both populations display significant immunomodulatory properties, including, but not limited to, a significant increase in mRNA expression of programmed death-ligand 1 and indoleamine 2,3-dioxygenase. BMDCs and Dexa BMDCs display a profound impaired capacity to stimulate allogeneic lymphocytes. Moreover, in a fully MHC I/II mismatched rat corneal transplantation model, injection of donor-derived, untreated BMDC or Dexa BMDCs (1 × 10(6) cells, day -7) significantly prolonged corneal allograft survival without the need for additional immunosuppression. Although neovascularization was not reduced and evidence of donor-specific alloantibody response was detected, a significant reduction in allograft cellular infiltration combined with a significant increase in the ratio of intragraft FoxP3-expressing regulatory cells was observed. Our comprehensive analysis demonstrates the novel cellular therapeutic approach and significant effect of donor-derived, untreated BMDCs and Dexa BMDCs in preventing corneal allograft rejection.

  11. Recovery of neurological function of ischemic stroke by application of conditioned medium of bone marrow mesenchymal stem cells derived from normal and cerebral ischemia rats

    OpenAIRE

    2014-01-01

    Background Several lines of evidence have demonstrated that bone marrow-derived mesenchymal stem cells (BM-MSC) release bioactive factors and provide neuroprotection for CNS injury. However, it remains elusive whether BM-MSC derived from healthy donors or stroke patients provides equal therapeutic potential. The present work aims to characterize BM-MSC prepared from normal healthy rats (NormBM-MSC) and cerebral ischemia rats (IschBM-MSC), and examine the effects of their conditioned medium (C...

  12. A robust and reproducible animal serum-free culture method for clinical-grade bone marrow-derived mesenchymal stromal cells

    OpenAIRE

    Laitinen, Anita; Oja, Sofia; Kilpinen, Lotta; Kaartinen, Tanja; Möller, Johanna; Laitinen, Saara; Korhonen, Matti; Nystedt, Johanna

    2015-01-01

    Efficient xenofree expansion methods to replace fetal bovine serum (FBS)-based culture methods are strongly encouraged by the regulators and are needed to facilitate the adoption of mesenchymal stromal cell (MSC)-based therapies. In the current study we established a clinically-compliant and reproducible animal serum-free culture protocol for bone marrow-(BM-) MSCs based on an optimized platelet-derived supplement. Our study compared two different platelet-derived supplements, platelet lysate...

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

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Suna, E-mail: wangs3@mail.nih.gov; 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.

  14. An Improved Harvest and in Vitro Expansion Protocol for Murine Bone Marrow-Derived Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Song Xu

    2010-01-01

    Full Text Available Compared to bone marrow (BM derived mesenchymal stem cells (MSCs from human origin or from other species, the in vitro expansion and purification of murine MSCs (mMSCs is much more difficult because of the low MSC yield and the unwanted growth of non-MSCs in the in vitro expansion cultures. We describe a modified protocol to isolate and expand murine BM derived MSCs based on the combination of mechanical crushing and collagenase digestion at the moment of harvest, followed by an immunodepletion step using microbeads coated with CD11b, CD45 and CD34 antibodies. The number of isolated mMSCs as estimated by colony forming unit-fibroblast (CFU-F assay showed that this modified isolation method could yield 70.0% more primary colonies. After immunodepletion, a homogenous mMSC population could already be obtained after two passages. Immunodepleted mMSCs (ID-mMSCs are uniformly positive for stem cell antigen-1 (Sca-1, CD90, CD105 and CD73 cell surface markers, but negative for the hematopoietic surface markers CD14, CD34 and CD45. Moreover the immunodepleted cell population exhibits more differentiation potential into adipogenic, osteogenic and chondrogenic lineages. Our data illustrate the development of an efficient and reliable expansion protocol increasing the yield and purity of mMSCs and reducing the overall expansion time.

  15. Human ESC-Derived MSCs Outperform Bone Marrow MSCs in the Treatment of an EAE Model of Multiple Sclerosis

    Directory of Open Access Journals (Sweden)

    Xiaofang Wang

    2014-07-01

    Full Text Available Current therapies for multiple sclerosis (MS are largely palliative, not curative. Mesenchymal stem cells (MSCs harbor regenerative and immunosuppressive functions, indicating a potential therapy for MS, yet the variability and low potency of MSCs from adult sources hinder their therapeutic potential. MSCs derived from human embryonic stem cells (hES-MSCs may be better suited for clinical treatment of MS because of their unlimited and stable supply. Here, we show that hES-MSCs significantly reduce clinical symptoms and prevent neuronal demyelination in a mouse experimental autoimmune encephalitis (EAE model of MS, and that the EAE disease-modifying effect of hES-MSCs is significantly greater than that of human bone-marrow-derived MSCs (BM-MSCs. Our evidence also suggests that increased IL-6 expression by BM-MSCs contributes to the reduced anti-EAE therapeutic activity of these cells. A distinct ability to extravasate and migrate into inflamed CNS tissues may also be associated with the robust therapeutic effects of hES-MSCs on EAE.

  16. An electromagnetic compressive force by cell exciter stimulates chondrogenic differentiation of bone marrow-derived mesenchymal stem cells.

    Science.gov (United States)

    Park, Sang-Hyug; Sim, Woo Young; Park, Sin Wook; Yang, Sang Sik; Choi, Byung Hyune; Park, So Ra; Park, Kwideok; Min, Byoung-Hyun

    2006-11-01

    In this study, we present a biological micro-electromechanical system and its application to the chondrogenic differentiation of rabbit bone marrow-derived mesenchymal stem cells (MSCs). Actuated by an electromagnetic force, the micro cell exciter was designed to deliver a cyclic compressive load (CCL) with various magnitudes. Two major parts in the system are an actuator and a cartridge-type chamber. The former has a permanent magnet and coil, and the latter is equipped with 7 sample dishes and 7 metal caps. Mixed with a 2.4% alginate solution, the alginate/MSC layers were positioned in the sample dishes; the caps contained chondrogenic defined medium without transforming growth factor-beta (TGF-beta). Once powered, the actuator coil-derived electromagnetic force pulled the metal caps down, compressing the samples. The cyclic load was given at 1-Hz frequency for 10 min twice a day. Samples in the dishes without a cap served as a control. The samples were analyzed at 3, 5, and 7 days after stimulation for cell viability, biochemical assays, histologic features, immunohistochemistry, and gene expression of the chondrogenic markers. Applied to the alginate/MSC layer, the CCL system enhanced the synthesis of cartilage-specific matrix proteins and the chondrogenic markers, such as aggrecan, type II collagen, and Sox9. We found that the micromechanically exerted CCL by the cell exciter was very effective in enhancing the chondrogenic differentiation of MSCs, even without using exogenous TGF-beta.

  17. Curcumin-functionalized silk materials for enhancing adipogenic differentiation of bone marrow-derived human mesenchymal stem cells.

    Science.gov (United States)

    Li, Chunmei; Luo, Tingting; Zheng, Zhaozhu; Murphy, Amanda R; Wang, Xiaoqin; Kaplan, David L

    2015-01-01

    Curcumin, a natural phenolic compound derived from the plant Curcuma longa, was physically entrapped and stabilized in silk hydrogel films, and its influence on human bone marrow-derived mesenchymal stem cells (hBMSC) was assessed related to adipogenic differentiation. The presence of curcumin significantly reduced the silk gelation time and changed the porous morphology of gel matrix, but did not change the formation of the silk beta-sheet structure. Based on spectrofluorimetric analysis, curcumin most likely interacted with hydrophobic residues in silk, interacting with the beta-sheet domains formed in the hydrogels. The antioxidant activity of silk film-associated curcumin remained functional over at least one month in both the dry and hydrated state. Negligible curcumin was released from silk hydrogel films over 48 h incubation in aqueous solution. For hBMSC cultured on silk films containing more than 0.25 mg ml(-1) curcumin, cell proliferation was inhibited, while adipogenesis was significantly promoted based on transcripts as well as Oil Red O staining. When hBMSC were cultured in media containing free curcumin, both proliferation and adipogenesis of hBMSC were inhibited when curcumin concentrations exceeded 5 μM, which is more than 1000 times higher than the level of curcumin released from the films in aqueous solution. Thus, silk film-associated curcumin exhibited different effects on hBMSC proliferation and differentiation compared with curcumin in solution.

  18. Retention of human bone marrow-derived cells in murine lungs following bleomycin-induced lung injury.

    Science.gov (United States)

    Liebler, Janice M; Lutzko, Carolyn; Banfalvi, Agnes; Senadheera, Dinithi; Aghamohammadi, Neema; Crandall, Edward D; Borok, Zea

    2008-08-01

    We studied the capacity of adult human bone marrow-derived cells (BMDC) to incorporate into distal lung of immunodeficient mice following lung injury. Immunodeficient NOD/SCID and NOD/SCID/beta(2) microglobulin (beta(2)M)(null) mice were administered bleomycin (bleo) or saline intranasally. One, 2, 3 and 4 days after bleo or saline, human BMDC labeled with CellTracker Green CMFDA (5-chloromethylfluorescein diacetate) were infused intravenously. Retention of CMFDA(+) cells was maximal when delivered 4 days after bleo treatment. Seven days after bleo, cells from NOD/SCID mice were CMFDA(+), which increased 10- to 100-fold in NOD/SCID/beta(2)M(null) mice. Preincubation of BMDC with Diprotin A, a reversible inhibitor of CD26 peptidase activity that enhances the stromal-derived factor-1 (SDF-1/CXCL12)/CXCR4 axis, resulted in a 30% increase in the percentage of CMFDA(+) cells retained in the lung. These data indicate that human BMDC can be identified in lungs of mice following injury, albeit at low levels, and this may be modestly enhanced by manipulation of the SDF-1/CXCR4 axis. Given the overall low number of human cells detected, methods to increase homing and retention of adult BMDC, and consideration of other stem cell populations, will likely be required to facilitate engraftment in the treatment of lung injury.

  19. Umbilical cord-derived stem cells (MODULATISTTM show strong immunomodulation capacity compared to adipose tissue-derived or bone marrow-derived mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Phuc Van Pham

    2016-06-01

    Full Text Available Introduction: Mesenchymal stem cells (MSCs show great promise in regenerative medicine. Clinical applications of MSCs have recently increased significantly, especially for immune diseases. Autologous transplantation is considered a safe therapy. However, its main disadvantages are poor stability and quality of MSCs from patient to patient, and labor-intensive and time-consuming culture procedures. Therefore, allogeneic MSC transplantation has recently emerged as a potential replacement for autologous transplantation. and ldquo;Off the shelf and rdquo; MSC products, or so-called and ldquo;stem cell drugs and rdquo;, have rapidly developed; these products have already been approved in various countries, including Canada, Korea and Japan. This study aims to evaluate a new stem cell product or and ldquo;drug and rdquo;, termed ModulatistTM, derived from umbilical cord mesenchymal stem cells (UCMSCs, which have strong immunomodulatory properties, compared to bone marrow-derived MSCs (BMMSCs or adipose tissue-derived stem cells (ADSCs. Methods: ModulatistTM was produced from MSCs derived from whole umbilical cord (UC tissue (which includes Wharton's jelly and UC, according to GMP compliant procedures. Bone marrow- and adipose tissue-derived MSCs were isolated and proliferated in standard conditions, according to GMP compliant procedures. Immunomodulation mediated by MSCs was assessed by allogenic T cell suppression and cytokine release; role of prostaglandin E2 in the immunomodulation was also evaluated. Results: The results showed that ModulatistTM exhibited stronger immunomodulation than BMMSC and ADSC in vitro. ModulatistTM strongly suppressed allogeneic T cells proliferation and decreased cytokine production, compared to BMMSCs and ADSCs. Conclusion: ModulatistTM is a strong immunomodulator and promising MSC product. It may be useful to modulate or treat autoimmune diseases. [Biomed Res Ther 2016; 3(6.000: 687-696

  20. Human Induced Pluripotent Cell-Derived Sensory Neurons for Fate Commitment of Bone Marrow-Derived Schwann Cells: Implications for Remyelination Therapy.

    Science.gov (United States)

    Cai, Sa; Han, Lei; Ao, Qiang; Chan, Ying-Shing; Shum, Daisy Kwok-Yan

    2016-09-14

    : Strategies that exploit induced pluripotent stem cells (iPSCs) to derive neurons have relied on cocktails of cytokines and growth factors to bias cell-signaling events in the course of fate choice. These are often costly and inefficient, involving multiple steps. In this study, we took an alternative approach and selected 5 small-molecule inhibitors of key signaling pathways in an 8-day program to induce differentiation of human iPSCs into sensory neurons, reaching ≥80% yield in terms of marker proteins. Continuing culture in maintenance medium resulted in neuronal networks immunopositive for synaptic vesicle markers and vesicular glutamate transporters suggestive of excitatory neurotransmission. Subpopulations of the derived neurons were electrically excitable, showing tetrodotoxin-sensitive action potentials in patch-clamp experiments. Coculture of the derived neurons with rat Schwann cells under myelinating conditions resulted in upregulated levels of neuronal neuregulin 1 type III in conjunction with the phosphorylated receptors ErbB2 and ErbB3, consistent with amenability of the neuritic network to myelination. As surrogates of embryonic dorsal root ganglia neurons, the derived sensory neurons provided contact-dependent cues to commit bone marrow-derived Schwann cell-like cells to the Schwann cell fate. Our rapid and efficient induction protocol promises not only controlled differentiation of human iPSCs into sensory neurons, but also utility in the translation to a protocol whereby human bone marrow-derived Schwann cells become available for autologous transplantation and remyelination therapy.

  1. Imbalances in Mobilization and Activation of Pro-Inflammatory and Vascular Reparative Bone Marrow-Derived Cells in Diabetic Retinopathy.

    Science.gov (United States)

    Chakravarthy, Harshini; Beli, Eleni; Navitskaya, Svetlana; O'Reilly, Sandra; Wang, Qi; Kady, Nermin; Huang, Chao; Grant, Maria B; Busik, Julia V

    2016-01-01

    Diabetic retinopathy is a sight-threatening complication of diabetes, affecting 65% of patients after 10 years of the disease. Diabetic metabolic insult leads to chronic low-grade inflammation, retinal endothelial cell loss and inadequate vascular repair. This is partly due to bone marrow (BM) pathology leading to increased activity of BM-derived pro-inflammatory monocytes and impaired function of BM-derived reparative circulating angiogenic cells (CACs). We propose that diabetes has a significant long-term effect on the nature and proportion of BM-derived cells that circulate in the blood, localize to the retina and home back to their BM niche. Using a streptozotocin mouse model of diabetic retinopathy with GFP BM-transplantation, we have demonstrated that BM-derived circulating pro-inflammatory monocytes are increased in diabetes while reparative CACs are trapped in the BM and spleen, with impaired release into circulation. Diabetes also alters activation of splenocytes and BM-derived dendritic cells in response to LPS stimulation. A majority of the BM-derived GFP cells that migrate to the retina express microglial markers, while others express endothelial, pericyte and Müller cell markers. Diabetes significantly increases infiltration of BM-derived microglia in an activated state, while reducing infiltration of BM-derived endothelial progenitor cells in the retina. Further, control CACs injected into the vitreous are very efficient at migrating back to their BM niche, whereas diabetic CACs have lost this ability, indicating that the in vivo homing efficiency of diabetic CACs is dramatically decreased. Moreover, diabetes causes a significant reduction in expression of specific integrins regulating CAC migration. Collectively, these findings indicate that BM pathology in diabetes could play a role in both increased pro-inflammatory state and inadequate vascular repair contributing to diabetic retinopathy.

  2. Imbalances in Mobilization and Activation of Pro-Inflammatory and Vascular Reparative Bone Marrow-Derived Cells in Diabetic Retinopathy.

    Directory of Open Access Journals (Sweden)

    Harshini Chakravarthy

    Full Text Available Diabetic retinopathy is a sight-threatening complication of diabetes, affecting 65% of patients after 10 years of the disease. Diabetic metabolic insult leads to chronic low-grade inflammation, retinal endothelial cell loss and inadequate vascular repair. This is partly due to bone marrow (BM pathology leading to increased activity of BM-derived pro-inflammatory monocytes and impaired function of BM-derived reparative circulating angiogenic cells (CACs. We propose that diabetes has a significant long-term effect on the nature and proportion of BM-derived cells that circulate in the blood, localize to the retina and home back to their BM niche. Using a streptozotocin mouse model of diabetic retinopathy with GFP BM-transplantation, we have demonstrated that BM-derived circulating pro-inflammatory monocytes are increased in diabetes while reparative CACs are trapped in the BM and spleen, with impaired release into circulation. Diabetes also alters activation of splenocytes and BM-derived dendritic cells in response to LPS stimulation. A majority of the BM-derived GFP cells that migrate to the retina express microglial markers, while others express endothelial, pericyte and Müller cell markers. Diabetes significantly increases infiltration of BM-derived microglia in an activated state, while reducing infiltration of BM-derived endothelial progenitor cells in the retina. Further, control CACs injected into the vitreous are very efficient at migrating back to their BM niche, whereas diabetic CACs have lost this ability, indicating that the in vivo homing efficiency of diabetic CACs is dramatically decreased. Moreover, diabetes causes a significant reduction in expression of specific integrins regulating CAC migration. Collectively, these findings indicate that BM pathology in diabetes could play a role in both increased pro-inflammatory state and inadequate vascular repair contributing to diabetic retinopathy.

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

    Directory of Open Access Journals (Sweden)

    Khanabdali R

    2015-12-01

    Full Text Available Ramin Khanabdali,1 Anbarieh Saadat,1 Maizatul Fazilah,1 Khairul Fidaa’ Khairul Bazli,1 Rida-e-Maria Qazi,2 Ramla Sana Khalid,2 Durriyyah Sharifah Hasan Adli,1 Soheil Zorofchian Moghadamtousi,1 Nadia Naeem,2 Irfan Khan,2 Asmat Salim,2 ShamsulAzlin Ahmad Shamsuddin,1 Gokula Mohan1 1Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia; 2Dr Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan Abstract: 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

  4. Stem Cell Ophthalmology Treatment Study (SCOTS):improvement in serpiginous choroidopathy following autologous bone marrow derived stem cell treatment

    Institute of Scientific and Technical Information of China (English)

    Jeffrey N Weiss; Susan C Benes; Steven Levy

    2016-01-01

    We report results in a 77-year-old male patient with visual loss from long-standing serpiginous choroidop-athy treated with bone marrow derived stem cells (BMSC) within the Stem Cell Ophthalmology Treatment Study (SCOTS). SCOTS is an Institutional Review Board approved clinical trial and the largest ophthal-mology stem cell study registered at the National Institutes of Health to date (ClinicalTrials.gov Identiifer:NCT01920867). Eight months after treatment by a combination of retrobulbar, subtenon, intravitreal and intravenous injection of BMSC, the patient’s best corrected Snellen acuity improved from 20/80– to 20/60+1 in the right eye and from 20/50– to 20/20–3 in the left eye. The Early Treatment of Diabetic Retinopathy Study (ETDRS) visual acuity continued to improve over the succeeding 8 months and the optical coherence tomography macular volume increased. The increases in visual acuity and macular volume are encouraging and suggest that the use of BMSC as provided in SCOTS may be a viable approach to treating serpiginous choroidopathy.

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

    Directory of Open Access Journals (Sweden)

    Maryam Mohammadian

    2016-01-01

    Full Text Available 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.

  6. Application of Bone Marrow-Derived Mesenchymal Stem Cells in the Treatment of Intrauterine Adhesions in Rats

    Directory of Open Access Journals (Sweden)

    Jianmei Wang

    2016-09-01

    Full Text Available Aims: To investigate the therapeutic effects of bone marrow-derived mesenchymal stem cells (BMSCs transplantation on intrauterine adhesions (IUA. Methods: BMSCs were isolated and labeled by green fluorescence protein. IUA model was established by mechanical injury. 48 rats were randomly divided into control, IUA model, BMSCs vein injection and BMSCs intrauterine injection groups (n=12 in each group. The third generation of BMSCs was injected through tail vein or intrauterine. Three rats were killed at time 0 h, 7 d, 14 d and 28 d and bilateral uterus were obtained at each time points for the subseqent experiments. Morphological changes were determined by hematoxylin-eosin staining or Masson staining. Estrogen receptor (ER and progesterone receptor (PR were detected by immunohistochemistry. Results: BMSCs were specifically stained by CD44 and CD90, but not by CD45. Before treatment, the numbers of endometrial glands were significantly decreased, while fibrosis area rate was increased in IUA model group (PConclusion: BMSCs transplantation was effective to repair the damaged endometrium likely through promoting the ER and PR expressions.

  7. Classically and alternatively activated bone marrow derived macrophages differ in cytoskeletal functions and migration towards specific CNS cell types

    Directory of Open Access Journals (Sweden)

    Dijkstra Christine D

    2011-05-01

    Full Text Available Abstract Background Macrophages play an important role in neuroinflammatory diseases such as multiple sclerosis (MS and spinal cord injury (SCI, being involved in both damage and repair. The divergent effects of macrophages might be explained by their different activation status: classically activated (CA/M1, pro-inflammatory, macrophages and alternatively activated (AA/M2, growth promoting, macrophages. Little is known about the effect of macrophages with these phenotypes in the central nervous system (CNS and how they influence pathogenesis. The aim of this study was therefore to determine the characteristics of these phenotypically different macrophages in the context of the CNS in an in vitro setting. Results Here we show that bone marrow derived CA and AA macrophages have a distinct migratory capacity towards medium conditioned by various cell types of the CNS. AA macrophages were preferentially attracted by the low weight ( Conclusion In conclusion, since AA macrophages are more motile and are attracted by NCM, they are prone to migrate towards neurons in the CNS. CA macrophages have a lower motility and a stronger adhesion to ECM. In neuroinflammatory diseases the restricted migration and motility of CA macrophages might limit lesion size due to bystander damage.

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

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Zhenyang [Department of Neurology, The First Affiliated Hospital, Sun Yat-sen University, No. 58, Zhongshan 2nd Road, Guangzhou 510080, Guangdong Province (China); Leng, Yan [Department of Rehabilitation, The First Affiliated Hospital, Sun Yat-sen University, No. 58, Zhongshan 2nd Road, Guangzhou 510080, Guangdong Province (China); Zhou, Chen; Ma, Zhenyu; Zhong, Zhigang [Department of Neurology, The First Affiliated Hospital, Sun Yat-sen University, No. 58, Zhongshan 2nd Road, Guangzhou 510080, Guangdong Province (China); Shi, Xing-Ming [Institute of Molecular Medicine and Genetics, Georgia Health Sciences University, Augusta, GA 30912 (United States); Zhang, Weixi, E-mail: weixizhang@qq.com [Department of Neurology, The First Affiliated Hospital, Sun Yat-sen University, No. 58, Zhongshan 2nd Road, Guangzhou 510080, Guangdong Province (China)

    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.

  9. Implants composed of carbon fiber mesh and bone-marrow-derived, chondrocyte-enriched cultures for joint surface reconstruction.

    Science.gov (United States)

    Robinson, D; Efrat, M; Mendes, D G; Halperin, N; Nevo, Z

    1993-01-01

    The current study integrates two distinct approaches in joint resurfacing into a combined type of implant, composed of carbon fiber mesh impregnated and coated with a hyaluronic-acid-based delivery substance containing cultured cells. Rabbit autogeneic chondrocyte-enriched cultures obtained from mesenchymal stem cells (chondroprogenitor cells) derived from adult rabbit bone marrow were grown in vitro under conditions favoring chondrogenesis. The improvement in quality of repair when a combined implant containing both cells and a carbon scaffold was used, in comparison to the utilization of carbon fiber mesh alone, was clearly demonstrated using clinical, histological, biochemical, and biomechanical examinations. Evaluations of the joints were performed at 6 weeks and 6 months after implantation. The repair tissue in the cell-implanted joints consisted of a typical hyaline cartilage, which was more cellular and thicker than the repair tissue in the hyaluronic-acid-impregnated carbon-fiber-implanted control joints. The hyaline cartilage in the experimental group formed a superficial layer above the carbon fibers, flush with the joint surface. In the controls, in which carbon fiber and the delivery substance alone were implanted, a histologically and biochemically fibrous tissue that was inferior biomechanically to the new cartilage was formed by the cells containing implants.

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

    Energy Technology Data Exchange (ETDEWEB)

    Luan Xiying [Institute of Medical Biotechnology, Jiangsu Province Key Laboratory of Stem Cell, Suzhou University, Suzhou 215007 (China); Wang Yong [Institute of Medical Biotechnology, Jiangsu Province Key Laboratory of Stem Cell, Suzhou University, Suzhou 215007 (China); Duan Xiang [Institute of Medical Biotechnology, Jiangsu Province Key Laboratory of Stem Cell, Suzhou University, Suzhou 215007 (China); Duan Qiaoyan [Institute of Medical Biotechnology, Jiangsu Province Key Laboratory of Stem Cell, Suzhou University, Suzhou 215007 (China); Li Mingzhong [School of Materials Engineering, Suzhou University, Suzhou 215006 (China); Lu Shenzhou [School of Materials Engineering, Suzhou University, Suzhou 215006 (China); Zhang Huanxiang [Institute of Medical Biotechnology, Jiangsu Province Key Laboratory of Stem Cell, Suzhou University, Suzhou 215007 (China); Zhang Xueguang [Institute of Medical Biotechnology, Jiangsu Province Key Laboratory of Stem Cell, Suzhou University, Suzhou 215007 (China)

    2006-12-15

    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.

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

    Science.gov (United States)

    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.

  12. Sonic Hedgehog Produced by Bone Marrow-Derived Mesenchymal Stromal Cells Supports Cell Survival in Myelodysplastic Syndrome

    Directory of Open Access Journals (Sweden)

    Jixue Zou

    2015-01-01

    Full Text Available The role of marrow microenvironment in the pathogenesis of myelodysplastic syndrome (MDS remains controversial. Therefore, we studied the influence of bone marrow-derived mesenchymal stromal cells (BMSCs from patients with different risk types of MDS on the survival of the MDS cell lines SKM-1 and MUTZ-1. We first demonstrated that the expression of Sonic hedgehog (Shh, smoothened (Smo, and glioma-associated oncogene homolog 1 (Gli1 was increased in MDS patients n=23; the increase in expression was positively correlated with the presence of high-risk factors. The Shh signaling inhibitor, cyclopamine, inhibited high-risk MDS BMSC-induced survival of SKM-1 and MUTZ-1 cells, suggesting a role for Shh signaling in MDS cell survival. Furthermore, cyclopamine-mediated inhibition of Shh signaling in SKM-1 and MUTZ-1 cells resulted in decreased DNMT1 expression and cell survival; however, exogenous Shh peptide had the opposite effect, suggesting that Shh signaling could regulate the expression of DNMT1, thereby modulating cell survival in MDS. In addition, the apoptosis of SKM-1 and MUTZ-1 cell increased significantly when cultured with cyclopamine and a demethylation agent, 5-Aza-2′-deoxycytidine. These findings suggest that Shh signaling from BMSCs is important in the pathogenesis of MDS and could play a role in disease progression by modulating methylation.

  13. Collection of Macaca fascicularis cDNAs derived from bone marrow, kidney, liver, pancreas, spleen, and thymus

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    Kameoka Yosuke

    2009-09-01

    Full Text Available Abstract Background Consolidating transcriptome data of non-human primates is essential to annotate primate genome sequences, and will facilitate research using non-human primates in the genomic era. Macaca fascicularis is a macaque monkey that is commonly used for biomedical and ecological research. Findings We constructed cDNA libraries of Macaca fascicularis, derived from tissues obtained from bone marrow, liver, pancreas, spleen, and thymus of a young male, and kidney of a young female. In total, 5'-end sequences of 56,856 clones were determined. Including the previously established cDNA libraries from brain and testis, we have isolated 112,587 cDNAs of Macaca fascicularis, which correspond to 56% of the curated human reference genes. Conclusion These sequences were deposited in the public sequence database as well as in-house macaque genome database http://genebank.nibio.go.jp/qfbase/. These data will become valuable resources for identifying functional parts of the genome of macaque monkeys in future studies.

  14. The cellular prion protein negatively regulates phagocytosis and cytokine expression in murine bone marrow-derived macrophages.

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    Min Wang

    Full Text Available The cellular prion protein (PrP(C is a glycosylphosphatidylinositol (GPI-anchored glycoprotein on the cell surface. Previous studies have demonstrated contradictory roles for PrP(C in connection with the phagocytic ability of macrophages. In the present work, we investigated the function of PrP(C in phagocytosis and cytokine expression in bone marrow-derived macrophages infected with Escherichia coli. E. coli infection induced an increase in the PRNP mRNA level. Knockout of PrP(C promoted bacterial uptake; upregulated Rab5, Rab7, and Eea1 mRNA expression; and increased the recruitment of lysosomal-associated membrane protein-2 to phagosomes, suggesting enhanced microbicidal activity. Remarkably, knockout of PrP(C suppressed the proliferation of internalized bacteria and increased the expression of cytokines such as interleukin-1β. Collectively, our data reveal an important role of PrP(C as a negative regulator for phagocytosis, phagosome maturation, cytokine expression, and macrophage microbicidal activity.

  15. Osteogenic induction of bone marrow-derived stromal cells on simvastatin-releasing, biodegradable, nano- to microscale fiber scaffolds.

    Science.gov (United States)

    Wadagaki, Ryu; Mizuno, Daiki; Yamawaki-Ogata, Aika; Satake, Makoto; Kaneko, Hiroaki; Hagiwara, Sumitaka; Yamamoto, Noriyuki; Narita, Yuji; Hibi, Hideharu; Ueda, Minoru

    2011-07-01

    Tissue engineering is an effective approach for the treatment of bone defects. Statins have been demonstrated to promote osteoblastic differentiation of bone marrow-derived stromal cells (BMSCs). Electrospun biodegradable fibers have also shown applicability to drug delivery in the form of bone tissue engineered scaffolds with nano- to microscale topography and high porosity similar to the natural extracellular matrix (ECM). The aim of this study was to investigate the feasibility of a simvastatin-releasing, biodegradable, nano- to microscale fiber scaffold (SRBFS) for bone tissue engineering with BMSCs. Simvastatin was released from SRBFS slowly. BMSCs were observed to spread actively and rigidly adhere to SRBFS. BMSCs on SRBFS showed an increase in alkaline phosphatase activity 2 weeks after cell culture. Furthermore, osteoclastogenesis was suppressed by SRBFS in vitro. The new bone formation and mineralization in the SRBFS group were significantly better than in the biodegradable fiber scaffold (BFS) without simvastatin 12 weeks after implantation of the cell-scaffold construct into an ectopic site on the murine back. These results suggest that SRBFS promoted osteoblastic differentiation of BMSCs in vitro and in vivo, and demonstrate feasibility as a bone engineering scaffold.

  16. Gomisin N has anti-allergic effect and inhibits inflammatory cytokine expression in mouse bone marrow-derived mast cells.

    Science.gov (United States)

    Chae, Hee-Sung; Kang, Ok-Hwa; Oh, You-Chang; Choi, Jang-Gi; Keum, Joon-Ho; Kim, Sung-Bae; Kim, Yong-Sik; Mun, Su-Hyun; Shin, Dong-Won; Han, Sin-Hee; Kwon, Dong-Yeul

    2011-12-01

    Gomisin N is a bioactive compound and a prominent anti-allergic agent found in the fruits of tree Schizandra chinensis. However, its effects on the bone marrow-derived mast cell (BMMC)-mediated allergy and inflammation mechanism remain unknown. In this study, the biological effects of gomisin were evaluated while focusing on its effects on the allergic mediator in PMA + A23187-stimulated BMMCs. The anti-allergic effect of gomisin has shown that inhibited PMA + A23187-induced interleukin-6 (IL-6) production. An investigation was also conducted to determine its effects on the production of several allergic mediators including prostaglandin D(2) (PGD(2)), leukotriene C(4) (LTC(4)), β-hexosaminidase (β-Hex), and cyclooxygenase-2 (COX-2) protein. The results revealed that gomisin inhibited the PMA + A23187-induced production of IL-6, PGD(2), LTC(4), β-Hex, and COX-2 protein. Taken together, these findings indicate that gomisin N has the potential for use in the treatment of allergy.

  17. The Effect of EPO Gene Overexpression on Proliferation and Migration of Mouse Bone Marrow-Derived Mesenchymal Stem Cells.

    Science.gov (United States)

    Lin, Haihong; Luo, Xinping; Jin, Bo; Shi, Haiming; Gong, Hui

    2015-04-01

    The aim of this study is to investigate the effect of erythropoietin (EPO) gene overexpression on proliferation and migration of mouse bone marrow-derived mesenchymal stem cells (MSCs), and to determine the underlying signaling pathway. Mouse MSCs were cultured in vitro and EPO gene was transfected into the 6th generation of MSCs via lentivirus vector. The transfected cells were identified by flow cytometry and the EPO levels in supernatant were measured with ELISA. In addition, cell proliferation was assessed by CCK-8 assay and cell migration was evaluated by Transwell assay. The activation of Akt, ERK1/2, and p38MAPK signaling was detected by western blotting. The lentivirus vector containing EPO was successfully constructed and transfected into MSCs. No remarkable change was found in the cell surface markers after transfection while a significant increase of EPO level in supernatant was noticed in transfected MSCs compared to controls (P EPO modification enhanced the phosphorylation of PI3K/Akt and ERK signaling pathway, and suppressed the phosphorylation of p38MAPK without affecting the levels of total Akt, ERK1/2, and p38MAPK in MSCs. After transfection, MSCs secreted more EPO which enhanced the capability of proliferation and migration. Moreover, our results suggested that the enhanced proliferation and migration might be associated with activation of PI3K/Akt and ERK or inhibition of P38MAPK signaling pathway.

  18. Stem Cell Ophthalmology Treatment Study (SCOTS): bone marrow-derived stem cells in the treatment of Leber's hereditary optic neuropathy

    Science.gov (United States)

    Weiss, Jeffrey N.; Levy, Steven; Benes, Susan C.

    2016-01-01

    The Stem Cell Ophthalmology Treatment Study (SCOTS) is currently the largest-scale stem cell ophthalmology trial registered at ClinicalTrials.gov (identifier: NCT01920867). SCOTS utilizes autologous bone marrow-derived stem cells (BMSCs) to treat optic nerve and retinal diseases. Treatment approaches include a combination of retrobulbar, subtenon, intravitreal, intra-optic nerve, subretinal, and intravenous injection of autologous BMSCs according to the nature of the disease, the degree of visual loss, and any risk factors related to the treatments. Patients with Leber's hereditary optic neuropathy had visual acuity gains on the Early Treatment Diabetic Retinopathy Study (ETDRS) of up to 35 letters and Snellen acuity improvements from hand motion to 20/200 and from counting fingers to 20/100. Visual field improvements were noted. Macular and optic nerve head nerve fiber layer typically thickened. No serious complications were seen. The increases in visual acuity obtained in our study were encouraging and suggest that the use of autologous BMSCs as provided in SCOTS for ophthalmologic mitochondrial diseases including Leber's hereditary optic neuropathy may be a viable treatment option. PMID:27904503

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

    Science.gov (United States)

    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.

  20. Altered membrane dynamics of quantum dot-conjugated integrins during osteogenic differentiation of human bone marrow derived progenitor cells.

    Science.gov (United States)

    Chen, Hongfeng; Titushkin, Igor; Stroscio, Michael; Cho, Michael

    2007-02-15

    Functionalized quantum dots offer several advantages for tracking the motion of individual molecules on the cell surface, including selective binding, precise optical identification of cell surface molecules, and detailed examination of the molecular motion without photobleaching. We have used quantum dots conjugated with integrin antibodies and performed studies to quantitatively demonstrate changes in the integrin dynamics during osteogenic differentiation of human bone marrow derived progenitor cells (BMPCs). Consistent with the unusually strong BMPC adhesion previously observed, integrins on the surface of undifferentiated BMPC were found in clusters and the lateral diffusion was slow (e.g., approximately 10(-11) cm2/s). At times as early as those after a 3-day incubation in the osteogenic differentiation media, the integrin diffusion coefficients increased by an order of magnitude, and the integrin dynamics became indistinguishable from that measured on the surface of terminally differentiated human osteoblasts. Furthermore, microfilaments in BMPCs consisted of atypically thick bundles of stress fibers that were responsible for restricting the integrin lateral mobility. Studies using laser optical tweezers showed that, unlike fully differentiated osteoblasts, the BMPC cytoskeleton is weakly associated with its cell membrane. Based on these findings, it appears likely that the altered integrin dynamics is correlated with BMPC differentiation and that the integrin lateral mobility is restricted by direct links to microfilaments.

  1. Stromal Derived Factor-1/CXCR4 Axis Involved in Bone Marrow Mesenchymal Stem Cells Recruitment to Injured Liver

    Directory of Open Access Journals (Sweden)

    Kuai Xiao Ling

    2016-01-01

    Full Text Available The molecular mechanism of bone marrow mesenchymal stromal stem cells (BMSCs mobilization and migration to the liver was poorly understood. Stromal cell-derived factor-1 (SDF-1 participates in BMSCs homing and migration into injury organs. We try to investigate the role of SDF-1 signaling in BMSCs migration towards injured liver. The expression of CXCR4 in BMSCs at mRNA level and protein level was confirmed by RT-PCR, flow cytometry, and immunocytochemistry. The SDF-1 or liver lysates induced BMSCs migration was detected by transwell inserts. CXCR4 antagonist, AMD3100, and anti-CXCR4 antibody were used to inhibit the migration. The Sprague-Dawley rat liver injury model was established by intraperitoneal injection of thioacetamide. The concentration of SDF-1 increased as modeling time extended, which was determined by ELISA method. The Dir-labeled BMSCs were injected into the liver of the rats through portal vein. The cell migration in the liver was tracked by in vivo imaging system and the fluorescent intensity was measured. In vivo, BMSCs migrated into injured liver which was partially blocked by AMD3100 or anti-CXCR4 antibody. Taken together, the results demonstrated that the migration of BMSCs was regulated by SDF-1/CXCR4 signaling which involved in BMSCs recruitment to injured liver.

  2. Intratracheal therapy with autologous bone marrow-derived mononuclear cells reduces airway inflammation in horses with recurrent airway obstruction.

    Science.gov (United States)

    Barussi, Fernanda C M; Bastos, Fernanda Z; Leite, Lidiane M B; Fragoso, Felipe Y I; Senegaglia, Alexandra C; Brofman, Paulo R S; Nishiyama, Anita; Pimpão, Cláudia T; Michelotto, Pedro V

    2016-10-01

    This research evaluated the effects of bone marrow-derived mononuclear cells (BMMCs) on the inflammatory process in the equine recurrent airway obstruction (RAO). Eight horses in RAO clinical score were divided into cell therapy group (Gcel) treated with a single intratracheal dose of BMMCs, and dexamethasone group (Gdex) treated with 21days of oral dexamethasone. The horses were clinically revaluated on days 7 and 21, together with cytological evaluation of the BALF, and detection of inflammatory markers (interleukins [IL]-10, -4, and -17, and interferon γ and α). There were decreases in respiratory effort and clinical score on days 7 and 21(p<0.05) for both groups. The percentage of neutrophils decreased and macrophages increased on days 7 and 21 (p<0.005) in both groups. IL-10 levels increased in the Gcel group on day 21 compared to days 0 and 7 (p<0.05), but this was not observed in the Gdex group. The quantification of IL-4, IL-17, IFN-γ, and IFN-α did not change between evaluations in both groups. These preliminary results suggest that BMMCs may ameliorate the inflammatory response of RAO.

  3. Murine bone marrow-derived dendritic cells as a potential in vitro model for predictive identification of chemical sensitizers.

    Science.gov (United States)

    Pépin, Elsa; Goutet, Michèle; Ban, Masarin

    2007-12-10

    The identification of potential sensitizing chemicals is a key step in the safety assessment process. To this end, predictive tests that require no or few animals and that are reliable, inexpensive and easy to perform are needed. The aim of this study was to evaluate the performance of murine bone marrow-derived dendritic cells (BMDCs) in an in vitro skin sensitization model. BMDCs were exposed to six well-known allergens (dinitrochlorobenzene, DNCB; dinitrofluorobenzene, DNFB; Bandrowski's base, BB; paraphenylenediamine, PPD; nickel sulfate, NiSO(4); cinnamaldehyde, Cinn). Surface expression of MHC class II, CD40, CD54, and CD86 was measured by flow cytometry after 48h exposure to these chemicals. All the allergens tested induced a significant increase in marker expression, with an augmentation in the percentage of mature cells ranging from 2.3- to 10.5-fold change over control. The level of up-regulation was dependent on the concentration and the strength of the allergens. In contrast, the irritants (sodium dodecyl sulfate, SDS and 4-aminobenzoic acid, pABA) and the negative control (zinc sulfate, ZnSO(4)) tested induced either no modification or a down-regulation of membrane marker expression. Taken together, our data suggest that murine BMDCs may represent a new and valuable in vitro model to predict the sensitizing properties of chemicals.

  4. Bone marrow-derived mesenchymal stem cells enhance angiogenesis via their α6β1 integrin receptor.

    Science.gov (United States)

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

    2013-11-15

    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.

  5. Three-dimensional graphene foams loaded with bone marrow derived mesenchymal stem cells promote skin wound healing with reduced scarring.

    Science.gov (United States)

    Li, Zhonghua; Wang, Haiqin; Yang, Bo; Sun, Yukai; Huo, Ran

    2015-12-01

    The regeneration of functional skin remains elusive, due to poor engraftment, deficient vascularization, and excessive scar formation. Aiming to overcome these issues, the present study proposed the combination of a three-dimensional graphene foam (GF) scaffold loaded with bone marrow derived mesenchymal stem cells (MSCs) to improve skin wound healing. The GFs demonstrated good biocompatibility and promoted the growth and proliferation of MSCs. Meanwhile, the GFs loaded with MSCs obviously facilitated wound closure in animal model. The dermis formed in the presence of the GF structure loaded with MSCs was thicker and possessed a more complex structure at day 14 post-surgery. The transplanted MSCs correlated with upregulation of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), which may lead to neo-vascularization. Additionally, an anti-scarring effect was observed in the presence of the 3D-GF scaffold and MSCs, as evidenced by a downregulation of transforming growth factor-beta 1 (TGF-β1) and alpha-smooth muscle actin (α-SMA) together with an increase of TGF-β3. Altogether, the GF scaffold could guide the wound healing process with reduced scarring, and the MSCs were crucial to enhance vascularization and provided a better quality neo-skin. The GF scaffold loaded with MSCs possesses necessary bioactive cues to improve wound healing with reduced scarring, which may be of great clinical significance for skin wound healing.

  6. Altered microRNA expression profile in exosomes during osteogenic differentiation of human bone marrow-derived mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Ji-Feng Xu

    Full Text Available 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.

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

    Science.gov (United States)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    Francis, W.R., E-mail: w.francis@swansea.ac.uk [Institute of Life Science, College of Medicine, Swansea University (United Kingdom); Owens, S.E.; Wilde, C. [Institute of Life Science, College of Medicine, Swansea University (United Kingdom); Pallister, I. [Institute of Life Science, College of Medicine, Swansea University (United Kingdom); Trauma and Orthopaedics, Morriston Hospital, Swansea (United Kingdom); Kanamarlapudi, V. [Institute of Life Science, College of Medicine, Swansea University (United Kingdom); Zou, W., E-mail: weizou60@hotmail.com [College of Life Sciences, Liaoning Normal University, Dalian 116081 (China); Liaoning Key Laboratories of Biotechnology and Molecular Drug Research and Development, Dalian 116081 (China); Xia, Z. [Institute of Life Science, College of Medicine, Swansea University (United Kingdom)

    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.

  9. Bone marrow-derived cells participate in stromal remodeling of the lung following acute bacterial pneumonia in mice.

    Science.gov (United States)

    Serikov, Vladimir B; Mikhaylov, Viatcheslav M; Krasnodembskay, Anna D; Matthay, Michael A

    2008-01-01

    Bone marrow-derived cells (BMDC) have been shown to graft injured tissues, differentiate in specialized cells, and participate in repair. The importance of these processes in acute lung bacterial inflammation and development of fibrosis is unknown. The goal of this study was to investigate the temporal sequence and lineage commitment of BMDC in mouse lungs injured by bacterial pneumonia. We transplanted GFP-tagged BMDC into 5-Gy-irradiated C57BL/6 mice. After 3 months of recovery, mice were subjected to LD(50) intratracheal instillation of live E. coli (controls received saline) which produced pneumonia and subsequent areas of fibrosis. Lungs were investigated by immunohistology for up to 6 months. At the peak of lung inflammation, the predominant influx of BMDC were GFP(+) leukocytes. Postinflammatory foci of lung fibrosis were evident after 1-2 months. The fibrotic foci in lung stroma contained clusters of GFP(+) CD45(+) cells, GFP(+) vimentin-positive cells, and GFP(+) collagen I-positive fibroblasts. GFP(+) endothelial or epithelial cells were not identified. These data suggest that following 5-Gy irradiation and acute bacterial pneumonia, BMDC may temporarily participate in lung postinflammatory repair and stromal remodeling without long-term engraftment as specialized endothelial or epithelial cells.

  10. A double blind randomized placebo controlled phase I/II study assessing the safety and efficacy of allogeneic bone marrow derived mesenchymal stem cell in critical limb ischemia

    OpenAIRE

    Gupta, Pawan K; Chullikana, Anoop; Parakh, Rajiv; Desai, Sanjay; Das, Anjan; Gottipamula, Sanjay; Krishnamurthy, Sagar; Anthony, Naveen; Pherwani, Arun; Majumdar, Anish S

    2013-01-01

    Background Peripheral vascular disease of the lower extremities comprises a clinical spectrum that extends from no symptoms to presentation with critical limb ischemia (CLI). Bone marrow derived Mesenchymal Stem Cells (BM- MSCs) may ameliorate the consequences of CLI due to their combinatorial potential for inducing angiogenesis and immunomodulatory environment in situ. The primary objective was to determine the safety of BM- MSCs in patients with CLI. Methods Prospective, double blind random...

  11. The Effect of Bone Marrow-Derived Mesenchymal Stem Cells and Their Conditioned Media Topically Delivered in Fibrin Glue on Chronic Wound Healing in Rats

    OpenAIRE

    Mehanna, Radwa A.; Iman Nabil; Noha Attia; Bary, Amany A.; Razek, Khalid A.; Ahmed, Tamer A. E.; Fatma Elsayed

    2015-01-01

    Bone marrow-derived mesenchymal stem cells (BM-MSCs) represent a modern approach for management of chronic skin injuries. In this work, we describe BM-MSCs application versus their conditioned media (CM) when delivered topically admixed with fibrin glue to enhance the healing of chronic excisional wounds in rats. Fifty-two adult male rats were classified into four groups after induction of large-sized full-thickness skin wound: control group (CG), fibrin only group (FG), fibrin + MSCs group (...

  12. The osteogenic differentiation of human bone marrow MSCs on HUVEC-derived ECM and β-TCP scaffold.

    Science.gov (United States)

    Kang, Yunqing; Kim, Sungwoo; Bishop, Julius; Khademhosseini, Ali; Yang, Yunzhi

    2012-10-01

    Extracellular matrix (ECM) serves a key role in cell migration, attachment, and cell development. Here we report that ECM derived from human umbilical vein endothelial cells (HUVEC) promoted osteogenic differentiation of human bone marrow mesenchymal stem cells (hMSC). We first produced an HUVEC-derived ECM on a three-dimensional (3D) beta-tricalcium phosphate (β-TCP) scaffold by HUVEC seeding, incubation, and decellularization. The HUVEC-derived ECM was then characterized by SEM, FTIR, XPS, and immunofluorescence staining. The effect of HUVEC-derived ECM-containing β-TCP scaffold on hMSC osteogenic differentiation was subsequently examined. SEM images indicate a dense matrix layer deposited on the surface of struts and pore walls. FTIR and XPS measurements show the presence of new functional groups (amide and hydroxyl groups) and elements (C and N) in the ECM/β-TCP scaffold when compared to the β-TCP scaffold alone. Immunofluorescence images indicate that high levels of fibronectin and collagen IV and low level of laminin were present on the scaffold. ECM-containing β-TCP scaffolds significantly increased alkaline phosphatase (ALP) specific activity and up-regulated expression of osteogenesis-related genes such as runx2, alkaline phosphatase, osteopontin and osteocalcin in hMSC, compared to β-TCP scaffolds alone. This increased effect was due to the activation of MAPK/ERK signaling pathway since disruption of this pathway using an ERK inhibitor PD98059 results in down-regulation of these osteogenic genes. Cell-derived ECM-containing calcium phosphate scaffolds is a promising osteogenic-promoting bone void filler in bone tissue regeneration.

  13. Activin receptor-like kinase receptors ALK5 and ALK1 are both required for TGFβ-induced chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells

    NARCIS (Netherlands)

    L.M.G. De Kroon (Laurie); R. Narcisi (Roberto); E.N. Blaney Davidson (Esmeralda); M.A. Cleary (Mairéad); H.M. van Beuningen (Henk); W.J.L.M. Koevoet (Wendy J.L.M.); G.J.V.M. van Osch (Gerjo); P.M. van der Kraan (Peter)

    2015-01-01

    textabstractIntroduction Bone marrow-derived mesenchymal stem cells (BMSCs) are promising for cartilage regeneration because BMSCs can differentiate into cartilage tissue-producing chondrocytes. Transforming Growth Factor beta; (TGFbeta;) is crucial for inducing chondrogenic differentiation of BMSCs

  14. Epidermis–dermis junction as a novel location for bone marrow-derived cells to reside in response to ionizing radiation

    Energy Technology Data Exchange (ETDEWEB)

    Okano, Junko, E-mail: jokano@belle.shiga-med.ac.jp [Division of Anatomy and Cell Biology, Shiga University of Medical Science, Shiga (Japan); Kojima, Hideto; Katagi, Miwako [Department of Stem Cell Biology and Regenerative Medicine, Shiga University of Medical Science, Shiga (Japan); Nakae, Yuki [Department of Internal Medicine, Shiga University of Medical Science, Shiga (Japan); Terashima, Tomoya [Department of Stem Cell Biology and Regenerative Medicine, Shiga University of Medical Science, Shiga (Japan); Nakagawa, Takahiko [TMK Project, Medical Innovation Center, Kyoto University Graduate School of Medicine, Kyoto (Japan); Kurakane, Takeshi; Okamoto, Naoki; Morohashi, Keita [Division of Anatomy and Cell Biology, Shiga University of Medical Science, Shiga (Japan); Maegawa, Hiroshi [Department of Internal Medicine, Shiga University of Medical Science, Shiga (Japan); Udagawa, Jun [Division of Anatomy and Cell Biology, Shiga University of Medical Science, Shiga (Japan)

    2015-06-12

    Bone marrow-derived cells (BMDCs) can migrate into the various organs in the mice irradiated by ionizing radiation (IR). However, it may not be the case in the skin. While IR is used for bone marrow (BM) transplantation, studying with the epidermal sheets demonstrated that the BMDC recruitment is extraordinarily rare in epidermis in the mouse. Herein, using the chimera mice with BM from green fluorescent protein (GFP) transgenic mice, we simply examined if BMDCs migrate into any layers in the total skin, as opposed to the epidermal sheets, in response to IR. Interestingly, we identified the presence of GFP-positive (GFP{sup +}) cells in the epidermis-dermis junction in the total skin sections although the epidermal cell sheets failed to have any GFP cells. To examine a possibility that the cells in the junction could be mechanically dissociated during separating epidermal sheets, we then salvaged such dissociated cells and examined its characteristics. Surprisingly, some GFP{sup +} cells were found in the salvaged cells, indicating that these cells could be derived from BM. In addition, such BMDCs were also associated with inflammation in the junction. In conclusion, BMDCs can migrate to and reside in the epidermis-dermis junction after IR. - Highlights: • Bone marrow-derived cells (BMDCs) migrate in the epidermis due to ionizing radiation (IR). • BMDCs dissociate from the epidermis-dermis junction in preparing epidermal sheets. • The doses of IR determine the location and the number of migrating BMDCs in the skin.

  15. Good manufacturing practice-compliant animal-free expansion of human bone marrow derived mesenchymal stroma cells in a closed hollow-fiber-based bioreactor.

    Science.gov (United States)

    Nold, Philipp; Brendel, Cornelia; Neubauer, Andreas; Bein, Gregor; Hackstein, Holger

    2013-01-01

    Mesenchymal stroma cells (MSC) are increasingly recognized for various applications of cell-based therapies such as regenerative medicine or immunomodulatory treatment strategies. Standardized large-scale expansions of MSC under good manufacturing practice (GMP)-compliant conditions avoiding animal derived components are mandatory for further evaluation of these novel therapeutic approaches in clinical trials. We applied a novel automated hollow fiber cell expansion system (CES) for in vitro expansion of human bone marrow derived MSC employing a GMP-compliant culture medium with human platelet lysate (HPL). Between 8 and 32 ml primary bone marrow aspirate were loaded into the hollow fiber CES and cultured for 15-27 days. 2-58 million MSC were harvested after primary culture. Further GMP-compliant cultivation of second passage MSC for 13 days led to further 10-20-fold enrichment. Viability, surface antigen expression, differentiation capacity and immunosuppressive function of MSC cultured in the hollow fiber CES were in line with standard criteria for MSC definition. We conclude that MSC can be enriched from primary bone marrow aspirate in a GMP-conform manner within a closed hollow fiber bioreactor and maintain their T lymphocyte inhibitory capacity. Standardized and reliable conditions for large scale MSC expansion pave the way for safe applications in humans in different therapeutic approaches.

  16. Synergistic and Superimposed Effect of Bone Marrow-Derived Mesenchymal Stem Cells Combined with Fasudil in Experimental Autoimmune Encephalomyelitis.

    Science.gov (United States)

    Yu, Jing-Wen; Li, Yan-Hua; Song, Guo-Bin; Yu, Jie-Zhong; Liu, Chun-Yun; Liu, Jian-Chun; Zhang, Hai-Fei; Yang, Wan-Fang; Wang, Qing; Yan, Ya-Ping; Xiao, Bao-Guo; Ma, Cun-Gen

    2016-12-01

    Bone marrow-derived mesenchymal stem cells (MSCs) are the ideal transplanted cells of cellular therapy for promoting neuroprotection and neurorestoration. However, the optimization of transplanted cells and the improvement of microenvironment around implanted cells are still two critical challenges for enhancing therapeutic effect. In the current study, we observed the therapeutic potential of MSCs combined with Fasudil in mouse model of experimental autoimmune encephalomyelitis (EAE) and explored possible mechanisms of action. The results clearly show that combined intervention of MSCs and Fasudil further reduced the severity of EAE compared with MSCs or Fasudil alone, indicating a synergistic and superimposed effect in treating EAE. The addition of Fasudil inhibited MSC-induced inflammatory signaling TLR-4/MyD88 and inflammatory molecule IFN-γ, IL-1β, and TNF-α but did not convert M1 microglia to M2 phenotype. The delivery of MSCs enhanced the expression of glial cell-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) compared with that of Fasudil. Importantly, combined intervention of MSCs and Fasudil further increased the expression of BDNF and GDNF compared with the delivery of MSCs alone, indicating that combined intervention of MSCs and Fasudil synergistically contributes to the expression of neurotrophic factors which should be related to the expression of increased galactocerebroside (GalC) compared with mice treated with Fasudil and MSCs alone. However, a lot of investigation is warranted to further elucidate the cross talk of MSCs and Fasudil in the therapeutic potential of EAE/multiple sclerosis.

  17. Mouse bone marrow-derived dendritic cells can phagocytize the Sporothrix schenckii, and mature and activate the immune response by secreting interleukin-12 and presenting antigens to T lymphocytes.

    Science.gov (United States)

    Kusuhara, Masahiro; Qian, Hua; Li, Xiaoguang; Tsuruta, Daisuke; Tsuchisaka, Atsunari; Ishii, Norito; Ohata, Chika; Furumura, Minao; Hashimoto, Takashi

    2014-05-01

    In sporotrichosis, dermal dendritic cells were considered to participate in induction of the immune responses against Sporothrix schenckii infection. However, it is still unclear whether and how dermal dendritic cells were involved in the progress. To clarify the pathogenic role of dermal dendritic cells (DC) in sporotrichosis, we examined the phagocytosis, maturation stages, cytokine production and antigen-presenting ability of mouse bone marrow-derived DC after stimulation with S. schenckii. By analysis of flow cytometry, electron microscope and confocal microscope, mouse bone marrow-derived DC were proved to be able to phagocytize the S. schenckii. The increased expression of CD40, CD80 and CD86 on the surface of S. schenckii-pulsed mouse bone marrow-derived DC was detected by flow cytometer, indicating that the S. schenckii-pulsed mouse bone marrow-derived DC underwent the maturation program. The secretory enhancement of interleukin (IL)-12, but not IL-4, was found in S. schenckii-pulsed mouse bone marrow-derived DC, suggesting the possible activation of T-helper 1 prone immune responses. Furthermore, S. schenckii-pulsed mouse bone marrow-derived DC were demonstrated to be capable of inducing the proliferation of T lymphocytes from BALB/c mice that were pre-sensitized with S. schenckii. Together, all the results implied that dermal DC may participate in the induction of immune responses against S. schenckii infection in sporotrichosis.

  18. Isolation, expansion and characterization of bone marrow-derived mesenchymal stromal cells in serum-free conditions.

    Science.gov (United States)

    Gottipamula, Sanjay; Ashwin, K M; Muttigi, Manjunatha S; Kannan, Suresh; Kolkundkar, Udaykumar; Seetharam, Raviraja N

    2014-04-01

    Bone marrow-derived mesenchymal stromal cells (BM-MSCs) heralded a new beginning for regenerative medicine and generated tremendous interest as the most promising source for therapeutic application. Most cell therapies require stringent regulatory compliance and prefer the use of serum-free media (SFM) or xeno-free media (XFM) for the MSC production process, starting from the isolation onwards. Here, we report on serum-free isolation and expansion of MSCs and compare them with cells grown in conventional fetal bovine serum (FBS)-containing media as a control. The isolation, proliferation and morphology analysis demonstrated significant differences between MSCs cultured in various SFM/XFM in addition to their difference with FBS controls. BD Mosaic™ Mesenchymal Stem Cell Serum-Free media (BD-SFM) and Mesencult-XF (MSX) supported the isolation, sequential passaging, tri-lineage differentiation potential and acceptable surface marker expression profile of BM-MSCs. Further, MSCs cultured in SFM showed higher immune suppression and hypo-immunogenicity properties, making them an ideal candidate for allogeneic cell therapy. Although cells cultured in control media have a significantly higher proliferation rate, BM-MSCs cultured in BD-SFM or MSX media are the preferred choice to meet regulatory requirements as they do not contain bovine serum. While BM-MSCs cultured in BD-SFM and MSX media adhered to all MSC characteristics, in the case of few parameters, the performance of cells cultured in BD-SFM was superior to that of MSX media. Pre-clinical safety and efficiency studies are required before qualifying SFM or XFM media-derived MSCs for therapeutic applications.

  19. Human bone marrow-derived adult stem cells for post-myocardial infarction cardiac repair: current status and future directions.

    Science.gov (United States)

    Wei, H M; Wong, P; Hsu, L F; Shim, W

    2009-10-01

    Stem cell-based cell therapy has emerged as a potentially therapeutic option for patients with acute myocardial infarction (AMI) and heart failure. With the completion of a number of trials using bone marrow (BM)-derived adult stem cells, critical examination of the overall clinical benefits, limitations and potential side effects of this revolutionary treatment will pave the way for future clinical research. At present, clinical trials have been conducted almost exclusively using BM stem cells. The primary endpoints of these trials are mainly safety and feasibility, with secondary endpoints in the efficacy of post-myocardial infarction (MI) cardiac repair. Intervention with BM-derived cells was mainly carried out by endogenously-mobilised BM cells with granulocyte-colony stimulating factor, and more frequently, by intracoronary infusion or direct intramyocardial injection of autologous BM cells. While these studies have been proven safe and feasible without notable side effects, mixed outcomes in terms of clinical benefits have been reported. The major clinical benefits observed are improved cardiac contractile function and suppressed left ventricular negative remodelling, including reduced infarct size and improved cardiac perfusion of infarct zone. Moderate and transient clinical benefits have been mostly observed in studies with intracoronary infusion or direct intramyocardial injection of BM cells. These effects are widely considered to be indirect effects of implanted cells in association with paracrine factors, cell fusion, passive ventricular remodelling, or the responses of endogenous cardiac stem cells. In contrast, evidence of cardiac regeneration characterised by differentiation of implanted stem cells into cardiomyocytes and other cardiac cell lineages, is weak or lacking. To elucidate a clear risk-benefit of this exciting therapy, future studies on the mechanisms of cardiac cell therapy will need to focus on confirming the ideal cell types in relation

  20. IDENTIFICATION AND KINETICS OF 2 RECENTLY BONE-MARROW-DERIVED B-CELL POPULATIONS IN PERIPHERAL LYMPHOID-TISSUES

    NARCIS (Netherlands)

    KROESE, FGM; DEBOER, NK; DEBOER, T; NIEUWENHUIS, P; KANTOR, AB; DEENEN, GJ

    1995-01-01

    In rats, the glycoprotein Thy-1 is expressed on recently bone marrow (BM)-generated B cells but not on mature recirculating follicular (RF) B cells. Here we demonstrate that Thy-1(+) B cells consist of two phenotypically distinct, but developmentally related, populations: a population of newly forme

  1. Bone marrow-derived myofibroblasts are the providers of pro-invasive matrix metalloproteinase 13 in primary tumor

    DEFF Research Database (Denmark)

    Lecomte, Julie; Masset, Anne; Blacher, Silvia;

    2012-01-01

    Carcinoma-associated fibroblasts are key contributors of the tumor microenvironment that regulates carcinoma progression. They consist of a heterogeneous cell population with diverse origins, phenotypes, and functions. In the present report, we have explored the contribution of bone marrow (BM)-d...

  2. Microscopic examination and cytokine expression of bone marrow-derived dendritic cells following exposure to low pathogenic avian ionfluenza

    Science.gov (United States)

    Dendritic cells (DC) function as professional antigen presenting cells, and act as sentinels of the immune system. They are a part of the primary immune response to pathogens and help bridge the innate and adaptive immune responses. They are believed to migrate from bone marrow into the blood stre...

  3. Elutriated stem cells derived from the adult bone marrow differentiate into insulin-producing cells in vivo and reverse chemical diabetes.

    Science.gov (United States)

    Iskovich, Svetlana; Goldenberg-Cohen, Nitza; Stein, Jerry; Yaniv, Isaac; Fabian, Ina; Askenasy, Nadir

    2012-01-01

    An ongoing debate surrounds the existence of stem cells in the adult endowed with capacity to differentiate into multiple lineages. We examined the possibility that adult bone marrow cells participate in recovery from chemical diabetes through neogenesis of insulin-producing cells. Small-sized cells negative for lineage markers derived by counterflow centrifugal elutriation from the bone marrow were transplanted into mice made diabetic with streptozotocin and sublethal irradiation. These cells homed efficiently to the injured islets and contributed to tissue revascularization. Islet-homed CD45-negative donor cells identified by sex chromosomes downregulated GFP, expressed PDX-1 and proinsulin, and converted the hormone precursor to insulin. An estimated 7.6% contribution of newly formed insulin-producing cells to islet cellularity increased serum insulin and stabilized glycemic control starting at 5 weeks post-transplant and persisting for 20 weeks. Newly differentiated cells displayed normal diploid genotype and there was no evidence of fusion between the grafted stem cells or their myeloid progeny and injured β-cells. Considering the extensive functional incorporation of insulin-producing donor cells in the injured islets, we conclude that the adult bone marrow contains a subset of small cells endowed with plastic developmental capacity.

  4. Induction of chromosomal aberrations in rat bone marrow cells and mutations in Salmonella typhimurium by benz[a]anthracene derivatives.

    Science.gov (United States)

    Ito, Y; Ueda, N; Maeda, S; Murao, S; Sugiyama, T; Lee, H; Harvey, R G

    1988-09-01

    Benz[a]anthracene (BA) and its derivatives containing methyl and/or ethyl groups in the 7 and/or 12 positions were tested for their ability to induce chromosome aberrations (CA) in rat bone marrow cells and for their mutagenicity to Salmonella typhimurium TA100 or TA98. The incidence of aberrant cells induced by the BA derivatives, given in lipid emulsion as a single-pulse dose of 50 mg/kg body weight into the caudal vein, was in the order: DMBA greater than EMBA greater than MEBA greater than other BA derivatives = control. The alkyl groups, at least 1 methyl group, at the 7 and 12 positions of BA seemed to be necessary to induce CA, although DEBA having ethyl groups at both the 7 and 12 positions of BA did not induce CA. DMBA or EMBA induced not only gaps and breaks but also exchanges and multiple CA, while the CA induced by other BA derivatives consisted of only gaps and breaks. 7MBA and 12MBA which exhibit carcinogenic activity intermediate between that of DMBA and BA induced few CA in the present system. However, the correlation coefficient between the logarithm incidence of aberrant cells and the carcinogenicity index calculated from the data of 9 BA derivatives including both 7MBA and 12MBA was 0.792. The relative mutagenicities of the BA derivatives with TA100 in the presence of hepatic S9 from polychlorinated biphenyl (PCB)-treated rats were in the order: BA greater than 7MBA greater than DMBA greater than 12MBA greater than 7EBA greater than EMBA greater than MEBA greater than 12EBA = DEBA = control. The results with TA98 were essentially the same as those with TA100. The results with TA100 in the presence of hepatic S9 from phenobarbital (PB)-treated rats were in the order: DMBA greater than 12MBA greater than 7MBA greater than 7EBA greater than BA greater than EMBA = MEBA greater than 12EBA = DEBA = control. These findings reveal no obvious relation between the mutagenic activities of the BA derivatives with the PCB-S9 or PB-S9 activating systems and

  5. Comparative evaluation of differentiation potential of menstrual blood- versus bone marrow-derived stem cells into hepatocyte-like cells.

    Directory of Open Access Journals (Sweden)

    Sayeh Khanjani

    Full Text Available Menstrual blood has been introduced as an easily accessible and refreshing stem cell source with no ethical consideration. Although recent works have shown that menstrual blood stem cells (MenSCs possess multi lineage differentiation capacity, their efficiency of hepatic differentiation in comparison to other stem cell resources has not been addressed so far. The aim of this study was to investigate hepatic differentiation capacity of MenSCs compared to bone marrow-derived stem cells (BMSCs under protocols developed by different concentrations of hepatocyte growth factor (HGF and oncostatin M (OSM in combination with other components in serum supplemented or serum-free culture media. Such comparison was made after assessment of immunophenotye, trans-differentiation potential, immunogenicity and tumorigeicity of these cell types. The differential expression of mature hepatocyte markers such as albumin (ALB, cytokeratin 18 (CK-18, tyrosine aminotransferase and cholesterol 7 alpha-hydroxylase activities (CYP7A1 at both mRNA and protein levels in differentiating MenSCs was significantly higher in upper concentration of HGF and OSM (P1 compared to lower concentration of these factors (P2. Moreover, omission of serum during differentiation process (P3 caused typical improvement in functions assigned to hepatocytes in differentiated MenSCs. While up-regulation level of ALB and CYP7A1 was higher in differentiated MenSCs compared to driven BMSCs, expression level of CK-18, detected level of produced ALB and glycogen accumulation were lower or not significantly different. Therefore, based on the overall comparable hepatic differentiation ability of MenSCs with BMSCs, and also accessibility, refreshing nature and lack of ethical issues of MenSCs, these cells could be suggested as an apt and safe alternative to BMSCs for future stem cell therapy of chronic liver diseases.

  6. Bone Marrow-Derived Stem Cell (BMDSC transplantation improves fertility in a murine model of Asherman's syndrome.

    Directory of Open Access Journals (Sweden)

    Feryal Alawadhi

    Full Text Available Asherman's Syndrome is characterized by intrauterine adhesions or fibrosis resulting as a consequence of damage to the basal layer of endometrium and is associated with infertility due to loss of normal endometrium. We have previously shown that bone marrow derived stem cells (BMDSCs engraft the endometrium in mice and humans and Ischemia/reperfusion injury of uterus promoted BMDSCs migration to the endometrium; however, the role of BMDSCs in Asherman's syndrome has not been characterized. Here a murine model of Asherman's syndrome was created by traumatizing the uterus. We evaluate stem cell recruitment and pregnancy after BMDSCs transplantation in a model of Asherman's syndrome. In the Asheman's syndrome model, after BMDSC transplant, the Y chromosome bearing CD45-cells represented less than 0.1% of total endometrial cells. Twice the number of Y+CD45- cells was identified in the damaged uterus compared to the uninjured controls. There was no significant difference between the damaged and undamaged uterine horns in mice that received injury to a single horn. In the BMDSC transplant group, 9 of the 10 mice conceived, while only 3 of 10 in the non-transplanted group conceived (Chi-Square p = 0.0225; all mice in an uninjured control group conceived. The time to conception and mean litter size were not different between groups. Taken together, BMDSCs are recruited to endometrium in response to injury. Fertility improves after BMDSC transplant in Asherman's Syndrome mice, demonstrating a functional role for these cells in uterine repair. BMDSC transplantation is a potential novel treatment for Asherman's Syndrome and may also be useful to prevent Asherman's syndrome after uterine injury.

  7. Atherogenic Cytokines Regulate VEGF-A-Induced Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells into Endothelial Cells

    Science.gov (United States)

    Ikhapoh, Izuagie Attairu; Pelham, Christopher J.; Agrawal, Devendra K.

    2015-01-01

    Coronary artery stenting or angioplasty procedures frequently result in long-term endothelial dysfunction or loss and complications including arterial thrombosis and myocardial infarction. Stem cell-based therapies have been proposed to support endothelial regeneration. Mesenchymal stem cells (MSCs) differentiate into endothelial cells (ECs) in the presence of VEGF-A in vitro. Application of VEGF-A and MSC-derived ECs at the interventional site is a complex clinical challenge. In this study, we examined the effect of atherogenic cytokines (IL-6, TNFα, and Ang II) on EC differentiation and function. MSCs (CD44+, CD73+, CD90+, CD14−, and CD45−) were isolated from the bone marrow of Yucatan microswine. Naïve MSCs cultured in differentiation media containing VEGF-A (50 ng/mL) demonstrated increased expression of EC-specific markers (vWF, PECAM-1, and VE-cadherin), VEGFR-2 and Sox18, and enhanced endothelial tube formation. IL-6 or TNFα caused a dose-dependent attenuation of EC marker expression in VEGF-A-stimulated MSCs. In contrast, Ang II enhanced EC marker expression in VEGF-A-stimulated MSCs. Addition of Ang II to VEGF-A and IL-6 or TNFα was sufficient to rescue the EC phenotype. Thus, Ang II promotes but IL-6 and TNFα inhibit VEGF-A-induced differentiation of MSCs into ECs. These findings have important clinical implications for therapies intended to increase cardiac vascularity and reendothelialize coronary arteries following intervention. PMID:26106428

  8. Atherogenic Cytokines Regulate VEGF-A-Induced Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells into Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Izuagie Attairu Ikhapoh

    2015-01-01

    Full Text Available Coronary artery stenting or angioplasty procedures frequently result in long-term endothelial dysfunction or loss and complications including arterial thrombosis and myocardial infarction. Stem cell-based therapies have been proposed to support endothelial regeneration. Mesenchymal stem cells (MSCs differentiate into endothelial cells (ECs in the presence of VEGF-A in vitro. Application of VEGF-A and MSC-derived ECs at the interventional site is a complex clinical challenge. In this study, we examined the effect of atherogenic cytokines (IL-6, TNFα, and Ang II on EC differentiation and function. MSCs (CD44+, CD73+, CD90+, CD14−, and CD45− were isolated from the bone marrow of Yucatan microswine. Naïve MSCs cultured in differentiation media containing VEGF-A (50 ng/mL demonstrated increased expression of EC-specific markers (vWF, PECAM-1, and VE-cadherin, VEGFR-2 and Sox18, and enhanced endothelial tube formation. IL-6 or TNFα caused a dose-dependent attenuation of EC marker expression in VEGF-A-stimulated MSCs. In contrast, Ang II enhanced EC marker expression in VEGF-A-stimulated MSCs. Addition of Ang II to VEGF-A and IL-6 or TNFα was sufficient to rescue the EC phenotype. Thus, Ang II promotes but IL-6 and TNFα inhibit VEGF-A-induced differentiation of MSCs into ECs. These findings have important clinical implications for therapies intended to increase cardiac vascularity and reendothelialize coronary arteries following intervention.

  9. Bone marrow-derived mesenchymal stem cells maintain the resting phenotype of microglia and inhibit microglial activation.

    Directory of Open Access Journals (Sweden)

    Ke Yan

    Full Text Available Many studies have shown that microglia in the activated state may be neurotoxic. It has been proven that uncontrolled or over-activated microglia play an important role in many neurodegenerative disorders. Bone marrow-derived mesenchymal stem cells (BMSCs have been shown in many animal models to have a therapeutic effect on neural damage. Such a therapeutic effect is attributed to the fact that BMSCs have the ability to differentiate into neurons and to produce trophic factors, but there is little information available in the literature concerning whether BMSCs play a therapeutic role by affecting microglial activity. In this study, we triggered an inflammatory response situation in vitro by stimulating microglia with the bacterial endotoxin lipopolysaccharide (LPS, and then culturing these microglia with BMSC-conditioned medium (BMSC-CM. We found that BMSC-CM significantly inhibited proliferation and secretion of pro-inflammatory factors by activated microglia. Furthermore, we found that the phagocytic capacity of microglia was also inhibited by BMSC-CM. Finally, we investigated whether the induction of apoptosis and the production of nitric oxide (NO were involved in the inhibition of microglial activation. We found that BMSC-CM significantly induced apoptosis of microglia, while no apoptosis was apparent in the LPS-stimulated microglia. Our study also provides evidence that NO participates in the inhibitory effect of BMSCs. Our experimental results provide evidence that BMSCs have the ability to maintain the resting phenotype of microglia or to control microglial activation through their production of several factors, indicating that BMSCs could be a promising therapeutic tool for treatment of diseases associated with microglial activation.

  10. Transplantation of human bone marrow-derived mesenchymal stem cells transfected with ectodysplasin for regeneration of sweat glands

    Institute of Scientific and Technical Information of China (English)

    CAI Sa; PAN Yu; HAN Bing; SUN Tong-zhu; SHENG Zhi-yong; FU Xiao-bing

    2011-01-01

    Background Patients with severe full-thickness burn injury suffer from their inability to maintain body temperature through perspiration because the complete destructed sweat glands can not be regenerated. Bone marrow-derived mesenchymal stem cells (BM-MSCs) represent an ideal stem-cell source for cell therapy because of their easy purification and multipotency. In this study, we attempted to induce human BM-MSCs to differentiate into sweat gland cells for sweat gland regeneration through ectodysplasin (EDA) gene transfection. Methods The dynamic expression of EDA and EDA receptor (EDAR) were firstly observed in the sweat gland formation during embryological development. After transfection with EDA expression vector, human BM-MSCs were transplanted into the injured areas of burn animal models. The regeneration of sweat glands was identified by perspiration test and immunohistochemical analysis. Results Endogenous expression of EDA and EDAR correlated with sweat gland development in human fetal skin. After EDA transfection, BM-MSC acquired a sweat-gland-cell phenotype, evidenced by their expression of sweat gland markers by flow cytometry analysis. Immunohistochemical staining revealed a markedly contribution of EDA-transfected BM-MSCs to the regeneration of sweat glands in the scalded paws. Positive rate for perspiration test for the paws treated with EDA-transfected BM-MSCs was significantly higher than those treated with BM-MSCs or EDA expression vector (P <0.05). Conclusions Our results confirmed the important role of EDA in the development of sweat gland. BM-MSCs transfected with EDA significantly improved the sweat-gland regeneration. This study suggests the potential application of EDA-modified MSCs for the repair and regeneration of injured skin and its appendages.

  11. Hepatocyte growth factor modulates interleukin-6 production in bone marrow derived macrophages: implications for inflammatory mediated diseases.

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    Gina M Coudriet

    Full Text Available The generation of the pro-inflammatory cytokines IL-6, TNF-α, and IL-1β fuel the acute phase response (APR. To maintain body homeostasis, the increase of inflammatory proteins is resolved by acute phase proteins via presently unknown mechanisms. Hepatocyte growth factor (HGF is transcribed in response to IL-6. Since IL-6 production promotes the generation of HGF and induces the APR, we posited that accumulating HGF might be a likely candidate for quelling excess inflammation under non-pathological conditions. We sought to assess the role of HGF and how it influences the regulation of inflammation utilizing a well-defined model of inflammatory activation, lipopolysaccharide (LPS-stimulation of bone marrow derived macrophages (BMM. BMM were isolated from C57BL6 mice and were stimulated with LPS in the presence or absence of HGF. When HGF was present, there was a decrease in production of the pro-inflammatory cytokine IL-6, along with an increase in the anti-inflammatory cytokine IL-10. Altered cytokine production correlated with an increase in phosphorylated GSK3β, increased retention of the phosphorylated NFκB p65 subunit in the cytoplasm, and an enhanced interaction between CBP and phospho-CREB. These changes were a direct result of signaling through the HGF receptor, MET, as effects were reversed in the presence of a selective inhibitor of MET (SU11274 or when using BMM from macrophage-specific conditional MET knockout mice. Combined, these data provide compelling evidence that under normal circumstances, HGF acts to suppress the inflammatory response.

  12. Effect of pimecrolimus vs. corticosteroids on murine bone marrow-derived dendritic cell differentiation, maturation and function.

    Science.gov (United States)

    Krummen, Mathias B W; Varga, Georg; Steinert, Meike; Stuetz, Anton; Luger, Thomas A; Grabbe, Stephan

    2006-01-01

    Pimecrolimus (SDZ ASM981) is a non-steroid member of calcineurin inhibitors recently developed for the treatment of inflammatory skin diseases. In this study, we compared the effect of pimecrolimus and corticosteroids on the differentiation, maturation and function of murine bone marrow-derived dendritic cells (BM-DC). We added pimecrolimus at concentrations of 5-500 ng/ml or 0.5 ng/ml mometasone furoate at different timepoints to the BM-DC culture and checked (i) the number of matured cells, (ii) the expression of activation markers, (iii) the release of cytokines and (iv) the stimulatory capacity of the resulting BM-DC in vivo. Even at the highest concentration, pimecrolimus treatment resulted in only modest effects. In the pimecrolimus-treated culture, we observed a decrease in the numbers of matured cells but no significant effects on the expression of activation markers. The release of some inflammatory cytokines was reduced, but the stimulatory capacity in vivo was not affected. In contrast, mometasone furoate has pronounced effects on BM-DC at a concentration ten to 1000 times lower than those used with pimecrolimus. Furthermore, topical treatment of mice with clobetasole cream 0.05% resulted in almost complete depletion of splenic DC and a severe hyposplenia, while high-dose oral pimecrolimus treatment did not show any effects on the spleen or on splenic DC. These results support that pimecrolimus, unlike corticosteroids, has little effects on dendritic cells. To the best of our knowledge, this is the first study of this type with use of BM-DC.

  13. Soluble Jagged 1/Fc chimera protein induces the differentiation and maturation of bone marrow-derived dendritic cells

    Institute of Scientific and Technical Information of China (English)

    XING FeiYue; LIU Jing; YU Zhe; JI YuHua

    2008-01-01

    A soluble Jagged 1/Fc chimera protein (Jagged 1/Fc) was directly used to induce differentiation and maturation of bone marrow-derived dendritic cells (DCs) in mice in vitro. A model of inducing and am-plifying DCs in vitro was established. The effect of Jagged 1/Fc on morphology of DCs induced by both rmGM-CSF and rmlL-4 was observed under a confocal microscope. A fluorescein-labeled monoclonal antibody staining combined with flow cytometry was applied to detect the effect of Jagged 1/Fc on the expression of CD11c, MHC-Ⅱ, CD86, CD80 and CD40 molecules on the surface of DCs. The results showed that Jagged 1/Fc did not affect the morphological properties of DC differentiation induced by both rmGM-CSF and rmlL-4. But it could promote the differentiation and maturation of DCs induced by both. The effect of it was strikingly different in the expression profile of co-stimulating molecules and the morphologic properties of DCs from lipopolysaccharide (LPS). The levels of MHC-Ⅱ and CD40 molecule expression on the surface of DCs stimulated by Jagged 1/Fc were significantly lower than those stimulated by LPS, and the level of CD80 expression on the surface of DCs induced by Jagged 1/Fc was near to that induced by LPS. Jagged 1/Fc had no influence on the expression of CD86 mole-cule on the surface of DCs. Jagged 1/Fc when used alone could not maintain the growth, differentiation and maturation of DCs. All the findings indicate that Jagged 1/Fc influences the differentiation and maturation of DCs, which is not markedly similar to LPS, providing important evidence for its devel-opment and application as a novel immunosuppressant.

  14. The susceptive alendronate-treatment timing and dosage for osteogenesis enhancement in human bone marrow-derived stem cells.

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    Chih-Hsiang Chang

    Full Text Available Recent studies indicated that alendronate enhanced osteogenesis in osteoblasts and human bone marrow-derived stem cells. However, the time- and dose-dependent effects of Aln on osteogenic differentiation and cytotoxicity of hBMSCs remain undefined. In present study, we investigated the effective dose range and timing of hBMSCs. hBMSCs were treated with various Aln doses (1, 5 and 10 µM according to the following groups: group A was treated with Aln during the first five days of bone medium, groups B, C and D were treated during the first, second, and final five days of osteo-induction medium and group E was treated throughout the entire experiment. The mineralization level and cytotoxicity were measured by quantified Alizarin Red S staining and MTT assay. In addition, the reversal effects of farnesyl pyrophosphate and geranylgeranyl pyrophosphate replenishment in group B were also investigated. The results showed that Aln treatment in groups A, B and E enhanced hBMSC mineralization in a dose-dependent manner, and the most pronounced effects were observed in groups B and E. The higher dose of Aln simultaneously enhanced mineralization and caused cytotoxicity in groups B, C and E. Replenishment of FPP or GGPP resulted in partial or complete reverse of the Aln-induced mineralization respectively. Furthermore, the addition of FPP or GGPP also eliminated the Aln-induced cytotoxicity. We demonstrated that hBMSCs are susceptible to 5 µM Aln during the initiation stage of osteogenic differentiation and that a 10 µM dose is cytotoxic.

  15. Engineered myocardial tissues constructed in vivo using cardiomyocyte-like cells derived from bone marrow mesenchymal stem cells in rats

    Directory of Open Access Journals (Sweden)

    Xing Yujie

    2012-01-01

    Full Text Available Abstract Background To explore the feasibility of constructing engineered myocardial tissues (EMTs in vivo, using polylactic acid -co-glycolic acid (PLGA for scaffold and cardiomyocyte-like cells derived from bone marrow mesenchymal stem cells (BMMSCs for seeded cells. Methods BMMSCs were isolated from femur and tibia of Sprague-Dawley (SD rats by density-gradient centrifugation. The third passage cells were treated with 10 μmol/L 5-azacytidine (5-aza and 0.1 μmol/L angiotensin II (Ang II for 24 h, followed by culturing in complete medium for 3 weeks to differentiated into cardiomyocyte-like cells. The cardiomyocyte-like cells were seeded into PLGA scaffolds to form the grafts. The grafts were cultured in the incubator for three days and then implanted into the peritoneal cavity of SD rats. Four weeks later, routine hematoxylin-eosin (HE staining, immunohistochemical staining for myocardium-specific cardiac troponin I (cTnI, scanning electron microscopy and transmission electron microscopy were used to analyze the morphology and microconstruction of the EMTs in host rats. Results HE staining showed that the cardiomyocyte-like cells distributed equally in the PLGA scaffold, and the nuclei arranged in the spindle shape. Immunohistochemical staining revealed that majority of engrafted cells in the PLGA -Cardiomyocyte-like cells group were positive for cTnI. Scanning electron microscopy showed that the inoculated cells well attached to PLGA and grew in 3 dimensions in construct. Transmission electron microscopy showed that the EMTs contained well arranged myofilaments paralleled to the longitudinal cell axis, the cells were rich in endoplasmic reticulum and mitochondria, while desmosomes, gap junction and Z line-like substances were also can be observed as well within the engrafted cells. Conclusion We have developed an in vivo method to construct engineered myocardial tissue. The in vivo microenvironment helped engrafted cells/tissue survive and

  16. Response of human bone marrow-derived MSCs on triphasic Ca-P substrate with various HA/TCP ratio.

    Science.gov (United States)

    Bajpai, Indu; Kim, Duk Yeon; Kyong-Jin, Jung; Song, In-Hwan; Kim, Sukyoung

    2017-01-01

    Calcium phosphates (Ca-P) are used commonly as artificial bone substitutes to control the biodegradation rate of an implant in the body fluid. This study examined the in vitro proliferation of human bone marrow-derived mesenchymal stem cells (hBMSCs) on triphasic Ca-P samples. For this aspect, hydroxyapatite (HA), dicalcium phosphate dehydrate (DCPD), and calcium hydroxide (Ca(OH)2 ) were mixed at various ratios, cold compacted, and sintered at 1250°C in air. X-ray diffraction showed that the β-tricalcium phosphate (TCP) to α-TCP phase transformation increased with increasing DCPD/HA ratio. The micro-hardness deceased with increasing TCP content, whereas the mean grain size and porosity increased with increasing TCP concentration. To evaluate the in vitro degree of adhesion and proliferation on the HA/TCP samples, human BMSCs were incubated on the HA/TCP samples and analyzed by a cells proliferation assay, expression of the extracellular matrix (ECM) genes, such as α-smooth muscle actin (α-SMA) and fibronectin (FN), and FITC-phalloidin fluorescent staining. In terms of the interactions of human BMSCs with the triphasic Ca-P samples, H50T50 (Ca/P = 1.59) markedly enhanced cell spreading, proliferation, FN, and α-SMA compared with H100T0 (Ca/P = 1.67). Interestingly, these results show that among the five HA/TCP samples, H50T50 is the optimal Ca-P composition for in vitro cell proliferation. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 72-80, 2017.

  17. Bone marrow-derived mesenchymal stem cells enhance angiogenesis via their α6β1 integrin receptor

    Energy Technology Data Exchange (ETDEWEB)

    Carrion, Bita; Kong, Yen P. [Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109 (United States); Kaigler, Darnell [Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109 (United States); Department of Periodontics and Oral Medicine, University of Michigan, Ann Arbor, MI 48109 (United States); Putnam, Andrew J., E-mail: putnam@umich.edu [Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109 (United States)

    2013-11-15

    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. Highlights: • BMSCs stimulate angiogenesis, but the mechanisms remain unclear. • We silenced the expression of the α6 integrin subunit in BMSCs. • Silencing this receptor subunit significantly inhibited angiogenic sprouting. • Knocking down α6 integrin affected laminin and αSMA expression. • Silencing α6 integrin expression also reduced BMSC proliferation.

  18. Bone marrow-derived mesenchymal stem cells expressing the Shh transgene promotes functional recovery after spinal cord injury in rats.

    Science.gov (United States)

    Jia, Yijia; Wu, Dou; Zhang, Ruiping; Shuang, Weibing; Sun, Jiping; Hao, Haihu; An, Qijun; Liu, Qiang

    2014-06-24

    Spinal cord injury (SCI) is one of the most disabling diseases. Cell-based gene therapy is becoming a major focus for the treatment of SCI. Bone marrow-derived mesenchymal stem cells (BMSCs) are a promising stem cell type useful for repairing SCI. However, the effects of BMSCs transplants are likely limited because of low transplant survival after SCI. Sonic hedgehog (Shh) is a multifunctional growth factor which can facilitate neuronal and BMSCs survival, promote axonal growth, prevent activation of the astrocyte lineage, and enhance the delivery of neurotrophic factors in BMSCs. However, treatment of SCI with Shh alone also has limited effects on recovery, because the protein is cleared quickly. In this study, we investigated the use of BMSCs overexpressing the Shh transgene (Shh-BMSCs) in the treatment of rats with SCI, which could stably secrete Shh and thereby enhance the effects of BMSCs, in an attempt to combine the advantages of Shh and BMSCs and so to promote functional recovery. After Shh-BMSCs treatment of SCI via the subarachnoid, we detected significantly greater damage recovery compared with that seen in rats treated with phosphate-buffered saline (PBS) and BMSCs. Use of Shh-BMSCs increased the expression and secretion of Shh, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), improved the behavioral function, enhanced the BMSCs survival, promoted the expression level of neurofilament 200 (NF200), and reduced the expression of glial fibrillary acidic protein (GFAP). Thus, our results indicated that Shh-BMSCs enhanced recovery of neurological function after SCI in rats and could be a potential valuable therapeutic intervention for SCI in humans.

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

    Science.gov (United States)

    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

  20. Three-dimensional graphene foams loaded with bone marrow derived mesenchymal stem cells promote skin wound healing with reduced scarring

    Energy Technology Data Exchange (ETDEWEB)

    Li, Zhonghua [Department of Burn and Plastic Surgery, Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China); Department of Burn and Plastic Surgery, The Fourth People' s Hospital Of Jinan, Jinan 250031 (China); Wang, Haiqin [Department of Obstetrics and Gynecology, The Fifth People' s Hospital Of Jinan, Jinan 250022 (China); Yang, Bo; Sun, Yukai [Department of Burn and Plastic Surgery, The Fourth People' s Hospital Of Jinan, Jinan 250031 (China); Huo, Ran, E-mail: rhuo12@163.com [Department of Burn and Plastic Surgery, Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China)

    2015-12-01

    The regeneration of functional skin remains elusive, due to poor engraftment, deficient vascularization, and excessive scar formation. Aiming to overcome these issues, the present study proposed the combination of a three-dimensional graphene foam (GF) scaffold loaded with bone marrow derived mesenchymal stem cells (MSCs) to improve skin wound healing. The GFs demonstrated good biocompatibility and promoted the growth and proliferation of MSCs. Meanwhile, the GFs loaded with MSCs obviously facilitated wound closure in animal model. The dermis formed in the presence of the GF structure loaded with MSCs was thicker and possessed a more complex structure at day 14 post-surgery. The transplanted MSCs correlated with upregulation of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), which may lead to neo-vascularization. Additionally, an anti-scarring effect was observed in the presence of the 3D-GF scaffold and MSCs, as evidenced by a downregulation of transforming growth factor-beta 1 (TGF-β1) and alpha-smooth muscle actin (α-SMA) together with an increase of TGF-β3. Altogether, the GF scaffold could guide the wound healing process with reduced scarring, and the MSCs were crucial to enhance vascularization and provided a better quality neo-skin. The GF scaffold loaded with MSCs possesses necessary bioactive cues to improve wound healing with reduced scarring, which may be of great clinical significance for skin wound healing. - Highlights: • The GFs promoted the growth and proliferation of MSCs. • The GFs loaded with MSCs obviously facilitated wound closure in the animal model. • An anti-scarring effect was observed in the presence of 3D-GF scaffold and MSCs. • The GF scaffold loaded with MSCs has great effect on skin wound healing.

  1. Chondrogenic potential of mesenchymal stromal cells derived from equine bone marrow and umbilical cord blood

    DEFF Research Database (Denmark)

    Berg, Lise Charlotte; Koch, Thomas Gadegaard; Heerkens, T.

    2009-01-01

    Objective: Orthopaedic injury is the most common cause of lost training days or premature retirement in the equine athlete. Cell-based therapies are a potential new treatment option in musculo-skeletal diseases. Mesenthymal stromal cells (MSC) have been derived from multiple sources in the horse...

  2. Archival bone marrow samples

    DEFF Research Database (Denmark)

    Lund, Bendik; Najmi, Laeya A; Wesolowska-Andersen, Agata;

    2015-01-01

    AB Archival samples represent a significant potential for genetic studies, particularly in severe diseases with risk of lethal outcome, such as in cancer. In this pilot study, we aimed to evaluate the usability of archival bone marrow smears and biopsies for DNA extraction and purification, whole...... with samples stored for 4 to 10 years. Acceptable call rates for SNPs were detected for 7 of 42 archival samples. In conclusion, archival bone marrow samples are suitable for DNA extraction and multiple marker analysis, but WGA was less successful, especially when longer fragments were analyzed. Multiple SNP...

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

    Science.gov (United States)

    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.

  4. Microvesicles derived from adult human bone marrow and tissue specific mesenchymal stem cells shuttle selected pattern of miRNAs.

    Directory of Open Access Journals (Sweden)

    Federica Collino

    Full Text Available BACKGROUND: Cell-derived microvesicles (MVs have been described as a new mechanism of cell-to-cell communication. MVs after internalization within target cells may deliver genetic information. Human bone marrow derived mesenchymal stem cells (MSCs and liver resident stem cells (HLSCs were shown to release MVs shuttling functional mRNAs. The aim of the present study was to evaluate whether MVs derived from MSCs and HLSCs contained selected micro-RNAs (miRNAs. METHODOLOGY/PRINCIPAL FINDINGS: MVs were isolated from MSCs and HLSCs. The presence in MVs of selected ribonucleoproteins involved in the traffic and stabilization of RNA was evaluated. We observed that MVs contained TIA, TIAR and HuR multifunctional proteins expressed in nuclei and stress granules, Stau1 and 2 implicated in the transport and stability of mRNA and Ago2 involved in miRNA transport and processing. RNA extracted from MVs and cells of origin was profiled for 365 known human mature miRNAs by real time PCR. Hierarchical clustering and similarity analysis of miRNAs showed 41 co-expressed miRNAs in MVs and cells. Some miRNAs were accumulated within MVs and absent in the cells after MV release; others were retained within the cells and not secreted in MVs. Gene ontology analysis of predicted and validated targets showed that the high expressed miRNAs in cells and MVs could be involved in multi-organ development, cell survival and differentiation. Few selected miRNAs shuttled by MVs were also associated with the immune system regulation. The highly expressed miRNAs in MVs were transferred to target cells after MV incorporation. CONCLUSIONS: This study demonstrated that MVs contained ribonucleoproteins involved in the intracellular traffic of RNA and selected pattern of miRNAs, suggesting a dynamic regulation of RNA compartmentalization in MVs. The observation that MV-highly expressed miRNAs were transferred to target cells, rises the possibility that the biological effect of stem

  5. Dental pulp-derived stromal cells exhibit a higher osteogenic potency than bone marrow-derived stromal cells in vitro and in a porcine critical-size bone defect model

    DEFF Research Database (Denmark)

    Jensen, Jonas; Tvedesøe, Claus; Rölfing, Jan Hendrik Duedal;

    2016-01-01

    INTRODUCTION: The osteogenic differentiation of bone marrow-derived mesenchymal stromal cells (BMSCs) was compared with that of dental pulp-derived stromal cells (DPSCs) in vitro and in a pig calvaria critical-size bone defect model. METHODS: BMSCs and DPSCs were extracted from the tibia bone...

  6. Sphingosine-1-phosphate mediates proliferation maintaining the multipotency of human adult bone marrow and adipose tissue-derived stem cells.

    Science.gov (United States)

    He, Xiaoli; H'ng, Shiau-Chen; Leong, David T; Hutmacher, Dietmar W; Melendez, Alirio J

    2010-08-01

    High renewal and maintenance of multipotency of human adult stem cells (hSCs), are a prerequisite for experimental analysis as well as for potential clinical usages. The most widely used strategy for hSC culture and proliferation is using serum. However, serum is poorly defined and has a considerable degree of inter-batch variation, which makes it difficult for large-scale mesenchymal stem cells (MSCs) expansion in homogeneous culture conditions. Moreover, it is often observed that cells grown in serum-containing media spontaneously differentiate into unknown and/or undesired phenotypes. Another way of maintaining hSC development is using cytokines and/or tissue-specific growth factors; this is a very expensive approach and can lead to early unwanted differentiation. In order to circumvent these issues, we investigated the role of sphingosine-1-phosphate (S1P), in the growth and multipotency maintenance of human bone marrow and adipose tissue-derived MSCs. We show that S1P induces growth, and in combination with reduced serum, or with the growth factors FGF and platelet-derived growth factor-AB, S1P has an enhancing effect on growth. We also show that the MSCs cultured in S1P-supplemented media are able to maintain their differentiation potential for at least as long as that for cells grown in the usual serum-containing media. This is shown by the ability of cells grown in S1P-containing media to be able to undergo osteogenic as well as adipogenic differentiation. This is of interest, since S1P is a relatively inexpensive natural product, which can be obtained in homogeneous high-purity batches: this will minimize costs and potentially reduce the unwanted side effects observed with serum. Taken together, S1P is able to induce proliferation while maintaining the multipotency of different human stem cells, suggesting a potential for S1P in developing serum-free or serum-reduced defined medium for adult stem cell cultures.

  7. The Healing of Bone Marrow-Derived Stem Cells on Motor Functions in Acute Spinal Cord Injury of Mice

    Directory of Open Access Journals (Sweden)

    N Gashmardi

    2016-10-01

    Full Text Available Background & aim: Spinal cord injury is a devastating damage that can cause motor and sensory deficits reducing quality of life and life expectancy of patients. Stem cell transplantation can be one of the promising therapeutic strategies. Bone marrow is a rich source of stem cells that is able to differentiate into various cell types. In this study, bone marrow stem cells were transplanted into mice spinal cord injury model to evaluate the motor function test. Methods: Bone marrow stem cells were isolated from 3 mice. Thirty six mice were randomly divided into 3 groups: the control, sham and experimental. In sham group, mice were subjected to spinal cord compression. In experimental group, one day after lesion, isolated stem cells (200,000 were injected intravenously. Assessment of locomotor function was done by Toyama Mouse Score (TMS after 1, 2, 3, 4, 5 week post-injury. The data were analyzed using one-way Analysis of Variance and Tukey tests and statistical software Graph Pad and SPSS.P > 0/05 was considered as significant difference.  Results: The score of TMS after cell transplantation was higher in cell transplantation group (experimental, while it was significantly higher after fifth week when compared to other groups. Conclusion: The increase in TMS score in cell transplantation group showed that injection of stem cells in acute spinal cord injury can have a therapeutic effect and promote locomotor function.

  8. Allograftic bone marrow-derived mesenchymal stem cells transplanted into heart infarcted model of rabbit to renovate infarcted heart

    Institute of Scientific and Technical Information of China (English)

    王建安; 李长岭; 樊友启; 何红; 孙勇

    2004-01-01

    Objective: To investigate the directed transplantation of allograftic bone marrow-derived mesenchymal stem cells (MSCs) in myocardial infarcted (MI) model rabbits. Materials and Methods: Rabbits were divided into 3 groups, heart infarcted model with MSCs transplanted treatment (MSCs group, n=12), heart infarcted model with PBS injection (control group, n=20), sham operation with PBS injection (sham group, n=l 7). MSCs labelled by BrdUrd were injected into the MI area of the MSCs group. The same volume of PBS was injected into the MI area of the control group and sham group. The mortality, LVIDd, LVIDs and LVEF Of the two groups were compared 4 weeks later. Tropomyosin inhibitory component (Tn I) and BrdUrd immunohistochemistry identified the engrafted cells 4 weeks after transplantation. Result: The mortality of the MSCs group was 16.7% (2/12), and remarkably lower than the control group's mortality [35% (7/20) (P<0.05)].Among the animals that survived for 4 weeks, the LVIDd and LVIDs of the MSCs group after operation were 1.17±0.21 cm and 0.74±0.13 cm, and remarkably lower than those of the model group, which were 1.64±0.14 cm and 1.19±0.12 cm (P<0.05); the LVEF of the MSCs group after operation was 63±6%, and remarkably higher than that of the model group,which was 53±6% (P<0.05). Among the 10 cases of animals that survived for 4 weeks in the MSCs group, in 8 cases (80%),the transplanted cells survived in the non MI, MI region and its periphery, and even farther away; part of them differentiated into cardiomyocytes; in 7 cases (70%), the transplanted cells participated in the formation of blood vessel tissue in the MI region. Conclusion: Transplanted allograftic MSCs can survive and differentiate into cardiomyocytes, form the blood vessels in the MI region. MSCs transplantation could improve the heart function after MI.

  9. Effect of growth and differentiation factor 6 on the tenogenic differentiation of bone marrow-derived mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    CHAI Wei; NI Ming; RUI Yun-feng; ZHANG Kai-yi; ZHANG Qiang; XU Liang-liang; CHAN Kai-ming

    2013-01-01

    Background Recent studies showed that bone marrow-derived mesenchymal stem cells (BMSCs) had risk of ectopic bone formation.In this study,we aimed to investigate the effect of growth and differentiation factor 6 (GDF-6) on the tenogenic differentiation of BMSCs in vitro,and then combined with small intestine submucous (SIS) to promote tendon regeneration in vivo.Methods The BMSCs were isolated from the green fluorescent protein (GFP) rats,and were characterized by multi-differentiation assays following our previous study protocol.BMSCs cultured with different concentrations of GDF-6,without growth factors served as control.After 2 weeks,mRNA expression and protein expression of tendon specific markers were examined by qRT-PCR and Western blotting to define an optimal concentration of GDF-6.Mann-Whitney U-test was used to compare the difference in relative mRNA expression among all groups; P ≤0.05 was regarded as statistically significant.The GDF-6 treated BMSCs combined with SIS were implanted in nude mice and SD rat acute patellar tendon injury model,the BMSCs combined with SIS served as control.After 12 and 4 weeks in nude mice and tendon injury model,the samples were collected for histology.Results After the BMSCs were treated with different concentration of GDF-6 for 2 weeks,the fold changes of the specific markers (Tenomodulin and Scleraxis) mRNA expression were significantly higher in GDF-6 (20 ng/ml) group (P ≤0.05),which was also confirmed by Western blotting result.The BMSCs became parallel in orientation after GDF-6 (20 ng/ml) treatment,but the BMSCs in control group were randomly oriented.The GDF-6 (20 ng/ml) treated BMSCs were combined with SIS,and were implanted in nude mice for 12 weeks,the histology showed neo-tendon formation.In the SD rat patellar tendon window injury model,the histology also indicated the GDF-6 (20 ng/ml) treated BMSCs combined with SIS could promote tendon regeneration.Conclusions GDF-6 has tenogenic effect on the tenogenic

  10. GM-CSF Inhibits c-Kit and SCF Expression by Bone Marrow-Derived Dendritic Cells.

    Science.gov (United States)

    Barroeta Seijas, Amairelys Belen; Simonetti, Sonia; Vitale, Sara; Runci, Daniele; Quinci, Angela Caterina; Soriani, Alessandra; Criscuoli, Mattia; Filippi, Irene; Naldini, Antonella; Sacchetti, Federico Maria; Tarantino, Umberto; Oliva, Francesco; Piccirilli, Eleonora; Santoni, Angela; Di Rosa, Francesca

    2017-01-01

    Stem cell factor (SCF), the ligand of c-kit, is a key cytokine for hematopoiesis. Hematopoietic precursors express c-kit, whereas differentiated cells of hematopoietic lineage are negative for this receptor, with the exception of NK cells, mast cells, and a few others. While it has long been recognized that dendritic cells (DCs) can express c-kit, several questions remain concerning the SCF/c-kit axis in DCs. This is particularly relevant for DCs found in those organs wherein SCF is highly expressed, including the bone marrow (BM). We characterized c-kit expression by conventional DCs (cDCs) from BM and demonstrated a higher proportion of c-kit(+) cells among type 1 cDC subsets (cDC1s) than type 2 cDC subsets (cDC2s) in both humans and mice, whereas similar levels of c-kit expression were observed in cDC1s and cDC2s from mouse spleen. To further study c-kit regulation, DCs were generated with granulocyte-macrophage colony-stimulating factor (GM-CSF) from mouse BM, a widely used protocol. CD11c(+) cells were purified from pooled non-adherent and slightly adherent cells collected after 7 days of culture, thus obtaining highly purified BM-derived DCs (BMdDCs). BMdDCs contained a small fraction of c-kit(+) cells, and by replating them for 2 days with GM-CSF, we obtained a homogeneous population of c-kit(+) CD40(hi) MHCII(hi) cells. Not only did BMdDCs express c-kit but they also produced SCF, and both were striking upregulated if GM-CSF was omitted after replating. Furthermore, a small but significant reduction in BMdDC survival was observed upon SCF silencing. Incubation of BMdDCs with SCF did not modulate antigen presentation ability of these cells, nor it did regulate their membrane expression of the chemokine receptor CXCR4. We conclude that the SCF/c-kit-mediated prosurvival circuit may have been overlooked because of the prominent use of GM-CSF in DC cultures in vitro, including those human DC cultures destined for the clinics. We speculate that DCs more

  11. Allograftic bone marrow-derived mesenchymal stem cells transplanted into heart infarcted model of rabbit to renovate infarcted heart

    Institute of Scientific and Technical Information of China (English)

    王建安; 李长岭; 樊友启; 何红; 孙勇

    2004-01-01

    Objective: To investigate the directed transplantation of allograftic bone marrow-derived mesenchymal stem cells (MSCs) in myocardial infarcted (MI) model rabbits. Materials and Methods: Rabbits were divided into 3 groups, heart infarcted model with MSCs transplanted treatment (MSCs group, n=12), heart infarcted model with PBS injection (control group, n=20), sham operation with PBS injection (sham group, n=17). MSCs labelled by BrdUrd were injected into the MI area of the MSCs group. The same volume of PBS was injected into the MI area of the control group and sham group. The mortality, LVIDd, LVIDs and LVEF of the two groups were compared 4 weeks later. Tropomyosin inhibitory component (Tn Ⅰ) and BrdUrd immunohistochemistry identified the engrafted cells 4 weeks after transplantation. Result: The mortality of the MSCs group was 16.7% (2/12), and remarkably lower than the control group's mortality [35% (7/20) (P<0.05)]. Among the animals that survived for 4 weeks, the LVIDd and LVIDs of the MSCs group after operation were 1.17±0.21cm and 0.74±0.13cm, and remarkably lower than those of the model group, which were 1.64±0.14cm and 1.19±0.12cm (P<0.05); the LVEF of the MSCs group after operation was 63±6%, and remarkably higher than that of the model group, which was 53±6% (P<0.05). Among the 10 cases of animals that survived for 4 weeks in the MSCs group, in 8 cases (80%), the transplanted cells survived in the non MI, MI region and its periphery, and even farther away; part of them differentiated into cardiomyocytes; in 7 cases (70%), the transplanted cells participated in the formation of blood vessel tissue in the MI region. Conclusion: Transplanted allograftic MSCs can survive and differentiate into cardiomyocytes, form the blood vessels in the MI region. MSCs transplantation could improve the heart function after MI.

  12. GM-CSF Inhibits c-Kit and SCF Expression by Bone Marrow-Derived Dendritic Cells

    Science.gov (United States)

    Barroeta Seijas, Amairelys Belen; Simonetti, Sonia; Vitale, Sara; Runci, Daniele; Quinci, Angela Caterina; Soriani, Alessandra; Criscuoli, Mattia; Filippi, Irene; Naldini, Antonella; Sacchetti, Federico Maria; Tarantino, Umberto; Oliva, Francesco; Piccirilli, Eleonora; Santoni, Angela; Di Rosa, Francesca

    2017-01-01

    Stem cell factor (SCF), the ligand of c-kit, is a key cytokine for hematopoiesis. Hematopoietic precursors express c-kit, whereas differentiated cells of hematopoietic lineage are negative for this receptor, with the exception of NK cells, mast cells, and a few others. While it has long been recognized that dendritic cells (DCs) can express c-kit, several questions remain concerning the SCF/c-kit axis in DCs. This is particularly relevant for DCs found in those organs wherein SCF is highly expressed, including the bone marrow (BM). We characterized c-kit expression by conventional DCs (cDCs) from BM and demonstrated a higher proportion of c-kit+ cells among type 1 cDC subsets (cDC1s) than type 2 cDC subsets (cDC2s) in both humans and mice, whereas similar levels of c-kit expression were observed in cDC1s and cDC2s from mouse spleen. To further study c-kit regulation, DCs were generated with granulocyte-macrophage colony-stimulating factor (GM-CSF) from mouse BM, a widely used protocol. CD11c+ cells were purified from pooled non-adherent and slightly adherent cells collected after 7 days of culture, thus obtaining highly purified BM-derived DCs (BMdDCs). BMdDCs contained a small fraction of c-kit+ cells, and by replating them for 2 days with GM-CSF, we obtained a homogeneous population of c-kit+ CD40hi MHCIIhi cells. Not only did BMdDCs express c-kit but they also produced SCF, and both were striking upregulated if GM-CSF was omitted after replating. Furthermore, a small but significant reduction in BMdDC survival was observed upon SCF silencing. Incubation of BMdDCs with SCF did not modulate antigen presentation ability of these cells, nor it did regulate their membrane expression of the chemokine receptor CXCR4. We conclude that the SCF/c-kit-mediated prosurvival circuit may have been overlooked because of the prominent use of GM-CSF in DC cultures in vitro, including those human DC cultures destined for the clinics. We speculate that DCs more prominently rely

  13. Brain-derived neurotrophic factor genes transfect rat bone marrow mesenchymal stem cells based on cationic polymer vector

    Institute of Scientific and Technical Information of China (English)

    Zunsheng Zhang; Kun Zan; Yonghai Liu; Xia Shen

    2009-01-01

    BACKGROUND: Gene therapy is an effective expression of genes within target cells after transferring exogenous target genes. Both vector selection and transfection method are important factors for gene transfection. An ideal gene vector is required for a high transfusion of target gene and an exact introduction of target gene into specific target cells so as to express gene products. OBJECTIVE: To study the expression of mRNA and protein after transfecting rat bone marrow mesenchymal stem cells (BMSCs) with brain-derived neurotrophic factor (BDNF) genes based on cationic polymer vector. DESIGN, TIME AND SETTING: A randomized, controlled in vitro study using gene engineering, performed at the Neurobiology Laboratory, Xuzhou Medical College between October 2007 and April 2008. MATERIALS: PcDNA3.1 BDNF was obtained from Youbiai Biotechnological Company, Beijing and cationic polymer vector used was the SofastTM gene transfection reagent that was made by Taiyangma Biotechnological Co., Ltd., Xiamen. METHODS: BMSCs extracted from six Sprague Dawley (SD) rats aged 1 month were isolated and cultured in vitro. Third passage BMSCs were inoculated on a 6-well culture plate at the density of 1×106 cells/L. At about 80% confluence, BMSCs were transfected with PcDNA3.1-BDNF (2 μg) combined with SofastTM gene transfection reagent (6 μg) (BDNF group) or with PcDNA3.1 (2 μg) combined with SofastTM gene transfection reagent (6 μg) (blank vector group). Cells that were not transfected with any reagents but still cultured under primary culture conditions were used as a non-transfection group.MAIN OUTCOME MEASURES: Enzyme linked immunosorbent assay was used to measure time efficiency of BMSC-secreted BDNF protein. Twenty-four hours after gene transfection, RT-PCR was used to detect expression of BDNF mRNA in the BMSCs. Immunohistochemistry was used to determine expression of BDNF protein in the BMSCs.RESULTS: BDNF protein expression was detected at day 1 after gene transfection

  14. Immunomodulatory effects of bone marrow-derived mesenchymal stem cells in a swine hemi-facial allotransplantation model.

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    Yur-Ren Kuo

    Full Text Available BACKGROUND: In this study, we investigated whether the infusion of bone marrow-derived mesenchymal stem cells (MSCs, combined with transient immunosuppressant treatment, could suppress allograft rejection and modulate T-cell regulation in a swine orthotopic hemi-facial composite tissue allotransplantation (CTA model. METHODOLOGY/PRINCIPAL FINDINGS: Outbred miniature swine underwent hemi-facial allotransplantation (day 0. Group-I (n = 5 consisted of untreated control animals. Group-II (n = 3 animals received MSCs alone (given on days -1, +1, +3, +7, +14, and +21. Group-III (n = 3 animals received CsA (days 0 to +28. Group-IV (n = 5 animals received CsA (days 0 to +28 and MSCs (days -1, +1, +3, +7, +14, and +21. The transplanted face tissue was observed daily for signs of rejection. Biopsies of donor tissues and recipient blood sample were obtained at specified predetermined times (per 2 weeks post-transplant or at the time of clinically evident rejection. Our results indicated that the MSC-CsA group had significantly prolonged allograft survival compared to the other groups (P<0.001. Histological examination of the MSC-CsA group displayed the lowest degree of rejection in alloskin and lymphoid gland tissues. TNF-α expression in circulating blood revealed significant suppression in the MSC and MSC-CsA treatment groups, as compared to that in controls. IHC staining showed CD45 and IL-6 expression were significantly decreased in MSC-CsA treatment groups compared to controls. The number of CD4+/CD25+ regulatory T-cells and IL-10 expressions in the circulating blood significantly increased in the MSC-CsA group compared to the other groups. IHC staining of alloskin tissue biopsies revealed a significant increase in the numbers of foxp3(+T-cells and TGF-β1 positive cells in the MSC-CsA group compared to the other groups. CONCLUSIONS: These results demonstrate that MSCs significantly prolong hemifacial CTA survival. Our data indicate the MSCs did not

  15. Autologous Bone Marrow-Derived Mesenchymal Stem Cells Modulate Molecular Markers of Inflammation in Dogs with Cruciate Ligament Rupture

    Science.gov (United States)

    Muir, Peter; Hans, Eric C.; Racette, Molly; Volstad, Nicola; Sample, Susannah J.; Heaton, Caitlin; Holzman, Gerianne; Schaefer, Susan L.; Bloom, Debra D.; Bleedorn, Jason A.; Hao, Zhengling; Amene, Ermias; Suresh, M.; Hematti, Peiman

    2016-01-01

    Mid-substance rupture of the canine cranial cruciate ligament rupture (CR) and associated stifle osteoarthritis (OA) is an important veterinary health problem. CR causes stifle joint instability and contralateral CR often develops. The dog is an important model for human anterior cruciate ligament (ACL) rupture, where rupture of graft repair or the contralateral ACL is also common. This suggests that both genetic and environmental factors may increase ligament rupture risk. We investigated use of bone marrow-derived mesenchymal stem cells (BM-MSCs) to reduce systemic and stifle joint inflammatory responses in dogs with CR. Twelve dogs with unilateral CR and contralateral stable partial CR were enrolled prospectively. BM-MSCs were collected during surgical treatment of the unstable CR stifle and culture-expanded. BM-MSCs were subsequently injected at a dose of 2x106 BM-MSCs/kg intravenously and 5x106 BM-MSCs by intra-articular injection of the partial CR stifle. Blood (entry, 4 and 8 weeks) and stifle synovial fluid (entry and 8 weeks) were obtained after BM-MSC injection. No adverse events after BM-MSC treatment were detected. Circulating CD8+ T lymphocytes were lower after BM-MSC injection. Serum C-reactive protein (CRP) was decreased at 4 weeks and serum CXCL8 was increased at 8 weeks. Synovial CRP in the complete CR stifle was decreased at 8 weeks. Synovial IFNγ was also lower in both stifles after BM-MSC injection. Synovial/serum CRP ratio at diagnosis in the partial CR stifle was significantly correlated with development of a second CR. Systemic and intra-articular injection of autologous BM-MSCs in dogs with partial CR suppresses systemic and stifle joint inflammation, including CRP concentrations. Intra-articular injection of autologous BM-MSCs had profound effects on the correlation and conditional dependencies of cytokines using causal networks. Such treatment effects could ameliorate risk of a second CR by modifying the stifle joint inflammatory response

  16. Mouse bone marrow-derived mesenchymal stromal cells turn activated macrophages into a regulatory-like profile.

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    Julian Maggini

    Full Text Available In recent years it has become clear that the therapeutic properties of bone marrow-derived mesenchymal stromal cells (MSC are related not only to their ability to differentiate into different lineages but also to their capacity to suppress the immune response. We here studied the influence of MSC on macrophage function. Using mouse thioglycolate-elicited peritoneal macrophages (M stimulated with LPS, we found that MSC markedly suppressed the production of the inflammatory cytokines TNF-alpha, IL-6, IL-12p70 and interferon-gamma while increased the production of IL-10 and IL-12p40. Similar results were observed using supernatants from MSC suggesting that factor(s constitutively released by MSC are involved. Supporting a role for PGE(2 we observed that acetylsalicylic acid impaired the ability of MSC to inhibit the production of inflammatory cytokines and to stimulate the production of IL-10 by LPS-stimulated M. Moreover, we found that MSC constitutively produce PGE2 at levels able to inhibit the production of TNF-alpha and IL-6 by activated M. MSC also inhibited the up-regulation of CD86 and MHC class II in LPS-stimulated M impairing their ability to activate antigen-specific T CD4+ cells. On the other hand, they stimulated the uptake of apoptotic thymocytes by M. Of note, MSC turned M into cells highly susceptible to infection with the parasite Trypanosoma cruzi increasing more than 5-fold the rate of M infection. Using a model of inflammation triggered by s.c. implantation of glass cylinders, we found that MSC stimulated the recruitment of macrophages which showed a low expression of CD86 and the MHC class II molecule Ia(b and a high ability to produce IL-10 and IL-12p40, but not IL-12 p70. In summary, our results suggest that MSC switch M into a regulatory profile characterized by a low ability to produce inflammatory cytokines, a high ability to phagocyte apoptotic cells, and a marked increase in their susceptibility to infection by

  17. The role of growth factors in maintenance of stemness in bone marrow-derived mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Eom, Young Woo; Oh, Ji-Eun [Cell Therapy and Tissue Engineering Center, Wonju College of Medicine, Yonsei Univ., Wonju (Korea, Republic of); Lee, Jong In [Department of Hematology-Oncology, Wonju College of Medicine, Yonsei Univ., Wonju (Korea, Republic of); Baik, Soon Koo [Cell Therapy and Tissue Engineering Center, Wonju College of Medicine, Yonsei Univ., Wonju (Korea, Republic of); Department of Internal Medicine, Wonju College of Medicine, Yonsei Univ., Wonju (Korea, Republic of); Rhee, Ki-Jong [Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei Univ., Wonju (Korea, Republic of); Shin, Ha Cheol; Kim, Yong Man [Pharmicell Co., Ltd., Sungnam (Korea, Republic of); Ahn, Chan Mug [Department of Basic Science, Wonju College of Medicine, Yonsei Univ., Wonju (Korea, Republic of); Kong, Jee Hyun [Department of Hematology-Oncology, Wonju College of Medicine, Yonsei Univ., Wonju (Korea, Republic of); Kim, Hyun Soo, E-mail: khsmd@pharmicell.com [Pharmicell Co., Ltd., Sungnam (Korea, Republic of); Shim, Kwang Yong, E-mail: kyshim@yonsei.ac.kr [Department of Hematology-Oncology, Wonju College of Medicine, Yonsei Univ., Wonju (Korea, Republic of)

    2014-02-28

    Highlights: • Expression of FGF-2, FGF-4, EGF, and HGF decreased during long-term culture of BMSCs. • Loss of growth factors induced autophagy, senescence and decrease of stemness. • FGF-2 increased proliferation potential via AKT and ERK activation in BMSCs. • FGF-2 suppressed LC3-II expression and down-regulated senescence of BMSCs. • HGF was important in maintenance of the differentiation potential of BMSCs. - Abstract: Mesenchymal stem cells (MSCs) are an active topic of research in regenerative medicine due to their ability to secrete a variety of growth factors and cytokines that promote healing of damaged tissues and organs. In addition, these secreted growth factors and cytokines have been shown to exert an autocrine effect by regulating MSC proliferation and differentiation. We found that expression of EGF, FGF-4 and HGF were down-regulated during serial passage of bone marrow-derived mesenchymal stem cells (BMSCs). Proliferation and differentiation potentials of BMSCs treated with these growth factors for 2 months were evaluated and compared to BMSCs treated with FGF-2, which increased proliferation of BMSCs. FGF-2 and -4 increased proliferation potentials at high levels, about 76- and 26-fold, respectively, for 2 months, while EGF and HGF increased proliferation of BMSCs by less than 2.8-fold. Interestingly, differentiation potential, especially adipogenesis, was maintained only by HGF treatment. Treatment with FGF-2 rapidly induced activation of AKT and later induced ERK activation. The basal level of phosphorylated ERK increased during serial passage of BMSCs treated with FGF-2. The expression of LC3-II, an autophagy marker, was gradually increased and the population of senescent cells was increased dramatically at passage 7 in non-treated controls. But FGF-2 and FGF-4 suppressed LC3-II expression and down-regulated senescent cells during long-term (i.e. 2 month) cultures. Taken together, depletion of growth factors during serial passage

  18. [Effect of laminar shear stress on the expression of matrix metalloproteinases-9 in rat bone marrow-derived mesenchymal stem cells].

    Science.gov (United States)

    Chen, Longju; Sun, Xiaodong; Tang, Jie; Ding, Yan; Li, Jing; Li, Wenchun; Gong, Jian; Wang, Hanqin

    2010-12-01

    This paper was designed to investigate the effect of laminar shear stress on matrix metalloproteinase -9 (MMP-9) expression in rat bone marrow-derived mesenchymal stem cells (MSCs), and the possible signal transduction mechanism involved. Rat bone marrow MSCs were isolated and cultured, then, exposed to laminar shear stress at indicated strengths such as low (5dyne/cm2), medium (15 dyne/cm2) and high (30 dyne/cm2) via parallel plate flow chamber. RT-PCR was used to analyze the expression of MMP-9. The signaling inhibitors such as Wortmannin (PI3K specific inhabitor), SB202190 (p38MAPK specific inhabitor), and PD98059 (ERK1/2 specific inhabitor) were used to investigate the possible mechanical signal transduction pathway. The results showed: (1) The expression of MMP-9 was weak in static state, however, MMP-9 expression increased when MSCs were exposed to 15 dyne/cm2 shear stress for 2 hours, and MMP-9 expression increased with the extension of stimulating time, and it reached the peak at 24 h; (2) MSCs were stimulated by shear stress for 2 hours at different strengths (5 dyne/cm2, 15 dyne/cm2, 30 dyne/cm2), and under all these conditions, the expression of MMP-9 increased, and reached the peak at 15 dyne/cm2; (3) After MSCs were pretreated by three kinds of signal pathway inhibitors, the expression of MMP-9 did not change obviously in Wortmannin group and PD98059 group, but it was significantly inhibited in SB202190 group. This study demonstrated that shear stress could induce the expression of MMP-9 in rat bone marrow-derived mesenchymal stem cells; the amount of MMP-9 expression was closely related to stimulating time and the strengths of shear stress; and p38MAPK signal pathway played a critical role during the process.

  19. Bone Formation by Sheep Stem Cells in an Ectopic Mouse Model: Comparison of Adipose and Bone Marrow Derived Cells and Identification of Donor-Derived Bone by Antibody Staining

    DEFF Research Database (Denmark)

    Kjærgaard, Kristian; Dreyer, Chris Halling; Ditzel, Nicholas;

    2016-01-01

    Background. Scaffolds for bone tissue engineering (BTE) can be loaded with stem and progenitor cells (SPC) from different sources to improve osteogenesis. SPC can be found in bone marrow, adipose tissue, and other tissues. Little is known about osteogenic potential of adipose-derived culture...

  20. Bone marrow-derived cells mobilized by granulocyte-colony stimulating factor facilitate vascular regeneration in mouse kidney after ischemia/reperfusion injury.

    Science.gov (United States)

    Akihama, Susumu; Sato, Kazunari; Satoh, Shigeru; Tsuchiya, Norihiko; Kato, Tetsuro; Komatsuda, Atsushi; Hirokawa, Makoto; Sawada, Kenichi; Nanjo, Hiroshi; Habuchi, Tomonori

    2007-12-01

    Bone marrow-derived cells (BMDC) play crucial roles in tissue regeneration. Granulocyte-colony stimulating factor (G-CSF) mobilizes BMDC and may facilitate the repair of kidney tissues after ischemia/reperfusion (I/R) injury. The tissue protective action of resveratrol, an antioxidant, might modify the regenerating potential of BMDC in I/R renal injury. This study examined whether G-CSF and/or resveratrol affect the recruitment of BMDC into vascular endothelial cells and renal tubular cells and the kidney function after I/R injury. I/R renal injury was induced in female mice that had been lethally irradiated and transplanted with male bone marrow cells. The mice were given saline, resveratrol or G-CSF, daily for 7 days. Non-irradiated and non-bone-marrow-transplanted female mice, which underwent the same kidney injury, were included as control. White blood cell (WBC) count and serum creatinine were monitored. Immunohistologic evaluation for renal tubular cells (cytokeratin) and endothelial cells (factor VIII-related antigen), and fluorescence in situ hybridization for mouse Y chromosome were performed. Although WBC was significantly higher in the G-CSF group, there was no significant difference in creatinine levels among all groups. Factor VIII-related antigen-positive cells with a Y-chromosome signal were identified in the capillary wall between renal tubuli and most frequently seen in the G-CSF group (p cells having a Y-chromosome signal were identified. In conclusion, BMDC are recruited into endothelial cell in I/R renal injury without apparent renal tubular cell regeneration, and G-CSF facilitates the endothelial cell regeneration.

  1. Systemic preconditioning by a prolyl hydroxylase inhibitor promotes prevention of skin flap necrosis via HIF-1-induced bone marrow-derived cells.

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    Mitsuru Takaku

    Full Text Available BACKGROUND: Local skin flaps often present with flap necrosis caused by critical disruption of the blood supply. Although animal studies demonstrate enhanced angiogenesis in ischemic tissue, no strategy for clinical application of this phenomenon has yet been defined. Hypoxia-inducible factor 1 (HIF-1 plays a pivotal role in ischemic vascular responses, and its expression is induced by the prolyl hydroxylase inhibitor dimethyloxalylglycine (DMOG. We assessed whether preoperative stabilization of HIF-1 by systemic introduction of DMOG improves skin flap survival. METHODS AND RESULTS: Mice with ischemic skin flaps on the dorsum were treated intraperitoneally with DMOG 48 hr prior to surgery. The surviving area with neovascularization of the ischemic flaps was significantly greater in the DMOG-treated mice. Significantly fewer apoptotic cells were present in the ischemic flaps of DMOG-treated mice. Interestingly, marked increases in circulating endothelial progenitor cells (EPCs and bone marrow proliferative progenitor cells were observed within 48 hr after DMOG treatment. Furthermore, heterozygous HIF-1α-deficient mice exhibited smaller surviving flap areas, fewer circulating EPCs, and larger numbers of apoptotic cells than did wild-type mice, while DMOG pretreatment of the mutant mice completely restored these parameters. Finally, reconstitution of wild-type mice with the heterozygous deficient bone marrow cells significantly decreased skin flap survival. CONCLUSION: We demonstrated that transient activation of the HIF signaling pathway by a single systemic DMOG treatment upregulates not only anti-apoptotic pathways but also enhances neovascularization with concomitant increase in the numbers of bone marrow-derived progenitor cells.

  2. Origin of osteoclasts: Mature monocytes and macrophages are capable of differentiating into osteoclasts under a suitable microenvironment prepared by bone marrow-derived stromal cells

    Energy Technology Data Exchange (ETDEWEB)

    Udagawa, Nobuyuki; Takahashi, Naoyuki; Akatsu, Takuhiko; Tanaka, Hirofumi; Sasaki, Takahisa; Suda, Tatsuo (Showa Univ., Tokyo (Japan)); Nishihara, Tatsuji; Koga, Toshihiko (National Inst. of Health, Tokyo (Japan)); Martin, T.J. (Saint Vincent' s Inst. of Medical Research, Melbourne (Australia))

    1990-09-01

    The authors previously reported that osteoclast-like cells were formed in cocultures of a mouse marrow-derived stromal cell line (ST2) with mouse spleen cells in the presence of 1{alpha},25-dihydroxyvitamin D{sub 3} and dexamethasone. In this study, they developed a new coculture system to determine the origin of osteoclasts. When relatively small numbers of mononuclear cells obtained from mouse bone marrow, spleen, thymus, or peripheral blood were cultured for 12 days on the ST2 cell layers, they formed colonies with a linear relationship between the number of colonies formed and the number of hemopoietic cells inoculated. Tartrate-resistant acid phosphatase (TRAPase)-positive monoculear and multinucleated cells appeared in the colonies (TRAPase-positive colonies) in response to 1{alpha},25-dihydroxyvitamin D{sub 3} and dexamethasone. When hemopoietic cells suspended in a collagen-gel solution were cultured on the ST2 cell layers to prevent their movement, TRAPase-positive colonies were similarly formed, indicating that each colony originated from a single cell. Salmon {sup 125}I-labeled calcitonin specifically bound to the TRAPase-positive cells. Resorption lacunae were formed on dentine slices on which cocultures were performed. These results indicate that osteoclasts are also derived from the mature monocytes and macrophages when a suitable microenvironment is provided by bone marrow-derived stromal cells.

  3. After injection into the striatum, in vitro-differentiated microglia- and bone marrow-derived dendritic cells can leave the central nervous system via the blood stream.

    Science.gov (United States)

    Hochmeister, Sonja; Zeitelhofer, Manuel; Bauer, Jan; Nicolussi, Eva-Maria; Fischer, Marie-Therese; Heinke, Bernhard; Selzer, Edgar; Lassmann, Hans; Bradl, Monika

    2008-12-01

    The prototypic migratory trail of tissue-resident dendritic cells (DCs) is via lymphatic drainage. Since the central nervous system (CNS) lacks classical lymphatic vessels, and antigens and cells injected into both the CNS and cerebrospinal fluid have been found in deep cervical lymph nodes, it was thought that CNS-derived DCs exclusively used the cerebrospinal fluid pathway to exit from tissues. It has become evident, however, that DCs found in peripheral organs can also leave tissues via the blood stream. To study whether DCs derived from microglia and bone marrow can also use this route of emigration from the CNS, we performed a series of experiments in which we injected genetically labeled DCs into the striata of rats. We show here that these cells migrated from the injection site to the perivascular space, integrated into the endothelial lining of the CNS vasculature, and were then present in the lumen of CNS blood vessels days after the injection. Moreover, we also found these cells in both mesenteric lymph nodes and spleens. Hence, microglia- and bone marrow-derived DCs can leave the CNS via the blood stream.

  4. Bone marrow stromal cell : mediated neuroprotection for spinal cord repair

    NARCIS (Netherlands)

    Ritfeld, Gaby Jane

    2014-01-01

    Currently, there is no treatment available that restores anatomy and function after spinal cord injury. This thesis explores transplantation of bone marrow-derived mesenchymal stem cells (bone marrow stromal cells; BMSCs) as a therapeutic approach for spinal cord repair. BMSCs secrete neurotrophic f

  5. Comparison of the Treatment Efficiency of Bone Marrow-Derived Mesenchymal Stem Cell Transplantation via Tail and Portal Veins in CCl4-Induced Mouse Liver Fibrosis.

    Science.gov (United States)

    Truong, Nhung Hai; Nguyen, Nam Hai; Le, Trinh Van; Vu, Ngoc Bich; Huynh, Nghia; Nguyen, Thanh Van; Le, Huy Minh; Phan, Ngoc Kim; Pham, Phuc Van

    2016-01-01

    Because of self-renewal, strong proliferation in vitro, abundant sources for isolation, and a high differentiation capacity, mesenchymal stem cells are suggested to be potentially therapeutic for liver fibrosis/cirrhosis. In this study, we evaluated the treatment effects of mouse bone marrow-derived mesenchymal stem cells (BM-MSCs) on mouse liver cirrhosis induced by carbon tetrachloride. Portal and tail vein transplantations were examined to evaluate the effects of different injection routes on the liver cirrhosis model at 21 days after transplantation. BM-MSCs transplantation reduced aspartate aminotransferase/alanine aminotransferase levels at 21 days after injection. Furthermore, BM-MSCs induced positive changes in serum bilirubin and albumin and downregulated expression of integrins (600- to 7000-fold), transforming growth factor, and procollagen-α1 compared with the control group. Interestingly, both injection routes ameliorated inflammation and liver cirrhosis scores. All mice in treatment groups had reduced inflammation scores and no cirrhosis. In conclusion, transplantation of BM-MSCs via tail or portal veins ameliorates liver cirrhosis in mice. Notably, there were no differences in treatment effects between tail and portal vein administrations. In consideration of safety, we suggest transfusion of bone marrow-derived mesenchymal stem cells via a peripheral vein as a potential method for liver fibrosis treatment.

  6. Novel characterization of monocyte-derived cell populations in the meninges and choroid plexus and their rates of replenishment in bone marrow chimeric mice.

    Science.gov (United States)

    Chinnery, Holly R; Ruitenberg, Marc J; McMenamin, Paul G

    2010-09-01

    The mouse dura mater, pia mater, and choroid plexus contain resident macrophages and dendritic cells (DCs). These cells participate in immune surveillance, phagocytosis of cellular debris, uptake of antigens from the surrounding cerebrospinal fluid and immune regulation in many pathologic processes. We used Cx3cr1 knock-in, CD11c-eYFP transgenic and bone marrow chimeric mice to characterize the phenotype, density and replenishment rate of monocyte-derived cells in the meninges and choroid plexus and to assess the role of the chemokine receptor CX3CR1 on their number and tissue distribution. Iba-1 major histocompatibility complex (MHC) Class II CD169 CD68 macrophages and CD11c putative DCs were identified in meningeal and choroid plexus whole mounts. Comparison of homozygous and heterozygous Cx3cr1 mice did not reveal CX3CR1-dependancy on density, distribution or phenotype of monocyte-derived cells. In turnover studies, wild type lethally irradiated mice were reconstituted with Cx3cr1/-positive bone marrow and were analyzed at 3 days, 1, 2, 4 and 8 weeks after transplantation. There was a rapid replenishment of CX3CR1-positive cells in the dura mater (at 4 weeks) and the choroid plexus was fully reconstituted by 8 weeks. These data provide the foundation for future studies on the role of resident macrophages and DCs in conditions such as meningitis, autoimmune inflammatory disease and in therapies involving irradiation and hematopoietic or stem cell transplantation.

  7. Epidermis-dermis junction as a novel location for bone marrow-derived cells to reside in response to ionizing radiation.

    Science.gov (United States)

    Okano, Junko; Kojima, Hideto; Katagi, Miwako; Nakae, Yuki; Terashima, Tomoya; Nakagawa, Takahiko; Kurakane, Takeshi; Okamoto, Naoki; Morohashi, Keita; Maegawa, Hiroshi; Udagawa, Jun

    2015-06-12

    Bone marrow-derived cells (BMDCs) can migrate into the various organs in the mice irradiated by ionizing radiation (IR). However, it may not be the case in the skin. While IR is used for bone marrow (BM) transplantation, studying with the epidermal sheets demonstrated that the BMDC recruitment is extraordinarily rare in epidermis in the mouse. Herein, using the chimera mice with BM from green fluorescent protein (GFP) transgenic mice, we simply examined if BMDCs migrate into any layers in the total skin, as opposed to the epidermal sheets, in response to IR. Interestingly, we identified the presence of GFP-positive (GFP(+)) cells in the epidermis-dermis junction in the total skin sections although the epidermal cell sheets failed to have any GFP cells. To examine a possibility that the cells in the junction could be mechanically dissociated during separating epidermal sheets, we then salvaged such dissociated cells and examined its characteristics. Surprisingly, some GFP(+) cells were found in the salvaged cells, indicating that these cells could be derived from BM. In addition, such BMDCs were also associated with inflammation in the junction. In conclusion, BMDCs can migrate to and reside in the epidermis-dermis junction after IR.

  8. Reliable and inexpensive expression of large, tagged, exogenous proteins in murine bone marrow-derived macrophages using a second generation lentiviral system

    Directory of Open Access Journals (Sweden)

    Matthew R. Miller

    2015-08-01

    Full Text Available Over the past two decades, researchers have struggled to efficiently express foreign DNA in primary macrophages, impeding research progress. The applications of lipofection, electroporation, microinjection, and viral-mediated transfer typically result in disruptions in macrophage differentiation and function, low expression levels of exogenous proteins, limited efficiency and high cell mortality. In this report, after extensive optimization, we present a method of expressing large tagged proteins at high efficiency, consistency, and low cost using lentiviral infection. This method utilizes laboratory-propagated second generation plasmids to produce efficient virus that can be stored for later use. The expression of proteins up to 150 kDa in size is achieved in 30–70% of cells while maintaining normal macrophage differentiation and morphology as determined by fluorescence microscopy and Western blot analysis. This manuscript delineates the reagents and methods used to produce lentivirus to express exogenous DNA in murine bone marrow-derived macrophages sufficient for single cell microscopy as well as functional assays requiring large numbers of murine bone marrow-derived macrophages.

  9. Ex vivo differentiation of human bone marrow-derived stem cells into neuronal cell-like lineages

    Directory of Open Access Journals (Sweden)

    Al-Zoubi A

    2016-06-01

    Full Text Available Adeeb Al-Zoubi,1,2 Feras Altwal,3 Farah Khalifeh,2 Jamil Hermas,4 Ziad Al-Zoubi,5 Emad Jafar,5 Mohammed El-Khateeb,6,7 1Department of Surgery, University of Illinois College of Medicine at Peoria, Peoria, IL, USA; 2Stem Cells of Arabia, Amman, Jordan; 3Department of Neuroscience, School of Graduate and Postdoctoral Studies, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA; 4Stem Cell Division, Al-Yamama Company, 5Jordan Orthopedic and Spinal Center, 6National Center for Diabetes, Endocrinology and Genetics, 7Department of Pathology, Faculty of Medicine, University of Jordan, Amman, Jordan Background: Methods to obtain safe and practical populations of stem cells (SCs at a clinical grade that are able to differentiate into neuronal cell lineages are yet to be developed. In a previous study, we showed that mouse bone marrow-derived SCs (BM-SCs differentiated into neuronal cell-like lineages when put in a neuronal-like environment, which is a special media supplemented with the necessary growth factors needed for the differentiation of SCs into neuronal cell-like lineages. Aim: In this study, we aim to assess the potentials of adult human CD34+ and CD133+ SCs to differentiate into neuronal cell-like lineages ex vivo when placed in a neuronal-like microenvironment. Methods: The neuronal-like microenvironment was created by culturing cells in nonhematopoietic expansion media (NHEM supplemented with growth factors that favor differentiation into neuronal cell lineages (low-affinity nerve growth factor [LNGF], mouse spinal cord extract [mSpE], or both. Cultured cells were assessed for neuronal differentiation by cell morphologies and by expression of GFAP. Results: Our results show that culturing unpurified human BM-derived mononuclear cells (hBM-MNCs in NHEM+LNGF+mSpE did not lead to neuronal differentiation. In contrast, culturing of purified CD34+ hBM-SCs in NHEM+LNGF+mSpE favored their differentiation into astrocyte

  10. The CD271 expression could be alone for establisher phenotypic marker in Bone Marrow derived mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Antonio Carrasco-Yalan

    2011-04-01

    Full Text Available Mesenchymal stem cells (MSCs are of great interest for their potential use in cellular therapies. To define the population more precisely, diverse surface markers have been used. We propose here to use CD271 as the sole marker for MSCs in fresh bone marrow. We compared CD271+ populations to the presence or absence of five defined markers for MSCs: CD90+, CD105+, CD45-, CD34- and CD79. The correlations between markers were evaluated and analyzed with a Pearson's correlation test. We found that the average percentage of cells expressing the combination of markers CD90+, CD105+, CD45-, CD34- and CD79- was 0.54%, and that the average percentage average of CD271+ cells was 0.53%. The results were significant (p<0.05. The exclusive use of CD271 as a marker for MSCs from fresh samples of bone marrow appears to be highly selective. Using CD271 as the sole identification marker for MSCs could reduce costs and accelerate the process of identifying MSCs for the field of cellular therapy.

  11. How does the supernatant of Lactobacillus acidophilus affect the proliferation and differentiation activities of rat bone marrow-derived stromal cells?

    Science.gov (United States)

    Samadikuchaksaraei, A; Gholipourmalekabadi, M; Saberian, M; Abdollahpour Alitappeh, M; Shahidi Delshad, E

    2016-08-31

    Low proliferation rate and unwanted differentiation of bone marrow-derived stromal cells (rBMSCs) during the frequent passages have limited the use of such cells in clinical cell therapy. Recently, the researchers have focused on the effects of the components produced by some bacteria on proliferation of the stem cells. In this study, we discussed the possible effects of the Lactobacillus acidophilus supernatant on proliferation and differentiation of the rBMSCs. For this aim, the cells were isolated from rat bone marrow, characterized by culturing on tissue specific differentiation media and stained. The cells (passage two) were treated with different concentrations of the L. acidophilus supernatant (0, 0.1, 0.3, 0.9, 3, 9 and 30 &mgr;l/ml) for 14 days. The proliferation and differentiation capacity of the cells were then determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT assay) and tissue specific staining. The results showed a positive effect of the supernatant on the cell proliferation in 3 and 9 &mgr;l/ml concentrations, while did not affect the differentiation capacity of the rBMSCs. The current study strongly suggests the L. acidophilus supernatant as an alternative material that could be added to the media with aim of improvement in the proliferation rate of the rBMSCs without affecting their differentiation capacity.

  12. Gr-1 Ab administered after bone marrow transplantation plus thymus transplantation suppresses tumor growth by depleting granulocytic myeloid-derived suppressor cells.

    Science.gov (United States)

    Shi, Ming; Li, Ming; Cui, Yunze; Adachi, Yasushi; Ikehara, Susumu

    2014-01-01

    It has been shown that allogeneic intra-bone marrow-bone marrow transplantation (IBM-BMT) plus thymus transplantation (TT) is effective in treating recipients with malignant tumors. Although TT increases the percentage of T cells in the early term after BMT, the myeloid-derived suppressor cells (MDSCs) are still the dominant population. We used the Gr-1 Ab to deplete the granulocytic MDSCs (G-MDSCs) in tumor-bearing mice that had received BMT+TT. Two weeks after the BMT, the mice injected with Gr-1 Ab showed smaller tumors than those in the control group. In addition, Gr-1 Ab significantly increased the percentages and numbers of CD4+ and CD8+ T cells, and decreased the percentages and numbers of MDSCs and G-MDSCs. No side effects of the Gr-1 Ab on recipient or donor thymus were observed. These findings indicate that Gr-1 Ab administered after BMT+TT may enhance the effectiveness of tumor suppression.

  13. Effects of Simian Betaretrovirus Serotype 1 (SRV1) Infection on the Differentiation of Hematopoietic Progenitor Cells (CD34+) Derived from Bone Marrow of Rhesus Macaques (Macaca mulatta)

    Science.gov (United States)

    Montiel, Nestor A; Todd, Patricia A; Yee, JoAnn; Lerche, Nicholas W

    2012-01-01

    Peripheral blood cytopenias, particularly persistent anemia and neutropenia, are commonly associated with simian betaretrovirus infection of Asian monkeys of the genus Macaca. The pathogenetic mechanisms underlying these hematologic abnormalities are not well understood. The current study investigated the in vitro tropism of simian betaretrovirus (SRV) for both hematopoietic progenitor (CD34+) and stromal cells obtained from rhesus macaque bone marrow and assessed the effects of infection on hematopoietic progenitor cell differentiation in vitro. After in vitro exposure, SRV proviral DNA could be demonstrated by real-time PCR in cells and the reverse transcriptase assay in supernatants from SRV-exposed progenitor-associated stroma, but not in differentiated colonies derived from SRV-exposed progenitors. Furthermore, in vitro exposure involving cell–cell contact of uninfected CD34+ progenitor cells with SRV-infected stromal cells resulted in a statistically significant reduction in granulocyte–macrophage colony formation in absence of detectable SRV-infection of progenitor cells. Reduction in colony formation occurred in a ‘dose-dependent’ fashion with increasing contact time. No effects on erythroid lineages and RBC differentiation were noted. Our results suggest that hematologic abnormalities observed during SRV disease (natural or experimental) of rhesus macaques may not result from direct effects of viral infection of progenitor cell populations, but rather be (at least in part) a consequence of SRV infection of supportive bone marrow stroma with secondary effects on differentiation of associated progenitor cells. PMID:22330653

  14. Two populations of ovine bone marrow-derived dendritic cells can be generated with recombinant GM-CSF and separated on CD11b expression.

    Science.gov (United States)

    Foulon, Eliane; Foucras, Gilles

    2008-11-30

    Whereas studies on dendritic cells in rodents rely largely on bone marrow-derived dendritic cells (BM-DCs), no data are available about BM-DCs in sheep, a species that is largely used for immunology and transplantation studies. We have developed a culture protocol to produce ovine BM-DCs, using 6x(His)-tagged recombinant GM-CSF which was purified from baculovirus-infected insect cells. When ovine bone marrow progenitors were cultured in the presence of recombinant GM-CSF, large numbers of CD11c-positive cells were generated after 6-7 days. The phenotypic appearance of BM-DCs was assessed by flow cytometry and electron microscopy. Two DC subsets were identified that expressed different levels of MHC class II molecules, differed in receptor-mediated endocytosis, and could be separated on CD11b expression. When separated cells were incubated with microbial products, they react differently to those that are considered the TLR2 and TLR4 agonists in other species. Indeed, although CD11b(int/hi) cells were partially resistant to maturation induced by lipoteichoic acid or lipopolysaccharide, MHC class II upregulation was observed on CD11b(dull) cells. Moreover, these cells had strong stimulatory capacity for CD4 T cells when assayed in allogeneic reactions. This protocol will help analyzing ovine DC interactions with pathogens, and enables future studies on the development of vaccines.

  15. High-affinity uptake of kynurenine and nitric oxide-mediated inhibition of indoleamine 2,3-dioxygenase in bone marrow-derived myeloid dendritic cells.

    Science.gov (United States)

    Hara, Toshiaki; Ogasawara, Nanako; Akimoto, Hidetoshi; Takikawa, Osamu; Hiramatsu, Rie; Kawabe, Tsutomu; Isobe, Ken-Ichi; Nagase, Fumihiko

    2008-02-15

    Indoleamine 2,3-dioxygenase (IDO)-initiated tryptophan metabolism along the kynurenine (Kyn) pathway in some dendritic cells (DC) such as plasmacytoid DC (pDC) regulates T-cell responses. It is unclear whether bone marrow-derived myeloid DC (BMDC) express functional IDO. The IDO expression was examined in CD11c(+)CD11b(+) BMDC differentiated from mouse bone marrow cells using GM-CSF. CpG oligodeoxynucleotides (CpG) induced the expression of IDO protein with the production of nitric oxide (NO) in BMDC in cultures for 24h. In the enzyme assay using cellular extracts of BMDC, the IDO activity of BMDC stimulated with CpG was enhanced by the addition of a NO synthase (NOS) inhibitor, suggesting that IDO activity was suppressed by NO production. On the other hand, the concentration of Kyn in the culture supernatant of BMDC was not increased by stimulation with CpG. Exogenously added Kyn was taken up by BMDC independently of CpG stimulation and NO production, and the uptake of Kyn was inhibited by a transport system L-specific inhibitor or high concentrations of tryptophan. The uptake of tryptophan by BMDC was markedly lower than that of Kyn. In conclusion, IDO activity in BMDC is down-regulated by NO production, whereas BMDC strongly take up exogenous Kyn.

  16. Effects of lateral ventricular transplantation of bone marrow-derived mesenchymal stem cells modified with brain-derived neurotrophic factor gene on cognition in a rat model of Alzheimer's disease

    Institute of Scientific and Technical Information of China (English)

    Ping Zhang; Gangyong Zhao; Xianjiang Kang; Likai Su

    2012-01-01

    In the present study, transplantation of bone marrow-derived mesenchymal stem cells modified with brain-derived neurotrophic factor gene into the lateral ventricle of a rat model of Alzheimer's disease, resulted in significant attenuation of nerve cell damage in the hippocampal CA1 region. Furthermore, brain-derived neurotrophic factor and tyrosine kinase B mRNA and protein levels were significantly increased, and learning and memory were significantly improved. Results indicate that transplantation of bone marrow-derived mesenchymal stem cells modified with brain-derived neurotrophic factor gene can significantly improve cognitive function in a rat model of Alzheimer's disease, possibly by increasing the levels of brain-derived neurotrophic factor and tyrosine kinase B in the hippocampus.

  17. Increased stromal-cell-derived factor 1 enhances the homing of bone marrow derived mesenchymal stem cells in dilated cardiomyopathy in rats

    Institute of Scientific and Technical Information of China (English)

    ZHOU Yan-li; Michael Fu; ZHANG Hai-feng; LI Xin-li; DI Ruo-min; YAO Wen-ming; LI Dian-fu; FENG Jian-lin; HUANG Jun; CAO Ke-jiang

    2010-01-01

    Background Stem cell transplantation has been shown to have beneficial effects on dilated cardiomyopathy. However,mechanism for stem cell homing to cardiac tissue in dilated cardiomyopathy has not yet been elucidated.Methods Mesenchymal stem cells were obtained from rat bone marrow, expanded in vitro, and labeled with 99mTc.Cardiomyopathy model was induced by doxorubicin in rats. 99mTc labeled cells were infused into the left ventricles in cardiomyopathy and control rats. Sixteen hours after injection, animals were sacrificed and different tissues were harvested to measure specific radioactivity. By use of real-time polymerase chain reaction and immunohistochemistry,Mrna and protein expressions for stromal-cell-derived factor 1 in cardiac tissue were measured.Results Labeling efficiency of mesenchymal stem cells was (70.0±11.2)%. Sixteen hours after mesenchymal stem cell transplantation, the heart-to-muscle radioactivity ratio was increased significantly in cardiomyopathy hearts as compared to control hearts. Both Mrna and rotein expressions of stromal-cell-derived factor 1 were up-regulated in cardiomyopathy hearts as compared with control hearts.Conclusion In dilated cardiomyopathy induced by doxorubicin up-regulated expression of stromal-cell-derived factor 1in heart may induce mesenchymal stem cells home to the heart.

  18. Bone marrow-derived stromal cells are more beneficial cell sources for tooth regeneration compared with adipose-derived stromal cells.

    Science.gov (United States)

    Ye, Lanfeng; Chen, Lin; Feng, Fan; Cui, Junhui; Li, Kaide; Li, Zhiyong; Liu, Lei

    2015-10-01

    Tooth loss is presently a global epidemic and tooth regeneration is thought to be a feasible and ideal treatment approach. Choice of cell source is a primary concern in tooth regeneration. In this study, the odontogenic differentiation potential of two non-dental-derived stem cells, adipose-derived stromal cells (ADSCs) and bone marrow-derived stromal cells (BMSCs), were evaluated both in vitro and in vivo. ADSCs and BMSCs were induced in vitro in the presence of tooth germ cell-conditioned medium (TGC-CM) prior to implantation into the omentum majus of rats, in combination with inactivated dentin matrix (IDM). Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the mRNA expression levels of odontogenic-related genes. Immunofluorescence and immunohistochemical assays were used to detect the protein levels of odontogenic-specific genes, such as DSP and DMP-1 both in vitro and in vivo. The results suggest that both ADSCs and BMSCs have odontogenic differentiation potential. However, the odontogenic potential of BMSCs was greater compared with ADSCs, showing that BMSCs are a more appropriate cell source for tooth regeneration.

  19. Immunomodulatory effects of bone marrow mesenchymal stem cells derived from homologous recipients in rats after heart transplantation

    Directory of Open Access Journals (Sweden)

    De-zhong LIU

    2012-03-01

    Full Text Available Objective To observe the immunomodulatory effects of homologous bone marrow mesenchymal stem cells (MSCs obtained from the bone marrow in rats after heart transplantation. Methods Twenty adult male Lewis rats were used as donors for the heart transplantation, whereas twenty adult male Wistar rats served as recipients. The recipients with cervical heart transplantation were randomly divided into two groups (10 each. Approximately 3ml 0.9% NaCl solution was injected through the tail vein 24h after heart transplantation in the control group (group A. About 2×106 MSCs (suspended in 3ml 0.9% NaCl solution were injected through the tail vein 24h after heart transplantation in the MSCs treatment group (group B. Four recipient rats from each group were randomly chosen one week after transplantation for determining proportion of CD4+ T, CD8+ T, CD4+CD25high T, and CD4+CD25highfoxp3+ T cells in the lymphocytes in the venous blood and grafts. Subsequently, the CD4+/CD8+ ratio was calculated. The survival time of the grafts were observed in the remaining six rats in each group. Results (1The survival time of the transplanted hearts was 7.2±1.3d in group A, and 14.8±2.9d in group B, showing a significant difference between the two groups (P 0.05. The ratios of CD4+CD25high T cells/total lymphocytes and CD4+CD25highFoxp3+ T cells/ total lymphocytes in the allografts were evidently higher in group B (2.74%±0.28%, 2.54%±0.31% than in group A (0.61%±0.06%, 0.53%±0.06%, showing a significant statistical difference (P < 0.01. Conclusion Intravenous infusion with MSCs from the bone marrow of the recipients can induce immune tolerance and prolong the survival time of transplanted heart in rats.

  20. Local transplantation of ex vivo expanded bone marrow-derived CD34-positive cells accelerates fracture healing.

    Science.gov (United States)

    Kawakami, Yohei; Ii, Masaaki; Alev, Cantas; Kawamoto, Atsuhiko; Matsumoto, Tomoyuki; Kuroda, Ryosuke; Shoji, Taro; Fukui, Tomoaki; Masuda, Haruchika; Akimaru, Hiroshi; Mifune, Yutaka; Kuroda, Tomoya; Horii, Miki; Yokoyama, Ayumi; Kurosaka, Masahiro; Asahara, Takayuki

    2012-01-01

    Transplantation of bone marrow (BM) CD34(+) cells, an endothelial/hematopoietic progenitor-enriched cell population, has shown therapeutic efficiency in the treatment of ischemic diseases enhancing neovascularization. However, the number of CD34(+) cells obtained from bone marrow is not sufficient for routine clinical application. To overcome this issue, we developed a more efficient and clinically applicable CD34(+) cell expansion method. Seven-day ex vivo expansion culture of BM CD34(+) cells with a cocktail of five growth factors containing VEGF, SCF, IL-6, Flt-3 ligand, and TPO resulted in reproducible more than 20-fold increase in cell number. The favorable effect of the local transplantation of culture expanded (cEx)-BM CD34(+) cells on rat unhealing fractures was equivalent or higher than that of nonexpanded (fresh) BM CD34(+) cells exhibiting sufficient therapeutic outcome with frequent vasculogenic/osteogenic differentiation of transplanted cEx-BM CD34(+) cells and fresh BM CD34(+) cells as well as intrinsic enhancement of angiogenesis/osteogenesis at the treated fracture sites. Specifically, cEx-BM CD34(+) cell treatment demonstrated the best blood flow recovery at fracture sites compared with the nonexpanded BM CD34(+) cells. In vitro, cEx-BM CD34(+) cells showed higher colony/tube-forming capacity than nonexpanded BM CD34(+) cells. Both cells demonstrated differentiation potential into osteoblasts. Since fresh BM CD34(+) cells can be easily collected from fracture sites at the time of primary operation and stored for future use, autologous cEx-BM CD34(+) cell transplantation would be not only a simple but also a promising therapeutic strategy for unhealing fractures in the field of orthopedic trauma surgery.

  1. Gene expression patterns related to osteogenic differentiation of bone marrow-derived mesenchymal stem cells during ex vivo expansion.

    Science.gov (United States)

    Granchi, Donatella; Ochoa, Gorka; Leonardi, Elisa; Devescovi, Valentina; Baglìo, Serena Rubina; Osaba, Lourdes; Baldini, Nicola; Ciapetti, Gabriela

    2010-06-01

    Bone marrow is commonly used as a source of adult multipotent mesenchymal stem cells (MSCs), defined for their ability to differentiate in vitro into multiple lineages. The ex vivo-expanded MSCs are currently being evaluated as a strategy for the restoration of function in damaged skeletal tissue, both in cell therapy and tissue engineering applications. The aim of this study was to define gene expression patterns underlying the differentiation of MSCs into mature osteoblasts during the expansion in vitro, and to explore a variety of cell functions that cannot be easily evaluated using morphological, cytochemical, and biochemical assays. Cell cultures were obtained from bone marrow samples of six individuals undergoing total hip replacement, and a large-scale transcriptome analysis, using Affymetrix HG-U133A Plus 2.0 array (Affymetrix((R)), Santa Clara, CA), was performed at the occurrence of specific events, including the appearance of MSC surface markers, formation of colonies, and deposition of mineral nodules. We focused our attention on 213 differentially upregulated genes, some belonging to well-known pathways and some having one or more Gene Ontology annotations related to bone cell biology, including angiogenesis, bone-related genes, cell communication, development and morphogenesis, transforming growth factor-beta signaling, and Wnt signaling. Twenty-nine genes, whose role in bone cell pathophysiology has not been described yet, were found. In conclusion, gene expression patterns that characterize the early, intermediate, and late phases of the osteogenic differentiation process of ex vivo-expanded MSCs were defined. These signatures represent a useful tool to monitor the osteogenic process, and to analyze a broad spectrum of functions of MSCs cultured on scaffolds, especially when the constructs are conceived for releasing growth factors or other signals to promote bone regeneration.

  2. Cryopreservation of Rat Bone Marrow Derived Mesenchymal Stem Cells by Two Conventional and Open-pulled Straw Vitrification Methods

    Directory of Open Access Journals (Sweden)

    Mohammad Hadi Bahadori

    2009-01-01

    Full Text Available Objective: Mesenchymal stem cells (MSCs are obtained from a variety of sources, mainlythe bone marrow. These cells have a great potential for clinical research, however they cannotstay alive for long periods in culture. The aim of this study is to determine whether vitrificationcan be a useful freezing method for the storage of MSCs.Materials and Methods: Mesenchymal stem cells were isolated from rat bone marrow basedon their capacity to adhere to plastic culture surfaces. MSCs were cryopreserved using boththe vitrification method and open-pulled straw (OPS vitrification and stored in liquid nitrogenwith ethylene glycol ficoll (EFS as a cryoprotectant for two months. The morphology andviability of thawed MSCs were evaluated by trypan blue staining. Furthermore, pre and postcryopreserved MSCs were induced to osteocyte and adipocyte with corresponding osteogenicand adipogenic medium.Results: After thawing, the viability rates were 81.33% ± 6.83 for the vitrification method and80.83% ± 6.4 for OPS vitrification, while the values in the pre-vitrification control group were88.16% ± 6.3 (Mean ± SD, n = 6. Post-cryopreserved cells from both the vitrification methodand OPS vitrification also had a similar cellular morphology and colony-formation that wasindistinguishable from non-vitrified fresh MSCs. In addition, the resuscitated cells cultured ininduction medium showed osteogenesis. Mineral production and deposition was detectableby alizarine red S staining. Moreover, by applying an adipogenic differentiation condition,both pre and post cryopreserved cells differentiated into adipocyte and lipid vacuole accumulationthat was stained by oil red O.Conclusion: Vitrification is a reliable and effective method for the cryopreservation of MSCs.

  3. Vascular remodeling and mobilization of bone marrow-derived cells in cuff-induced vascular injury in LDL receptor knockout muce

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Background Vascular remodeling is an important pathologic process in vascular injury for various vascular disorders such as atherosclerosis,postangioplasty restenosis and transplant arteriopathy.Recently,pathologic change and the role of bone marrow derived cells were wildly studied in atherosclerosis and restenosis.But the manner of lesion formation in neointima and cell recruitment in vascular remodeling lesion in the present of hypercholesterolemia is not Vet fully understood. Methods Double-transgenic mice knockout of LDL receptor gene (LDL-/-) and expressing ubiquitously green fluorescent protein (GFP) were obtained by cross-breeding LDL-/-mice with the GFP-expressing transgenic mice. LDL-/- mice (22-24 weeks of age) fed high fat diet containing 1.25% (w/w) cholesterol were subjected to 9Gy irradiation and received bone marrow (BM) cells from the double-transgenic mice.Four weeks later,a nonconstrictive cuff was Dlaced around the right femoral artery.After another 2 weeks,both right and left femoral arteries were harvested and subjected to histochemical analysis.Apoptosis was analyzed in situ using TUNEL assay.Resuits Two weeks after cuff placement,atherosclerotic lesions developed in the intima consisting of a massive accumulation of foam cells, The tissue stained with anti-α smooth muscle actin (SMA) antibody,showed a number of SMA-positive cells in the intimal lesion area.They were also positive for GFP,indicating that BM-derived cells can differentiate to SMCs in the intima in cuff-induced vascular remodeling lesions.Numerous small vessels in the adventitia as well as the endothelial lining of the intima were positive both for CD31 and GFP.The intima and media showed a larae number of TUNEL-positive signals after 2 weeks cuff injury,indicating the presence of apoptosis in vascular remodelina.Conclusions Atherosclerotic lesions in mice can be developed in the intima after 2 weeks of cuff-induced vascular inJury under the hypercholesterolemic conditions

  4. Hard tissue formation in a porous HA/TCP ceramic scaffold loaded with stromal cells derived from dental pulp and bone marrow.

    NARCIS (Netherlands)

    Zhang, W.; Walboomers, X.F.; Osch, G.J.V.M. van; Dolder, J. van den; Jansen, J.A.

    2008-01-01

    The aim of this study was to compare the ability of hard tissue regeneration of four types of stem cells or precursors under both in vitro and in vivo situations. Primary cultures of rat bone marrow, rat dental pulp, human bone marrow, and human dental pulp cells were seeded onto a porous ceramic sc

  5. Bone marrow edema syndrome

    Energy Technology Data Exchange (ETDEWEB)

    Korompilias, Anastasios V.; Lykissas, Marios G.; Beris, Alexandros E. [University of Ioannina, Department of Orthopaedic Surgery, School of Medicine, Ioannina (Greece); Karantanas, Apostolos H. [University of Crete School of Medicine, Department of Radiology, Heraklion (Greece)

    2009-05-15

    Bone marrow edema syndrome (BMES) refers to transient clinical conditions with unknown pathogenic mechanism, such as transient osteoporosis of the hip (TOH), regional migratory osteoporosis (RMO), and reflex sympathetic dystrophy (RSD). BMES is primarily characterized by bone marrow edema (BME) pattern. The disease mainly affects the hip, the knee, and the ankle of middle-aged males. Many hypotheses have been proposed to explain the pathogenesis of the disease. Unfortunately, the etiology of BMES remains obscure. The hallmark that separates BMES from other conditions presented with BME pattern is its self-limited nature. Laboratory tests usually do not contribute to the diagnosis. Histological examination of the lesion is unnecessary. Plain radiographs may reveal regional osseous demineralization. Magnetic resonance imaging is mainly used for the early diagnosis and monitoring the progression of the disease. Early differentiation from other aggressive conditions with long-term sequelae is essential in order to avoid unnecessary treatment. Clinical entities, such as TOH, RMO, and RSD are spontaneously resolving, and surgical treatment is not needed. On the other hand, early differential diagnosis and surgical treatment in case of osteonecrosis is of crucial importance. (orig.)

  6. Donor-Matched Comparison of Chondrogenic Potential of Equine Bone Marrow- and Synovial Fluid-Derived Mesenchymal Stem Cells: Implications for Cartilage Tissue Regeneration

    Science.gov (United States)

    Zayed, Mohammed; Caniglia, Christopher; Misk, Nabil; Dhar, Madhu S.

    2017-01-01

    Mesenchymal stem cells (MSCs) have been demonstrated to be useful for cartilage tissue regeneration. Bone marrow (BM) and synovial fluid (SF) are promising sources for MSCs to be used in cartilage regeneration. In order to improve the clinical outcomes, it is recommended that prior to clinical use, the cellular properties and, specifically, their chondrogenic potential must be investigated. The purpose of this study is to compare and better understand the in vitro chondrogenic potential of equine bone marrow-derived mesenchymal stem cells (BMMSCs) and synovial fluid-derived mesenchymal stem cells (SFMSCs) populated from the same equine donor. BM- and SF-derived MSCs cultures were generated from five equine donors, and the MSCs were evaluated in vitro for their morphology, proliferation, trilineage differentiation, and immunophenotyping. Differences in their chondrogenic potentials were further evaluated quantitatively using glycosaminoglycan (GAG) content and via immunofluorescence of chondrogenic differentiation protein markers, SRY-type HMG box9, Aggrecan, and collagen II. The BMMSCs and SFMSCs were similar in cellular morphology, viability, and immunophenotype, but, varied in their chondrogenic potential, and expression of the key chondrogenic proteins. The SFMSCs exhibited a significant increase in GAG content compared to the BMMSCs (P < 0.0001) in three donors, suggesting increased levels of chondrogenesis. The expression of the key chondrogenic proteins correlated positively with the GAG content, suggesting that the differentiation process is dependent on the expression of the target proteins in these three donors. Our findings suggest that even though SFMSCs were hypothesized to be more chondrogenic relative to BMMSCs, there was considerable donor-to-donor variation in the primary cultures of MSCs which can significantly affect their downstream application.

  7. Neuronal-like differentiation of bone marrow-derived mesenchymal stem cells induced by striatal extracts from a rat model of Parkinson's disease

    Institute of Scientific and Technical Information of China (English)

    Xiaoling Qin; Wang Han; Zhigang Yu

    2012-01-01

    A rat model of Parkinson's disease was established by 6-hydroxydopamine injection into the medial forebrain bundle. Bone marrow-derived mesenchymal stem cells (BMSCs) were isolated from the femur and tibia, and were co-cultured with 10% and 60% lesioned or intact striatal extracts. The results showed that when exposed to lesioned striatal extracts, BMSCs developed bipolar or multi-polar morphologies, and there was an increase in the percentage of cells that expressed glial fibrillary acidic protein (GFAP), nestin and neuron-specific enolase (NSE). Moreover, the percentage of NSE-positive cells increased with increasing concentrations of lesioned striatal extracts. However, intact striatal extracts only increased the percentage of GFAP-positive cells. The findings suggest that striatal extracts from Parkinson's disease rats induce BMSCs to differentiate into neuronal-like cells in vitro.

  8. Xenogeneic transfer of fetal liver and adult bone marrow-derived haemopoietic cells in rodents: changes in spleen colony differentials with increased doses of cells.

    Science.gov (United States)

    Gulya, E; Gábor Szabó, L; Kelemen, E

    1997-01-01

    The effect of very high haemopoietic cell doses were investigated on the composition of splenic cell colonies/clusters in irradiated animals under xenogeneic circumstances. Differential cluster/colony counts from serial histological sections of the spleen were investigated before, and 9-12 days after transplantation of fetal liver- or adult bone marrow-derived haemopoietic cells following 5.0 to 8.5 Gy total body irradiation. Syngeneic as well as xenogeneic (mouse to rat and rat to mouse) transplantations were carried out. Cluster/colony differentials changed with the increase of the injected cell mass from 10(5) to 10(6) and 10(7) or more, i.e. the overwhelming erythroid pattern became trilinear even with xenogeneic transplants.

  9. Pelleted bone marrow derived mesenchymal stem cells are better protected from the deleterious effects of arthroscopic heat shock

    Directory of Open Access Journals (Sweden)

    Gauthaman eKalamegam

    2016-05-01

    Full Text Available Introduction: The impact of arthroscopic temperature on joint tissues is poorly understood and it is not known how mesenchymal stem cells (MSCs respond to the effects of heat generated by the device during the process of arthroscopy assisted experimental cell-based therapy. In the present study, we isolated and phenotypically characterized human bone marrow mesenchymal stem cells (hBMMSCs from osteoarthritis (OA patients, and evaluated the effect of arthroscopic heat on cell viability in suspension and pellet cultures.Methods: Primary cultures of hBMMSCs were isolated from bone marrow aspirates of OA patients and cultured using DMEM supplemented with 10% FBS and characterized for their stemness. hBMMSCs (1 x 106 cells cultured as single cell suspensions or cell pellets were exposed to an illuminated arthroscope for 10, 20 or 30 min. This was followed by analysis of cellular proliferation and heat shock related gene expression. Results: hBMMSCs were viable and exhibited population doubling, short spindle morphology, MSC related CD surface markers expression and tri-lineage differentiation into adipocytes, chondrocytes and osteoblasts. Chondrogenic and osteogenic differentiation increased collagen production and alkaline phosphatase activity. Exposure of hBMMSCs to an illuminated arthroscope for 10, 20 or 30 min for 72 h decreased cell proliferation in cell suspensions (63.27% at 30 min and increased cell proliferation in cell pellets (62.86% at 10 min and 68.57% at 20 min. hBMMSCs exposed to 37C, 45C and 55C for 120 seconds demonstrated significant upregulation of BAX, P53, Cyclin A2, Cyclin E1, TNF-α, and HSP70 in cell suspensions compared to cell pellets. Conclusions: hBMMSC cell pellets are better protected from temperature alterations compared to cell suspensions. Transplantation of hBMMSCs as pellets rather than as cell suspensions to the cartilage defect site would therefore support their viability and may aid enhanced cartilage

  10. Differentiation of mesenchymal stem cells derived from pancreatic islets and bone marrow into islet-like cell phenotype.

    Directory of Open Access Journals (Sweden)

    Cristina Zanini

    Full Text Available BACKGROUND: Regarding regenerative medicine for diabetes, accessible sources of Mesenchymal Stem Cells (MSCs for induction of insular beta cell differentiation may be as important as mastering the differentiation process itself. METHODOLOGY/PRINCIPAL FINDINGS: In the present work, stem cells from pancreatic islets (human islet-mesenchymal stem cells, HI-MSCs and from human bone marrow (bone marrow mesenchymal stem cells, BM-MSCs were cultured in custom-made serum-free medium, using suitable conditions in order to induce differentiation into Islet-like Cells (ILCs. HI-MSCs and BM-MSCs were positive for the MSC markers CD105, CD73, CD90, CD29. Following this induction, HI-MSC and BM-MSC formed evident islet-like structures in the culture flasks. To investigate functional modifications after induction to ILCs, ultrastructural analysis and immunofluorescence were performed. PDX1 (pancreatic duodenal homeobox gene-1, insulin, C peptide and Glut-2 were detected in HI-ILCs whereas BM-ILCs only expressed Glut-2 and insulin. Insulin was also detected in the culture medium following glucose stimulation, confirming an initial differentiation that resulted in glucose-sensitive endocrine secretion. In order to identify proteins that were modified following differentiation from basal MSC (HI-MSCs and BM-MSCs to their HI-ILCs and BM-ILCs counterparts, proteomic analysis was performed. Three new proteins (APOA1, ATL2 and SODM were present in both ILC types, while other detected proteins were verified to be unique to the single individual differentiated cells lines. Hierarchical analysis underscored the limited similarities between HI-MSCs and BM-MSCs after induction of differentiation, and the persistence of relevant differences related to cells of different origin. CONCLUSIONS/SIGNIFICANCE: Proteomic analysis highlighted differences in the MSCs according to site of origin, reflecting spontaneous differentiation and commitment. A more detailed understanding of

  11. Effects of histamine and its antagonists on murine T-cells and bone marrow-derived dendritic cells.

    Science.gov (United States)

    Hu, Xiufen; Zafar, Mohammad Ishraq; Gao, Feng

    2015-01-01

    We determined the effects of histamine and its antagonists on the surface marker expression of dendritic cells (DCs) and the influence of lipopolysaccharide (LPS), histamine, and histamine receptor antagonists on DCs and T-cells. The bone marrow was extracted from the femurs and tibiae of 6- to 8-week-old female Balb/c mice and cultured in medium containing penicillin, streptomycin, L-glutamine, fetal calf serum, or granulocyte macrophage colony-stimulating factor (GM-CSF) alone or with interleukin (IL)-4. The cells received three different doses of LPS and histamine, plus three different doses of descarboethoxyloratadine (DCL). We assayed the supernatant for various cytokines. The spleen cells of DO11.10 mice were examined by flow cytometry, which included labeling and sorting CD4+ T-cells, as well as coculture of DCs and T-cells with ovalbumin (OVA)323-339 peptide. Histamine or histamine plus DCL did not affect the expression of major histocompatibility complex class II, CD11c, CD11b, CD86, and CD80. However, GM-CSF increased the expression of all markers except CD80. Histamine increased interferon-γ production in GM-CSF + IL-4-cultured cells; it also enhanced IL-10 production, but suppressed IL-12 production in LPS-stimulated DCs with no DCL. Cimetidine inhibited IL-10 production and restored IL-12 secretion in LPS-treated DCs. LPS increased IL-10 and decreased IL-12 levels. GM-CSF + IL-4-generated DCs had a stronger stimulatory effect on DO11.10 T-cell proliferation than GM-CSF-generated DCs. Inducible costimulator ligand expression was higher in GM-CSF + IL-4- than in GM-CSF-generated DC groups after 2 days of coculture, but decreased 4 days later. IL-13 production was higher in bone marrow DCs generated with GM-CSF than in those generated with GM-CSF + IL-4. OVA-pulsed DCs and OVA-plus-DCL DCs showed increased IL-12 levels. OVA plus LPS increased both IL-10 and interferon-α. Although histamine or histamine receptor-1 antagonists did not influence DC LPS

  12. Antigen-pulsed bone marrow-derived and pulmonary dendritic cells promote Th2 cell responses and immunopathology in lungs during the pathogenesis of murine Mycoplasma pneumonia.

    Science.gov (United States)

    Dobbs, Nicole A; Zhou, Xia; Pulse, Mark; Hodge, Lisa M; Schoeb, Trenton R; Simecka, Jerry W

    2014-08-01

    Mycoplasmas are a common cause of pneumonia in humans and animals, and attempts to create vaccines have not only failed to generate protective host responses, but they have exacerbated the disease. Mycoplasma pulmonis causes a chronic inflammatory lung disease resulting from a persistent infection, similar to other mycoplasma respiratory diseases. Using this model, Th1 subsets promote resistance to mycoplasma disease and infection, whereas Th2 responses contribute to immunopathology. The purpose of the present study was to evaluate the capacity of cytokine-differentiated dendritic cell (DC) populations to influence the generation of protective and/or pathologic immune responses during M. pulmonis respiratory disease in BALB/c mice. We hypothesized that intratracheal inoculation of mycoplasma Ag-pulsed bone marrow-derived DCs could result in the generation of protective T cell responses during mycoplasma infection. However, intratracheal inoculation (priming) of mice with Ag-pulsed DCs resulted in enhanced pathology in the recipient mice when challenged with mycoplasma. Inoculation of immunodeficient SCID mice with Ag-pulsed DCs demonstrated that this effect was dependent on lymphocyte responses. Similar results were observed when mice were primed with Ag-pulsed pulmonary, but not splenic, DCs. Lymphocytes generated in uninfected mice after the transfer of either Ag-pulsed bone marrow-derived DCs or pulmonary DCs were shown to be IL-13(+) Th2 cells, known to be associated with immunopathology. Thus, resident pulmonary DCs most likely promote the development of immunopathology in mycoplasma disease through the generation of mycoplasma-specific Th2 responses. Vaccination strategies that disrupt or bypass this process could potentially result in a more effective vaccination.

  13. Sonic hedgehog protein promotes bone marrow-derived endothelial progenitor cell proliferation, migration and VEGF production via PI 3-kinase/ Akt signaling pathways

    Institute of Scientific and Technical Information of China (English)

    Jin-rong FU; Wen-li LIU; Jian-feng ZHOU; Han-ying SUN; Hui-zhen XU; Li LUO; Heng ZHANG; Yu-feng ZHOU

    2006-01-01

    Aim: To investigate the effects of Sonic hedgehog (shh) protein on bone marrowderived endothelial progenitor cells (BM-EPC) proliferation, migration and vascular endothelial growth factor (VEGF) production, and the potential signaling pathways involved in these effects. Methods: Bone marrow-derived Flk-l+ cells were enriched using the MACS system from adult Kunming mice and then BM-EPC was cultured in gelatin-coated culture dishes. The effects of shh N-terminal peptide on BM-EPC proliferation were evaluated using the MTT colorimetric assay. Cell migration was assayed using a modified Boyden chamber technique. The production of VEGF was determined by ELIS A and immunofluorescence analysis. The potential involvement of PKC and PI3K signaling pathways was explored using selective inhibitor or Western blot. Results: The proliferation, migration and VEGF production in BM-EPC could be promoted by endogenous shh Nterminal peptide at concentrations of 0.1 μg/mL to 10 ug/mL, and could be inhibited by anti-shh antibodies. Shh-mediated proliferation and migration in BM-EPC could be partly attenuated by anti-VEGF. Phospho-PI3-kinase expression in newly separated BM-EPC was low, and it increased significantly when exogenous shh N-terminal peptide was added, but could be attenuated by anti-human/mouse shh N-terminal peptide antibody. Moreover, the inhibitor of the PI3-kinase, but not the inhibitor of the PKC, significantly inhibited the shh-mediated proliferation, migration and VEGF production. Conclusion: Shh protein can stimulate bone marrow-derived BM-EPC proliferation, migration and VEGF production, which may promote neovascularization to ischemic tissues. This results also suggests that the PI3-kinase/Akt signaling pathways are involved in the angiogenic effects of shh.

  14. A robust and reproducible animal serum-free culture method for clinical-grade bone marrow-derived mesenchymal stromal cells.

    Science.gov (United States)

    Laitinen, Anita; Oja, Sofia; Kilpinen, Lotta; Kaartinen, Tanja; Möller, Johanna; Laitinen, Saara; Korhonen, Matti; Nystedt, Johanna

    2016-08-01

    Efficient xenofree expansion methods to replace fetal bovine serum (FBS)-based culture methods are strongly encouraged by the regulators and are needed to facilitate the adoption of mesenchymal stromal cell (MSC)-based therapies. In the current study we established a clinically-compliant and reproducible animal serum-free culture protocol for bone marrow-(BM-) MSCs based on an optimized platelet-derived supplement. Our study compared two different platelet-derived supplements, platelet lysate PL1 versus PL2, produced by two different methods and lysed with different amounts of freeze-thaw cycles. Our study also explored the effect of a low oxygen concentration on BM-MSCs. FBS-supplemented BM-MSC culture served as control. Growth kinetics, differentiation and immunomodulatory potential, morphology, karyotype and immunophenotype was analysed. Growth kinetics in long-term culture was also studied. Based on the initial results, we chose to further process develop the PL1-supplemented culture protocol at 20 % oxygen. The results from 11 individual BM-MSC batches expanded in the chosen condition were consistent, yielding 6.60 × 10(9) ± 4.74 × 10(9) cells from only 20 ml of bone marrow. The cells suppressed T-cell proliferation, displayed normal karyotype and typical MSC differentiation potential and phenotype. The BM-MSCs were, however, consistently HLA-DR positive when cultured in platelet lysate (7.5-66.1 %). We additionally show that culture media antibiotics and sterile filtration of the platelet lysate can be successfully omitted. We present a robust and reproducible clinically-compliant culture method for BM-MSCs based on platelet lysate, which enables high quantities of HLA-DR positive MSCs at a low passage number (p2) and suitable for clinical use.

  15. Rhus javanica Gall Extract Inhibits the Differentiation of Bone Marrow-Derived Osteoclasts and Ovariectomy-Induced Bone Loss

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    Tae-Ho Kim

    2016-01-01

    Full Text Available Inhibition of osteoclast differentiation and bone resorption is a therapeutic strategy for the management of postmenopausal bone loss. This study investigated the effects of Rhus javanica (R. javanica extracts on bone marrow cultures to develop agents from natural sources that may prevent osteoclastogenesis. Extracts of R. javanica (eGr cocoons spun by Rhus javanica (Bell. Baker inhibited the osteoclast differentiation and bone resorption. The effects of aqueous extract (aeGr or 100% ethanolic extract (eeGr on ovariectomy- (OVX- induced bone loss were investigated by various biochemical assays. Furthermore, microcomputed tomography (µCT was performed to study bone remodeling. Oral administration of eGr (30 mg or 100 mg/kg/day for 6 weeks augmented the inhibition of femoral bone mineral density (BMD, bone mineral content (BMC, and other factors involved in bone remodeling when compared to OVX controls. Additionally, eGr slightly decreased bone turnover markers that were increased by OVX. Therefore, it may be suggested that the protective effects of eGr could have originated from the suppression of OVX-induced increase in bone turnover. Collectively, the findings of this study indicate that eGr has potential to activate bone remodeling by inhibiting osteoclast differentiation and bone loss.

  16. Use of autologous mesenchymal stem cells derived from bone marrow for the treatment of naturally injured spinal cord in dogs.

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    Penha, Euler Moraes; Meira, Cássio Santana; Guimarães, Elisalva Teixeira; Mendonça, Marcus Vinícius Pinheiro; Gravely, Faye Alice; Pinheiro, Cláudia Maria Bahia; Pinheiro, Taiana Maria Bahia; Barrouin-Melo, Stella Maria; Ribeiro-Dos-Santos, Ricardo; Soares, Milena Botelho Pereira

    2014-01-01

    The use of stem cells in injury repair has been extensively investigated. Here, we examined the therapeutic effects of autologous bone marrow mesenchymal stem cells (MSC) transplantation in four dogs with natural traumatic spinal cord injuries. MSC were cultured in vitro, and proliferation rate and cell viability were evaluated. Cell suspensions were prepared and surgically administered into the spinal cord. The animals were clinically evaluated and examined by nuclear magnetic resonance. Ten days after the surgical procedure and MSC transplantation, we observed a progressive recovery of the panniculus reflex and diminished superficial and deep pain response, although there were still low proprioceptive reflexes in addition to a hyperreflex in the ataxic hind limb movement responses. Each dog demonstrated an improvement in these gains over time. Conscious reflex recovery occurred simultaneously with moderate improvement in intestine and urinary bladder functions in two of the four dogs. By the 18th month of clinical monitoring, we observed a remarkable clinical amelioration accompanied by improved movement, in three of the four dogs. However, no clinical gain was associated with alterations in magnetic resonance imaging. Our results indicate that MSC are potential candidates for the stem cell therapy following spinal cord injury.

  17. Use of Autologous Mesenchymal Stem Cells Derived from Bone Marrow for the Treatment of Naturally Injured Spinal Cord in Dogs

    Science.gov (United States)

    Penha, Euler Moraes; Meira, Cássio Santana; Guimarães, Elisalva Teixeira; Mendonça, Marcus Vinícius Pinheiro; Gravely, Faye Alice; Pinheiro, Cláudia Maria Bahia; Pinheiro, Taiana Maria Bahia; Barrouin-Melo, Stella Maria; Ribeiro-dos-Santos, Ricardo; Soares, Milena Botelho Pereira

    2014-01-01

    The use of stem cells in injury repair has been extensively investigated. Here, we examined the therapeutic effects of autologous bone marrow mesenchymal stem cells (MSC) transplantation in four dogs with natural traumatic spinal cord injuries. MSC were cultured in vitro, and proliferation rate and cell viability were evaluated. Cell suspensions were prepared and surgically administered into the spinal cord. The animals were clinically evaluated and examined by nuclear magnetic resonance. Ten days after the surgical procedure and MSC transplantation, we observed a progressive recovery of the panniculus reflex and diminished superficial and deep pain response, although there were still low proprioceptive reflexes in addition to a hyperreflex in the ataxic hind limb movement responses. Each dog demonstrated an improvement in these gains over time. Conscious reflex recovery occurred simultaneously with moderate improvement in intestine and urinary bladder functions in two of the four dogs. By the 18th month of clinical monitoring, we observed a remarkable clinical amelioration accompanied by improved movement, in three of the four dogs. However, no clinical gain was associated with alterations in magnetic resonance imaging. Our results indicate that MSC are potential candidates for the stem cell therapy following spinal cord injury. PMID:24723956

  18. Use of Autologous Mesenchymal Stem Cells Derived from Bone Marrow for the Treatment of Naturally Injured Spinal Cord in Dogs

    Directory of Open Access Journals (Sweden)

    Euler Moraes Penha

    2014-01-01

    Full Text Available The use of stem cells in injury repair has been extensively investigated. Here, we examined the therapeutic effects of autologous bone marrow mesenchymal stem cells (MSC transplantation in four dogs with natural traumatic spinal cord injuries. MSC were cultured in vitro, and proliferation rate and cell viability were evaluated. Cell suspensions were prepared and surgically administered into the spinal cord. The animals were clinically evaluated and examined by nuclear magnetic resonance. Ten days after the surgical procedure and MSC transplantation, we observed a progressive recovery of the panniculus reflex and diminished superficial and deep pain response, although there were still low proprioceptive reflexes in addition to a hyperreflex in the ataxic hind limb movement responses. Each dog demonstrated an improvement in these gains over time. Conscious reflex recovery occurred simultaneously with moderate improvement in intestine and urinary bladder functions in two of the four dogs. By the 18th month of clinical monitoring, we observed a remarkable clinical amelioration accompanied by improved movement, in three of the four dogs. However, no clinical gain was associated with alterations in magnetic resonance imaging. Our results indicate that MSC are potential candidates for the stem cell therapy following spinal cord injury.

  19. The CD271 expression could be alone for establisher phenotypic marker in Bone Marrow derived mesenchymal stem cells

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    Edgardo Flores-Torales

    2010-04-01

    Full Text Available Mesenchymal stem cells (MSCs are of great interest for their potential use in cellular therapies. To define thepopulation more precisely, diverse surface markers have been used. We propose here to use CD271 as the sole marker forMSCs in fresh bone marrow. We compared CD271+ populations to the presence or absence of five defined markers forMSCs: CD90+, CD105+, CD45-, CD34- and CD79. The correlations between markers were evaluated and analyzed with aPearson's correlation test. We found that the average percentage of cells expressing the combination of markers CD90+,CD105+, CD45-, CD34- and CD79- was 0.54%, and that the average percentage average of CD271+ cells was 0.53%. Theresults were significant (p<0.05. The exclusive use of CD271 as a marker for MSCs from fresh samples of bone marrowappears to be highly selective. Using CD271 as the sole identification marker for MSCs could reduce costs and acceleratethe process of identifying MSCs for the field of cellular therapy.

  20. Gene expression profiling of human mesenchymal stem cells derived from bone marrow during expansion and osteoblast differentiation

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    Windhager Reinhard

    2007-03-01

    Full Text Available Abstract Background Human mesenchymal stem cells (MSC with the capacity to differentiate into osteoblasts provide potential for the development of novel treatment strategies, such as improved healing of large bone defects. However, their low frequency in bone marrow necessitate ex vivo expansion for further clinical application. In this study we asked if MSC are developing in an aberrant or unwanted way during ex vivo long-term cultivation and if artificial cultivation conditions exert any influence on their stem cell maintenance. To address this question we first developed human oligonucleotide microarrays with 30.000 elements and then performed large-scale expression profiling of long-term expanded MSC and MSC during differentiation into osteoblasts. Results The results showed that MSC did not alter their osteogenic differentiation capacity, surface marker profile, and the expression profiles of MSC during expansion. Microarray analysis of MSC during osteogenic differentiation identified three candidate genes for further examination and functional analysis: ID4, CRYAB, and SORT1. Additionally, we were able to reconstruct the three developmental phases during osteoblast differentiation: proliferation, matrix maturation, and mineralization, and illustrate the activation of the SMAD signaling pathways by TGF-β2 and BMPs. Conclusion With a variety of assays we could show that MSC represent a cell population which can be expanded for therapeutic applications.

  1. Isolation, Culture, Differentiation, and Nuclear Reprogramming of Mongolian Sheep Fetal Bone Marrow-Derived Mesenchymal Stem Cells.

    Science.gov (United States)

    Su, Xiaohu; Ling, Yu; Liu, Chunxia; Meng, Fanhua; Cao, Junwei; Zhang, Li; Zhou, Huanmin; Liu, Zongzheng; Zhang, Yanru

    2015-08-01

    We have characterized the differentiation potentiality and the developmental potential of cloned embryos of fetal bone marrow mesenchymal stem cells (BMSCs) isolated from Mongolian sheep. BMSCs were harvested by centrifuging after the explants method and the mononuclear cells obtained were cultured. The isolated BMSCs were uniform, with a fibroblast-like spindle or stellate appearance, and we confirmed expression of OCT4, SOX2, and NANOG genes at passage 3 (P3) by RT-PCR. We measured the growth of the passage 1, 5, and 10 cultures and found exponential growth with a population doubling time of 29.7±0.05 h. We cultured the P3 BMSCs in vitro under inductive environments and were able to induce them to undergo neurogenesis and form cardiomyocytes and adipocytes. Donor cells at passages 3-4 were used for nuclear transfer (NT). We found the BMSCs could be expanded in vitro and used as nuclear donors for somatic cell nuclear transfer (SCNT). Thus, BMSCs are an attractive cell type for large-animal autologous studies and will be valuable material for somatic cell cloning and future transgenic research.

  2. Freezing and thawing of murine bone marrow-derived dendritic cells does not alter their immunophenotype and antigen presentation characteristics.

    Science.gov (United States)

    Mendoza, L; Bubeník, J; Indrová, M; Bieblová, J; Vonka, V; Símová, J

    2002-01-01

    The aim of this paper was to assess whether the BMDC after freezing and thawing are capable to retain the immunophenotype and antigen-presenting capacity. BMDC were generated from bone marrow precursor cells as described previously by culturing the cells in medium containing GM-CSF and IL-4. Afterwards, the cells were harvested, counted and used for phenotyping and priming of syngeneic spleen cells. For cryopreservation, the BMDC were frozen in the presence of 10% of dimethylsulphoxide (DMSO) and 90% foetal calf serum. Forty to fifty percent of both samples, frozen/thawed as well as fresh BMDC, exhibited characteristic DC morphology, and the DC obtained from the frozen/thawed samples expressed a similar level of MHC class I-, MHC class II-, CD80-, CD86-, CD11c-, CD11b-, CD54- and CD205-molecule as fresh DC. To examine the in vitro priming effect of cryopreserved BMDC on syngeneic non-adherent murine C57BL/6 (B6) spleen cells, the BMDC were thawed, pulsed with the lysate prepared from HPV 16-associated tumour MK16 and used for 3H-thymidine assay. The findings of the experiments indicate that fresh as well as cryopreserved murine BMDC preparations pulsed with tumour lysate were efficient to prime the mitogenic activity of syngeneic non-adherent splenocytes. Taken together, the results suggest that frozen/thawed BMDC are morphologically, phenotypically and functionally comparable with fresh BMDC and can be used for construction of dendritic cell-based tumour vaccines.

  3. Mesangiogenic Progenitor Cells Derived from One Novel CD64brightCD31brightCD14neg Population in Human Adult Bone Marrow

    Science.gov (United States)

    Barachini, Serena; Montali, Marina; Carnicelli, Vittoria; Fazzi, Rita; Parchi, Paolo; Petrini, Mario

    2016-01-01

    Mesenchymal stromal cells (MSCs) have been the object of extensive research for decades, due to their intrinsic clinical value. Nonetheless, the unambiguous identification of a unique in vivo MSC progenitor is still lacking, and the hypothesis that these multipotent cells could possibly arise from different in vivo precursors has been gaining consensus in the last years. We identified a novel multipotent cell population in human adult bone marrow that we first named Mesodermal Progenitor Cells (MPCs) for the ability to differentiate toward the mesenchymal lineage, while still retaining angiogenic potential. Despite extensive characterization, MPCs positioning within the differentiation pathway and whether they can be ascribed as possible distinctive progenitor of the MSC lineage is still unclear. In this study, we describe the ex vivo isolation of one novel bone marrow subpopulation (Pop#8) with the ability to generate MPCs. Multicolor flow cytometry in combination with either fluorescence-activated cell sorting or magnetic-activated cell sorting were applied to characterize Pop#8 as CD64brightCD31brightCD14neg. We defined Pop#8 properties in culture, including the potential of Pop#8-derived MPCs to differentiate into MSCs. Gene expression data were suggestive of Pop#8 in vivo involvement in hematopoietic stem cell niche constitution/maintenance. Pop#8 resulted over three logs more frequent than other putative MSC progenitors, corroborating the idea that most of the controversies regarding culture-expanded MSCs could be the consequence of different culture conditions that select or promote particular subpopulations of precursors. PMID:26975798

  4. Endometriosis impairs bone marrow-derived stem cell recruitment to the uterus whereas bazedoxifene treatment leads to endometriosis regression and improved uterine stem cell engraftment.

    Science.gov (United States)

    Sakr, Sharif; Naqvi, Hanyia; Komm, Barry; Taylor, Hugh S

    2014-04-01

    Endometriosis is a disease defined by the ectopic growth of uterine endometrium. Stem cells contribute to the generation of endometriosis as well as to repair and regeneration of normal endometrium. Here we demonstrate that the selective estrogen receptor modulator bazedoxifene (BZA), administered with conjugated estrogens (CEs), leads to regression of endometriosis lesions as well as reduction in stem cell recruitment to the lesions. Female mice underwent transplantation of male bone marrow. Endometrium was transplanted in the peritoneal cavity of half to create experimental endometriosis. Mice with or without experimental endometriosis were randomized to BZA/CE or vehicle treatment. Endometriosis lesions, bone marrow-derived mesenchymal stem cell engraftment of the lesions, and eutopic endometrium as well as ovarian stimulation were assessed. BZA treatment significantly reduced lesion size, gland number, and expression of proliferation marker proliferating cell nuclear antigen. Ovarian weight was not affected. Stem cells were recruited to the endometriosis lesions, and this recruitment was dramatically reduced by BZA/CE treatment. Stem cell engraftment was reduced in the uterus of animals with endometriosis; however the number of stem cells engrafting the uterus was completely restored by treatment with BZA/CE. Competition between endometriosis and the eutopic endometrium for a limited supply of stem cells and depletion of normal stem cells flux to the uterus is a novel mechanism by which endometriosis interferes with endometrial function and fertility. BZA/CE not only treats lesions of endometriosis, it also dramatically reduces stem cell recruitment to the lesions and restores stem cell engraftment of the uterine endometrium.

  5. Determining the minimum number of detectable cardiac-transplanted 111In-tropolone-labelled bone-marrow-derived mesenchymal stem cells by SPECT

    Science.gov (United States)

    Jin, Yuan; Kong, Huafu; Stodilka, Rob Z.; Wells, R. Glenn; Zabel, Pamela; Merrifield, Peter A.; Sykes, Jane; Prato, Frank S.

    2005-10-01

    In this work, we determined the minimum number of detectable 111In-tropolone-labelled bone-marrow-derived stem cells from the maximum activity per cell which did not affect viability, proliferation and differentiation, and the minimum detectable activity (MDA) of 111In by SPECT. Canine bone marrow mesenchymal cells were isolated, cultured and expanded. A number of samples, each containing 5 × 106 cells, were labelled with 111In-tropolone from 0.1 to 18 MBq, and cell viability was measured afterwards for each sample for 2 weeks. To determine the MDA, the anthropomorphic torso phantom (DataSpectrum Corporation, Hillsborough, NC) was used. A point source of 202 kBq 111In was placed on the surface of the heart compartment, and the phantom and all compartments were then filled with water. Three 111In SPECT scans (duration: 16, 32 and 64 min; parameters: 128 × 128 matrix with 128 projections over 360°) were acquired every three days until the 111In radioactivity decayed to undetectable quantities. 111In SPECT images were reconstructed using OSEM with and without background, scatter or attenuation corrections. Contrast-to-noise ratio (CNR) in the reconstructed image was calculated, and MDA was set equal to the 111In activity corresponding to a CNR of 4. The cells had 100% viability when incubated with no more than 0.9 MBq of 111In (80% labelling efficiency), which corresponded to 0.14 Bq per cell. Background correction improved the detection limits for 111In-tropolone-labelled cells. The MDAs for 16, 32 and 64 min scans with background correction were observed to be 1.4 kBq, 700 Bq and 400 Bq, which implies that, in the case where the location of the transplantation is known and fixed, as few as 10 000, 5000 and 2900 cells respectively can be detected.

  6. TNF-TNFR2/p75 signaling inhibits early and increases delayed nontargeted effects in bone marrow-derived endothelial progenitor cells.

    Science.gov (United States)

    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

    2014-05-16

    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.

  7. Primitive stem cells derived from bone marrow express glial and neuronal markers and support revascularization in injured retina exposed to ischemic and mechanical damage.

    Science.gov (United States)

    Goldenberg-Cohen, Nitza; Avraham-Lubin, Bat-Chen R; Sadikov, Tamilla; Goldstein, Ronald S; Askenasy, Nadir

    2012-06-10

    Ischemic or mechanical injury to the optic nerve is an irreversible cause of vision loss, associated with limited regeneration and poor response to neuroprotective agents. The aim of this study was to assess the capacity of adult bone marrow cells to participate in retinal regeneration following the induction of anterior ischemic optic neuropathy (AION) and optic nerve crush (ONC) in a rodent model. The small-sized subset of cells isolated by elutriation and lineage depletion (Fr25lin(-)) was found to be negative for the neuroglial markers nestin and glial fibrillary acidic protein (GFAP). Syngeneic donor cells, identified by genomic marker in sex-mismatched transplants and green fluorescent protein, incorporated into the injured retina (AION and ONC) at a frequency of 0.35%-0.45% after intravenous infusion and 1.8%-2% after intravitreous implantation. Perivascular cells with astrocytic morphology expressing GFAP and vimentin were of the predominant lineage that engrafted after AION injury; 10%-18% of the donor cells incorporated in the retinal ganglion cell layer and expressed NeuN, Thy-1, neurofilament, and beta-tubulin III. The Fr25lin(-) cells displayed an excellent capacity to migrate to sites of tissue disruption and developed coordinated site-specific morphological and phenotypic neural and glial markers. In addition to cellular reconstitution of the injured retinal layers, these cells contributed to endothelial revascularization and apparently supported remodeling by secretion of insulin-like growth factor-1. These results suggest that elutriated autologous adult bone marrow-derived stem cells may serve as an accessible source for cellular reconstitution of the retina following injury.

  8. Bone marrow: its contribution to heme catabolism.

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    Mähönen, Y; Anttinen, M; Vuopio, P; Tenhunen, R

    1976-01-01

    Heme oxygenase (HO) and biliverdin reductase (BR), the two NADPH-dependent enzymes involved in the degradation of hemoglobin and its derivatives, were measured in bone marrow aspirates from 5 hematologically normal persons, 4 patients with chronic leucemia (CL), 11 patients with acute leucemia (AL), 8 patients with refractory sideroblastic anemia (RA), 7 patients with iron-deficiency anemia (IA), 5 patients with hemolytic anemia (HA), and 7 patients with secondary anemia (SA) to determine the enzymatic capacity of the bone marrow in different hematologic disorders for heme catabolism. HO activity in the bone marrow of normal persons was 0.42 +/- 0.28 (SD) nmoles bilirubin/10 mg protein/min; in CL, 2.15 +/- 1.34; in AL, 0.39 +/- 0.25; in RA, 0.58 +/- 0.37; in IA, 0.41 +/- 0.28; in HA, 2.56 +/- 1.40; and in SA, 1.72 +/- 1.06. BR activity, respectively, was in normal persons 8.7 +/- 2.4 (SD) nmoles bilirubin/10 mg protein/min; in CL, 13.6 +/- 9.1; in AL, 3.8 +/- 3.1 in RA, 5.1 +/- 2.7; in IA, 5.5 +/- 3.7; in HA, 17.0 +/- 7.2; and in SA, 10.5 +/- 4.2. On the basis of these findings it seems evident that both oxygenase and biliverdin reductase activities of the bone marrow are capable of adaptive regulation. The physiologic role of bone marrow in heme catabolism seems to be of significant importance.

  9. The impact of cell source, culture methodology, culture location, and individual donors on gene expression profiles of bone marrow-derived and adipose-derived stromal cells

    NARCIS (Netherlands)

    Torensma, R.; Prins, H.J.; Schrama, E.; Verwiel, E.T.P.; Martens, A.C.M.; Roelofs, H.; Jansen, B.J.H.

    2013-01-01

    Bone marrow (BM) stromal cells (MSCs), also known as mesenchymal stem cells, display a high degree of heterogeneity. To shed light on the causes of this heterogeneity, MSCs were collected from either human BM (n=5) or adipose tissue (AT) (n=5), and expanded using 2 different culture methods: one bas

  10. Chondrogenic potential of adipose-derived stem cells versus bone marrow mesenchymal stem cells%脂肪干细胞与骨髓间充质干细胞成软骨能力的比较**

    Institute of Scientific and Technical Information of China (English)

    安荣泽; 赵俊延; 王兆杰

    2013-01-01

    BACKGROUND:Adipose-derived stem cel s and bone marrow mesenchymal stem cel s are used widely in cartilage tissue engineering, and there are many similarities in biological characteristics between two kinds of cel s. OBJECTIVE:To compare the chondrogenic potential of bone marrow mesenchymal stem cel s and adipose-derived stem cel s in vitro. METHODS:Adipose-derived stem cel s were isolated from the 3-month-old New Zealand white rabbits’ abdomen. Bilateral femurs of rabbits were obtained, and then the bone marrow mesenchymal stem cel s were separated with the adherence screening method. The growth curve of the passage 3 adipose-derived stem cel s and bone marrow mesenchymal stem cel s were drawn, and the doubling time of two kinds of cel s was compared. Then the passage 3 adipose-derived stem cel s and bone marrow mesenchymal stem cel s were treated with chondrogenic induction. After induced for 14 days, the adipose-derived stem cel s and bone marrow mesenchymal stem cel s were treated with toluidine blue staining and type Ⅱ immunohistochemistry staining respectively. RESULTS AND CONCLUSION:Primary bone marrow mesenchymal stem cel s showed aggregative growth, while the primary adipose-derived stem cel s were in single and scattered growth. The proliferation speed of adipose-derived stem cel s was faster than that of bone marrow mesenchymal stem cel s, while the doubling time of adipose-derived stem cel s was shorter than that of the bone marrow mesenchymal stem cel s. After chondrogenic induction for 14 days, both adipose-derived stem cel s and bone marrow mesenchymal stem cel s could express glycosaminoglycans and type Ⅱcol agen, and the expression level of type Ⅱ col agen in bone marrow mesenchymal stem cel s after chondrogenic induction was higher than that in the adipose-derived stem cel s. The in vitro proliferation of adipose-derived stem cel s and bone marrow mesenchymal stem cel s was rapid and stable, but the proliferative ability of adipose-derived

  11. Monosomy 7 in donor cell-derived leukemia after bone marrow transplantation for severe aplastic anemia: report of a new case and review of the literature

    Directory of Open Access Journals (Sweden)

    Luize Otero

    2012-01-01

    Full Text Available Monosomy 7 arises as a recurrent chromosome aberration in donor cell leukemia after hematopoietic stem cell transplantation. We report a new case of donor cell leukemia with monosomy 7 following HLA-identical allogenic bone marrow transplantation for severe aplastic anemia (SAA. The male patient received a bone marrow graft from his sister, and monosomy 7 was detected only in the XX donor cells, 34 months after transplantation. The patient's bone marrow microenvironment may have played a role in the leukemic transformation of the donor hematopoietic cells.

  12. Evaluation of transport conditions for autologous bone marrow-derived mesenchymal stromal cells for therapeutic application in horses

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    Miguel Espina

    2016-03-01

    Full Text Available Background. Mesenchymal stromal cells (MSCs are increasingly used for clinical applications in equine patients. For MSC isolation and expansion, a laboratory step is mandatory, after which the cells are sent back to the attending veterinarian. Preserving the biological properties of MSCs during this transport is paramount. The goal of the study was to compare transport-related parameters (transport container, media, temperature, time, cell concentration that potentially influence characteristics of culture expanded equine MSCs. Methods. The study was arranged in three parts comparing (I five different transport containers (cryotube, two types of plastic syringes, glass syringe, CellSeal, (II seven different transport media, four temperatures (4 °C vs. room temperature; −20 °C vs. −80 °C, four time frames (24 h vs. 48 h; 48 h vs. 72 h, and (III three MSC concentrations (5 × 106, 10 × 106, 20 × 106 MSC/ml. Cell viability (Trypan Blue exclusion; percent and total number viable cell, proliferation and trilineage differentiation capacity were assessed for each test condition. Further, the recovered volume of the suspension was determined in part I. Each condition was evaluated using samples of six horses (n = 6 and differentiation protocols were performed in duplicates. Results. In part I of the study, no significant differences in any of the parameters were found when comparing transport containers at room temperature. The glass syringe was selected for all subsequent evaluations (highest recoverable volume of cell suspension and cell viability. In part II, media, temperatures, or time frames had also no significant influence on cell viability, likely due to the large number of comparisons and small sample size. Highest cell viability was observed using autologous bone marrow supernatant as transport medium, and “transport” at 4 °C for 24 h (70.6% vs. control group 75.3%; this was not significant. Contrary, viability was unacceptably

  13. Migration of Bone Marrow-Derived Very Small Embryonic-Like Stem Cells toward An Injured Spinal Cord

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    Zoleikha Golipoor

    2016-02-01

    Full Text Available Objective: Bone marrow (BM is one of the major hematopoietic organs in postnatal life that consists of a heterogeneous population of stem cells which have been previously described. Recently, a rare population of stem cells that are called very small embryonic-like (VSEL stem cells has been found in the BM. These cells express several developmental markers of pluripotent stem cells and can be mobilized into peripheral blood (PB in response to tissue injury. In this study we have attempted to investigate the ability of these cells to migrate toward an injured spinal cord after transplantation through the tail vein in a rat model. Materials and Methods: In this experimental study, VSELs were isolated from total BM cells using a fluorescent activated cell sorting (FACS system and sca1 and stage specific embryonic antigen (SSEA-1 antibodies. After isolation, VSELs were cultured for 7 days on C2C12 as the feeder layer. Then, VSELs were labeled with 1,1´-dioctadecyl-3,3,3´,3´- tetramethylindocarbocyanine perchlorate (DiI and transplanted into the rat spinal cord injury (SCI model via the tail vein. Finally, we sought to determine the presence of VSELs in the lesion site. Results: We isolated a high number of VSELs from the BM. After cultivation, the VSELs colonies were positive for SSEA-1, Oct4 and Sca1. At one month after transplantation, real-time polymerase chain reaction analysis confirmed a significantly increased expression level of Oct4 and SSEA-1 positive cells at the injury site. Conclusion: VSELs have the capability to migrate and localize in an injured spinal cord after transplantation.

  14. Deficiency of ACE2 in Bone-Marrow-Derived Cells Increases Expression of TNF-α in Adipose Stromal Cells and Augments Glucose Intolerance in Obese C57BL/6 Mice

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    Sean E. Thatcher

    2012-01-01

    Full Text Available Deficiency of ACE2 in macrophages has been suggested to promote the development of an inflammatory M1 macrophage phenotype. We evaluated effects of ACE2 deficiency in bone-marrow-derived stem cells on adipose inflammation and glucose tolerance in C57BL/6 mice fed a high fat (HF diet. ACE2 activity was increased in the stromal vascular fraction (SVF isolated from visceral, but not subcutaneous adipose tissue of HF-fed mice. Deficiency of ACE2 in bone marrow cells significantly increased mRNA abundance of F4/80 and TNF-α in the SVF isolated from visceral adipose tissue of HF-fed chimeric mice, supporting increased presence of inflammatory macrophages in adipose tissue. Moreover, deficiency of ACE2 in bone marrow cells modestly augmented glucose intolerance in HF-fed chimeric mice and increased blood levels of glycosylated hemoglobin. In summary, ACE2 deficiency in bone marrow cells promotes inflammation in adipose tissue and augments obesity-induced glucose intolerance.

  15. Electrophysiological functional recovery in a rat model of spinal cord hemisection injury following bone marrow-derived mesenchymal stem cell transplantation under hypothermia

    Institute of Scientific and Technical Information of China (English)

    Dong Wang; Jianjun Zhang

    2012-01-01

    Following successful establishment of a rat model of spinal cord hemisection injury by resecting right spinal cord tissues, bone marrow stem cells were transplanted into the spinal cord lesions via the caudal vein while maintaining rectal temperature at 34 ± 0.5°C for 6 hours (mild hypothermia). Hematoxylin-eosin staining showed that astrocytes gathered around the injury site and formed scars at 4 weeks post-transplantation. Compared with rats transplanted with bone marrow stem cells under normal temperature, rats transplanted with bone marrow stem cells under hypothermia showed increased numbers of proliferating cells (bromodeoxyuridine-positive cells), better recovery of somatosensory-evoked and motor-evoked potentials, greater Basso, Beattie, and Bresnahan locomotor rating scores, and an increased degree of angle in the incline plate test. These findings suggested that hypothermia combined with bone marrow mesenchymal stem cells transplantation effectively promoted electrical conduction and nerve functional repair in a rat model of spinal cord hemisection injury.

  16. Blood and Bone Marrow Donation

    Science.gov (United States)

    ... waiting for a stem cell transplant. Bone marrow donation The most serious risk associated with donating bone ... you feel fully recovered. Peripheral blood stem cell donation The risks of this type of stem cell ...

  17. Evaluation of transport conditions for autologous bone marrow-derived mesenchymal stromal cells for therapeutic application in horses.

    Science.gov (United States)

    Espina, Miguel; Jülke, Henriette; Brehm, Walter; Ribitsch, Iris; Winter, Karsten; Delling, Uta

    2016-01-01

    Background. Mesenchymal stromal cells (MSCs) are increasingly used for clinical applications in equine patients. For MSC isolation and expansion, a laboratory step is mandatory, after which the cells are sent back to the attending veterinarian. Preserving the biological properties of MSCs during this transport is paramount. The goal of the study was to compare transport-related parameters (transport container, media, temperature, time, cell concentration) that potentially influence characteristics of culture expanded equine MSCs. Methods. The study was arranged in three parts comparing (I) five different transport containers (cryotube, two types of plastic syringes, glass syringe, CellSeal), (II) seven different transport media, four temperatures (4 °C vs. room temperature; -20 °C vs. -80 °C), four time frames (24 h vs. 48 h; 48 h vs. 72 h), and (III) three MSC concentrations (5 × 10(6), 10 × 10(6), 20 × 10(6) MSC/ml). Cell viability (Trypan Blue exclusion; percent and total number viable cell), proliferation and trilineage differentiation capacity were assessed for each test condition. Further, the recovered volume of the suspension was determined in part I. Each condition was evaluated using samples of six horses (n = 6) and differentiation protocols were performed in duplicates. Results. In part I of the study, no significant differences in any of the parameters were found when comparing transport containers at room temperature. The glass syringe was selected for all subsequent evaluations (highest recoverable volume of cell suspension and cell viability). In part II, media, temperatures, or time frames had also no significant influence on cell viability, likely due to the large number of comparisons and small sample size. Highest cell viability was observed using autologous bone marrow supernatant as transport medium, and "transport" at 4 °C for 24 h (70.6% vs. control group 75.3%); this was not significant. Contrary, viability was unacceptably low (cell

  18. Role of bone marrow-derived mesenchymal stem cells in a rat model of severe acute pancreatitis

    Institute of Scientific and Technical Information of China (English)

    Xiao-Huang Tu; Jing-Xiang Song; Xiao-Jun Xue; Xian-Wei Guo; Yun-Xia Ma; Zhi-Yao Chen; Zhong-Dong Zou; Lie Wang

    2012-01-01

    AIM:To investigate the role and potential mechanisms of bone marrow mesenchymal stem cells (MSCs) in severe acute peritonitis (SAP).METHODS:Pancreatic acinar cells from Sprague Dawley rats were randomly divided into three groups:nonsodium deoxycholate (SDOC) group (non-SODC group),SDOC group,and a MSCs intervention group (i.e.,a co-culture system of MSCs and pancreatic acinar cells + SDOC).The cell survival rate,the concentration of malonaldehyde (MDA),the density of superoxide dismutase (SOD),serum amylase (AMS) secretion rate and lactate dehydrogenase (LDH) leakage rate were detected at various time points.In a separate study,Sprague Dawley rats were randomly divided into either an SAP group or an SAP + MSCs group.Serum AMS,MDA and SOD,interleukin (IL)-6,IL-10,and tumor necrosis factor (TNF)-α levels,intestinal mucosa injury scores and proliferating cells of small intestinal mucosa were measured at various time points after injecting either MSCs or saline into rats.In both studies,the protective effect of MSCs was evaluated.RESULTS:In vitro,The cell survival rate of pancreatic acinar cells and the density of SOD were significantly reduced,and the concentration of MDA,AMS secretion rate and LDH leakage rate were significantly increased in the SDOC group compared with the MSCs intervention group and the Non-SDOC group at each time point.In vivo,Serum AMS,IL-6,TNF-α and MAD level in the SAP + MSCs group were lower than the SAP group;however serum IL-10 level was higher than the SAP group.Serum SOD level was higher than the SAP group at each time point,whereas a significant betweengroup difference in SOD level was only noted after 24 h.Intestinal mucosa injury scores was significantly reduced and the proliferating cells of small intestinal mucosa became obvious after injecting MSCs.CONCLUSION:MSCs can effectively relieve injury to pancreatic acinar cells and small intestinal epithelium,promote the proliferation of enteric epithelium and repair of the mucosa

  19. Leptin’s Pro-Angiogenic Signature in Breast Cancer

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    Gonzalez-Perez, Ruben Rene, E-mail: rgonzalez@msm.edu; Lanier, Viola; Newman, Gale [Department of Microbiology, Biochemistry and Immunology, Morehouse School of Medicine, 720 Westview Dr. SW., Atlanta, GA 30310 (United States)

    2013-09-06

    Obesity is linked to increased incidence of breast cancer. The precise causes and mechanisms of these morbid relationships are unknown. Contradictory data on leptin angiogenic actions have been published. However, accumulating evidence would suggest that leptin’s pro-angiogenic effects in cancer play an essential role in the disease. Leptin, the main adipokine secreted by adipose tissue, is also abnormally expressed together with its receptor (OB-R) by breast cancer cells. Leptin induces proliferation and angiogenic differentiation of endothelial cells upregulates VEGF/VEGFR2 and transactivates VEGFR2 independent of VEGF. Leptin induces two angiogenic factors: IL-1 and Notch that can increase VEGF expression. Additionally, leptin induces the secretion and synthesis of proteases and adhesion molecules needed for the development of angiogenesis. Leptin’s paracrine actions can further affect stromal cells and tumor associated macrophages, which express OB-R and secrete VEGF and IL-1, respectively. A complex crosstalk between leptin, Notch and IL-1 (NILCO) that induces VEGF/VEGFR2 is found in breast cancer. Leptin actions in tumor angiogenesis could amplify, be redundant and/or compensatory to VEGF signaling. Current failure of breast cancer anti-angiogenic therapies emphasizes the necessity of targeting the contribution of other pro-angiogenic factors in breast cancer. Leptin’s impact on tumor angiogenesis could be a novel target for breast cancer, especially in obese patients. However, more research is needed to establish the importance of leptin in tumor angiogenesis. This review is focused on updated information on how leptin could contribute to tumor angiogenesis.

  20. Chip-based comparison of the osteogenesis of human bone marrow- and adipose tissue-derived mesenchymal stem cells under mechanical stimulation.

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    Sang-Hyug Park

    Full Text Available Adipose tissue-derived stem cells (ASCs are considered as an attractive stem cell source for tissue engineering and regenerative medicine. We compared human bone marrow-derived mesenchymal stem cells (hMSCs and hASCs under dynamic hydraulic compression to evaluate and compare osteogenic abilities. A novel micro cell chip integrated with microvalves and microscale cell culture chambers separated from an air-pressure chamber was developed using microfabrication technology. The microscale chip enables the culture of two types of stem cells concurrently, where each is loaded into cell culture chambers and dynamic compressive stimulation is applied to the cells uniformly. Dynamic hydraulic compression (1 Hz, 1 psi increased the production of osteogenic matrix components (bone sialoprotein, oateopontin, type I collagen and integrin (CD11b and CD31 expression from both stem cell sources. Alkaline phosphatase and Alrizarin red staining were evident in the stimulated hMSCs, while the stimulated hASCs did not show significant increases in staining under the same stimulation conditions. Upon application of mechanical stimulus to the two types of stem cells, integrin (β1 and osteogenic gene markers were upregulated from both cell types. In conclusion, stimulated hMSCs and hASCs showed increased osteogenic gene expression compared to non-stimulated groups. The hMSCs were more sensitive to mechanical stimulation and more effective towards osteogenic differentiation than the hASCs under these modes of mechanical stimulation.

  1. Toxoplasma gondii is dependent on glutamine and alters migratory profile of infected host bone marrow derived immune cells through SNAT2 and CXCR4 pathways.

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    I-Ping Lee

    Full Text Available The obligate intracellular parasite, Toxoplasma gondii, disseminates through its host inside infected immune cells. We hypothesize that parasite nutrient requirements lead to manipulation of migratory properties of the immune cell. We demonstrate that 1 T. gondii relies on glutamine for optimal infection, replication and viability, and 2 T. gondii-infected bone marrow-derived dendritic cells (DCs display both "hypermotility" and "enhanced migration" to an elevated glutamine gradient in vitro. We show that glutamine uptake by the sodium-dependent neutral amino acid transporter 2 (SNAT2 is required for this enhanced migration. SNAT2 transport of glutamine is also a significant factor in the induction of migration by the small cytokine stromal cell-derived factor-1 (SDF-1 in uninfected DCs. Blocking both SNAT2 and C-X-C chemokine receptor 4 (CXCR4; the unique receptor for SDF-1 blocks hypermotility and the enhanced migration in T. gondii-infected DCs. Changes in host cell protein expression following T. gondii infection may explain the altered migratory phenotype; we observed an increase of CD80 and unchanged protein level of CXCR4 in both T. gondii-infected and lipopolysaccharide (LPS-stimulated DCs. However, unlike activated DCs, SNAT2 expression in the cytosol of infected cells was also unchanged. Thus, our results suggest an important role of glutamine transport via SNAT2 in immune cell migration and a possible interaction between SNAT2 and CXCR4, by which T. gondii manipulates host cell motility.

  2. Identification of New Rat Bone Marrow-Derived Population of Very Small Stem Cell with Oct-4A and Nanog Expression by Flow Cytometric Platforms

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    Anna Labedz-Maslowska

    2016-01-01

    Full Text Available Very small embryonic-like stem cells (VSELs represent a unique rare population of adult stem cells (SCs sharing several structural, genetic, biochemical, and functional properties with embryonic SCs and have been identified in several adult murine and human tissues. However, rat bone marrow- (BM- derived SCs closely resembling murine or human VSELs have not been described. Thus, we employed multi-instrumental flow cytometric approach including classical and imaging cytometry and we established that newly identified population of nonhematopoietic cells expressing CD106 (VCAM-I antigen contains SCs with very small size, expressing markers of pluripotency (Oct-4A and Nanog on both mRNA and protein levels that indicate VSEL population. Based on our experience in both murine and human VSEL isolation procedures by fluorescence-activated cell sorting (FACS, we also optimized sorting protocol for separation of CD45−/Lin−/CD106+ rat BM-derived VSELs from wild type and eGFP-expressing rats, which are often used as donor animals for cell transplantations in regenerative studies in vivo. Thus, this is a first study identifying multiantigenic phenotype and providing sorting protocols for isolation VSELs from rat BM tissue for further examining of their functional properties in vitro as well as regenerative capacity in distinct in vivo rat models of tissue injury.

  3. Prostaglandin E2 plays a key role in the immunosuppressive properties of adipose and bone marrow tissue-derived mesenchymal stromal cells

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    Yanez, Rosa, E-mail: rosamaria.yanez@ciemat.es; Oviedo, Alberto, E-mail: alberto.oviedo@ciemat.es; Aldea, Montserrat, E-mail: montserrat.aldea@ciemat.es; Bueren, Juan A., E-mail: juan.bueren@ciemat.es; Lamana, Maria L., E-mail: maruja.lamana@ciemat.es

    2010-11-15

    Mesenchymal stromal cells (MSCs) have important immunosuppressive properties, but the mechanisms and soluble factors involved in these effects remain unclear. We have studied prostaglandin-E2 (PGE2) as a possible candidate implied in adipose tissue-derived MSCs (Ad-MSCs) immunosuppressive properties over dendritic cells and T lymphocytes, compared to bone marrow derived MSCs (BM-MSCs). We found that both MSCs inhibited the maturation of myeloid-DCs and plasmocytoid-DCs. High levels of PGE2 were detected in DCs/MSCs co-cultures. Its blockade with indomethacin (IDM) allowed plasmocytoid-DCs but not myeloid-DCs maturation. Additionally, high levels of PGE2 were found in co-cultures in which Ad-MSCs or BM-MSCs inhibited activated T cells proliferation and pro-inflammatory cytokines production. PGE2 blockade by IDM preserved T lymphocytes proliferation but did not restore the pro-inflammatory cytokines secretion. However, an increased expression of transcription factors and cytokines genes involved in the Th1/Th2 differentiation pathway was detected in the T cells co-cultured with Ad-MSCs, but not with BM-MSCs. In conclusion, we propose that PGE2 is a soluble factor mediating most of the immunosuppressive effects of Ad-MSCs and BM-MSCs over p-DCs maturation and activated T lymphocytes proliferation and cytokine secretion.

  4. Migration and differentiation of bone marrow-derived multipotent adult progenitor cells through tail vein injection in a rat model of cerebral ischemia

    Institute of Scientific and Technical Information of China (English)

    Lei Lei; Ruixiang Zhou

    2009-01-01

    BACKGROUND: Multipotent adult progenitor cells (MAPCs) from the bone marrow have been shown to differentiate into neurons.OBJECTIVE: To observe migration, survival, and neuronal-like differentiation of MAPCs by tail vein injection.DESIGN, TIME AND SETTING: Randomized, controlled experiment of neural tissue engineering was performed at the Laboratory for Cardio-Cerebrovascular Disease, Hospital of Integrated Traditional and Western Medicine, Tongji Medical College of Huazhong University of Science and Technology between September 2006 and August 2007.MATERIALS: Eighty Sprague Dawley rats, 3-6 months old, underwent cerebral ischemia/reperfusion by thread technique, and were randomly divided into model and MAPCs groups (n = 40).METHODS: Mononuclear cells were harvested from bone marrow using the Ficoll-Paque density gradient centrifugation method. After removing CD45 and glycophorin A-positive cells (GLYA+) with immunomagnetic beads, CD45 GLYA adult progenitor cells were labeled with bromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU). A total of 1 mL cell suspension, containing 5 ×106 MAPCs, was injected into the MAPCs group through the tail vein. A total of 1 mL normal saline was injected into the model rats.MAIN OUTCOME MEASURES: After 60 days, BrdU and neuron-specific enolase double-positive cells were observed using immunofluorescence. Cell morphology was observed under electron microscopy, and nerve growth factor mRNA was measured through RT-PCR. In addition, rat neurological functions were measured with behavioral tests.RESULTS: Immunofluorescence revealed that MAPCs positive for BrdU and neuron specific enolase were found surrounding the ischemic focus in the MAPCs group. Microscopic observation suggested that MAPCs-derived neuronal-like cells connected with other nerve cells to form synapses, Compared with the model animals, the level of nerve growth factor mRNA was significantly upregulated in rats injected with MAPCs (P < 0.05). In addition, rats in the MAPCs

  5. Identification of stable reference genes for gene expression analysis of three-dimensional cultivated human bone marrow-derived mesenchymal stromal cells for bone tissue engineering.

    Science.gov (United States)

    Rauh, Juliane; Jacobi, Angela; Stiehler, Maik

    2015-02-01

    The principles of tissue engineering (TE) are widely used for bone regeneration concepts. Three-dimensional (3D) cultivation of autologous human mesenchymal stromal cells (MSCs) on porous scaffolds is the basic prerequisite to generate newly formed bone tissue. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is a specific and sensitive analytical tool for the measurement of mRNA-levels in cells or tissues. For an accurate quantification of gene expression levels, stably expressed reference genes (RGs) are essential to obtain reliable results. Since the 3D environment can affect a cell's morphology, proliferation, and gene expression profile compared with two-dimensional (2D) cultivation, there is a need to identify robust RGs for the quantification of gene expression. So far, this issue has not been adequately investigated. The aim of this study was to identify the most stably expressed RGs for gene expression analysis of 3D-cultivated human bone marrow-derived MSCs (BM-MSCs). For this, we analyzed the gene expression levels of n=31 RGs in 3D-cultivated human BM-MSCs from six different donors compared with conventional 2D cultivation using qRT-PCR. MSCs isolated from bone marrow aspirates were cultivated on human cancellous bone cube scaffolds for 14 days. Osteogenic differentiation was assessed by cell-specific alkaline phosphatase (ALP) activity and expression of osteogenic marker genes. Expression levels of potential reference and target genes were quantified using commercially available TaqMan(®) assays. mRNA expression stability of RGs was determined by calculating the coefficient of variation (CV) and using the algorithms of geNorm and NormFinder. Using both algorithms, we identified TATA box binding protein (TBP), transferrin receptor (p90, CD71) (TFRC), and hypoxanthine phosphoribosyltransferase 1 (HPRT1) as the most stably expressed RGs in 3D-cultivated BM-MSCs. Notably, genes that are routinely used as RGs, for example, beta actin

  6. Experimental study on cultivation and purification of bone marrow-derived mesenchymal stem cells and its co-culture with chitosan porous scaffolds in vitro

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    Feng YAN

    2014-12-01

    Full Text Available Background As commonly used scaffold material in tissue engineering, chitosan has many advantages, such as strong biodegradability, low antigenicity, good biocompatibility and no pyrogen reaction. This study aims to isolate, cultivate and purify Sprague-Dawley (SD rat bone marrow-derived mesenchymal stem cells (BMSCs, and to observe the growth of BMSCs when co-cultured with self-made chitosan porous scaffold in vitro and to test the biocompatibility of this tissue engineering scaffold, so as to lay the foundation for promoting nerve regeneration of transplant treatment.  Methods Three-week-old healthy male SD rats were used in this study, and BMSCs were isolated and purified through bone marrow adherent culture method. The surface markers of BMSCs at Passage 3 were detected and identified by flow cytometry (FCM and the BMSCs were three?dimensionally cultured in vitro on chitosan porous scaffolds produced by freeze-drying method. Ethanol alternative method was used to detect the chitosan scaffold porosity. Scanning electron microscope was used to explore the internal structure of the scaffold, measure the size of its aperture, and observe the morphology and development of the cells within the scaffold. Methyl thiazolyl tetrazolium (MTT method was used to determine the cells' proliferation.  Results The cultured BMSCs were uniform and similiar to fibrous arrangement, and mixed cells reduced obviously. The identification result of FCM showed the CD29 positive rate was 98.49% and CD45RA positive rate was only 0.85%. The chitosan scaffold had an interlinked, uniform similar three-dimensional porous structure and its aperture porosity was 90%. Some cells stretched out pseudopod and infiltrated into the porous structure of scaffold, even fusing with them. The BMSCs were seeded in the scaffold successfully. The chitosan scaffold had no obvious effect on BMSCs' proliferation. Conclusions Chitosan porous scaffolds have good structural character and

  7. Bone Marrow-Derived Cell Accumulation in the Spinal Cord Is Independent of Peripheral Mobilization in a Mouse Model of Amyotrophic Lateral Sclerosis

    Science.gov (United States)

    Peake, Kyle; Manning, John; Lewis, Coral-Ann; Tran, Kevin; Rossi, Fabio; Krieger, Charles

    2017-01-01

    Bone marrow-derived cells (BMDCs) are capable of migrating across the blood–brain barrier (BBB) and accumulating in the central nervous system (CNS) when transplanted into recipients conditioned with whole-body irradiation or chemotherapy. We used the chemotherapeutic agents busulfan and treosulfan to condition recipient mice for transplantation with bone marrow (BM) cells isolated from donor mice ubiquitously expressing green fluorescent protein. We attempted to increase the accumulation of BMDCs in the CNS by mobilization of BMDCs using either, or both, granulocyte colony-stimulating factor (GCSF) or plerixafor (AMD3100). We also used several concentrations of busulfan. We hypothesized that higher concentrations of busulfan and BMDC mobilization would increase numbers of GFP+ cells in the CNS. The doses of busulfan employed (60–125 mg/kg) all resulted in high levels of sustained chimerism (>85% 1 year post-transplant) in both the blood and BM of wild-type (WT) mice and an amyotrophic lateral sclerosis (ALS) mouse model. Moreover, cells accumulated within the CNS in a dose-, time-, and disease-dependent manner. Conditioning with the hydrophilic busulfan analog treosulfan, which is unable to cross the BBB efficiently, also resulted in a high degree of BM chimerism. However, few GFP+ BMDCs were found within the CNS of WT or ALS mice of treosulfan-conditioned mice. Mobilization of BMDCs into the circulation using GCSF and/or AMD3100 did not lead to increased accumulation of GFP+ BMDCs within the CNS of WT or ALS mice. Weekly analysis of BMDC accumulation revealed that BMDCs accumulated more rapidly and to a greater extent in the CNS of ALS mice conditioned with a high dose (125 mg/kg) of busulfan compared to a lower dose (80 mg/kg). The number of GFP+ BMDCs in the CNS labeling with the proliferation marker Ki67 increased in parallel with BMDC accumulation within the CNS. Our results indicate that establishment of high levels of blood and BM chimerism

  8. The active principle region of Buyang Huanwu decoction induced differentiation of bone marrow-derived mesenchymal stem cells into neural-like cells Superior effects over original formula of Buyang Huanwu decoction

    Institute of Scientific and Technical Information of China (English)

    Jinghui Zheng; Yi Wan; Jianhuai Chi; Dekai Shen; Tingting Wu; Weimin Li; Pengcheng Du

    2012-01-01

    The present study induced in vitro-cultured passage 4 bone marrow-derived mesenchymal stem cells to differentiate into neural-like cells with a mixture of alkaloid, polysaccharide, aglycone, glycoside, essential oils, and effective components of Buyang Huanwu decoction (active principle region of decoction for invigorating yang for recuperation). After 28 days, nestin and neuron-specific enolase were expressed in the cytoplasm. Reverse transcription-PCR and western blot analyses showed that nestin and neuron-specific enolase mRNA and protein expression was greater in the active principle region group compared with the original formula group. Results demonstrated that the active principle region of Buyang Huanwu decoction induced greater differentiation of rat bone marrow-derived mesenchymal stem cells into neural-like cells in vitro than the original Buyang Huanwu decoction formula.

  9. Arctigenin suppresses receptor activator of nuclear factor κB ligand (RANKL)-mediated osteoclast differentiation in bone marrow-derived macrophages.

    Science.gov (United States)

    Kim, A-Ram; Kim, Hyuk Soon; Lee, Jeong Min; Choi, Jung Ho; Kim, Se Na; Kim, Do Kyun; Kim, Ji Hyung; Mun, Se Hwan; Kim, Jie Wan; Jeon, Hyun Soo; Kim, Young Mi; Choi, Wahn Soo

    2012-05-05

    Osteoclasts, multinucleated bone-resorbing cells, are closely associated with bone diseases such as rheumatoid arthritis and osteoporosis. Osteoclasts are derived from hematopoietic precursor cells, and their differentiation is mediated by two cytokines, including macrophage colony stimulating factor and receptor activator of nuclear factor κB ligand (RANKL). Previous studies have shown that arctigenin exhibits an anti-inflammatory effect. However, the effect of arctigenin on osteoclast differentiation is yet to be elucidated. In this study, we found that arctigenin inhibited RANKL-mediated osteoclast differentiation in bone marrow macrophages in a dose-dependent manner and suppressed RANKL-mediated bone resorption. Additionally, the expression of typical marker proteins, such as NFATc1, c-Fos, TRAF6, c-Src, and cathepsin K, were significantly inhibited. Arctigenin inhibited the phosphorylation of Erk1/2, but not p38 and JNK, in a dose-dependent manner. Arctigenin also dramatically suppressed immunoreceptor tyrosine-based activation motif-mediated costimulatory signaling molecules, including Syk and PLCγ2, and Gab2. Notably, arctigenin inhibited the activation of Syk through RANKL stimulation. Furthermore, arctigenin prevented osteoclast differentiation in the calvarial bone of mice following stimulation with lipopolysaccharide. Our results show that arctigenin inhibits osteoclast differentiation in vitro and in vivo. Therefore, arctigenin may be useful for treating rheumatoid arthritis and osteoporosis.

  10. Anti-angiogenesis therapy based on the bone marrow-derived stromal cells genetically engineered to express sFlt-1 in mouse tumor model

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    Chen X-C

    2008-10-01

    Full Text Available Abstract Background Bone marrow-derived stromal cells (BMSCs are important for development, tissue cell replenishment, and wound healing in physiological and pathological conditions. BMSCs were found to preferably reach sites undergoing the process of cell proliferation, such as wound and tumor, suggesting that BMSCs may be used as a vehicle for gene therapy of tumor. Methods Mouse BMSCs were loaded with recombinant adenoviruses which express soluble Vascular Endothelial Growth Factor Receptor-1 (sFlt-1. The anti-angiogenesis of sFlt-1 in BMSCs was determined using endothelial cells proliferation inhibition assay and alginate encapsulation assay. The anti-tumor effects of BMSCs expressing sFlt-1 through tail-vein infusion were evaluated in two mouse tumor metastases models. Results BMSCs genetically modified with Adv-GFP-sFlt-1 could effectively express and secret sFlt-1. BMSCs loaded with sFlt-1 gene could preferentially home to tumor loci and decrease lung metastases and prolong lifespan in mouse tumor model through inducing anti-angiogenesis and apoptosis in tumors. Conclusion We demonstrated that BMSCs might be employed as a promising vehicle for tumor gene therapy which can effectively not only improve the concentration of anticancer therapeutics in tumors, but also modify the tumor microenvironment.

  11. Effect of Transplantation of Bone Marrow Derived Mesenchymal Stem Cells and Platelets Rich Plasma on Experimental Model of Radiation Induced Oral Mucosal Injury in Albino Rats

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    Basma Elsaadany

    2017-01-01

    Full Text Available Normal tissue damage following radiotherapy is still a major problem in cancer treatment. Therefore, the current work aimed at exploring the possible role of systemically injected bone marrow derived mesenchymal stem cells (BM-MSCs and/or locally injected platelet rich plasma (PRP in ameliorating the side effects of ionizing radiation on the rat’s tongue. Twelve rats served as control group (N and 48 rats received a single radiation dose of 13 Gy to the head and neck region; then, they were equally divided into 4 experimental groups: irradiated only (C, irradiated + MSCs (S, irradiated + (PRP (P, and combined group (PS. Animal scarification occurred in 3 and 7 days after radiation. Then, tongues were dissected and examined histologically and for expression of bcl-2 by RT-PCR. Histological examination of the treated groups (S, (P, and (PS revealed an obvious improvement in the histological structure of the tongue, compared to group (C, in addition to upregulated expression of bcl-2, indicating decreased apoptotic activity. Conclusion. BM-MSCs and PRP have shown positive effect in minimizing the epithelial atrophy of normal oral mucosa after regional radiotherapy, which was emphasized by decreasing apoptotic activity in these tissues. Nevertheless, combined use of BM-MSCs and PRP did not reveal the assumed synergetic effect in oral tissue protection.

  12. Chondrogenic Potency Analyses of Donor-Matched Chondrocytes and Mesenchymal Stem Cells Derived from Bone Marrow, Infrapatellar Fat Pad, and Subcutaneous Fat

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    John Garcia

    2016-01-01

    Full Text Available Autologous chondrocyte implantation (ACI is a cell-based therapy that has been used clinically for over 20 years to treat cartilage injuries more efficiently in order to negate or delay the need for joint replacement surgery. In this time, very little has changed in the ACI procedure, but now many centres are considering or using alternative cell sources for cartilage repair, in particular mesenchymal stem cells (MSCs. In this study, we have tested the chondrogenic potential of donor-matched MSCs derived from bone marrow (BM, infrapatellar fat pad (FP, and subcutaneous fat (SCF, compared to chondrocytes. We have confirmed that there is a chondrogenic potency hierarchy ranging across these cell types, with the most potent being chondrocytes, followed by FP-MSCs, BM-MSCs, and lastly SCF-MSCs. We have also examined gene expression and surface marker profiles in a predictive model to identify cells with enhanced chondrogenic potential. In doing so, we have shown that Sox-9, Alk-1, and Coll X expressions, as well as immunopositivity for CD49c and CD39, have predictive value for all of the cell types tested in indicating chondrogenic potency. The findings from this study have significant clinical implications for the refinement and development of novel cell-based cartilage repair strategies.

  13. Chondrogenic Potency Analyses of Donor-Matched Chondrocytes and Mesenchymal Stem Cells Derived from Bone Marrow, Infrapatellar Fat Pad, and Subcutaneous Fat

    Science.gov (United States)

    Garcia, John; McCarthy, Helen S.; Roberts, Sally; Richardson, James B.

    2016-01-01

    Autologous chondrocyte implantation (ACI) is a cell-based therapy that has been used clinically for over 20 years to treat cartilage injuries more efficiently in order to negate or delay the need for joint replacement surgery. In this time, very little has changed in the ACI procedure, but now many centres are considering or using alternative cell sources for cartilage repair, in particular mesenchymal stem cells (MSCs). In this study, we have tested the chondrogenic potential of donor-matched MSCs derived from bone marrow (BM), infrapatellar fat pad (FP), and subcutaneous fat (SCF), compared to chondrocytes. We have confirmed that there is a chondrogenic potency hierarchy ranging across these cell types, with the most potent being chondrocytes, followed by FP-MSCs, BM-MSCs, and lastly SCF-MSCs. We have also examined gene expression and surface marker profiles in a predictive model to identify cells with enhanced chondrogenic potential. In doing so, we have shown that Sox-9, Alk-1, and Coll X expressions, as well as immunopositivity for CD49c and CD39, have predictive value for all of the cell types tested in indicating chondrogenic potency. The findings from this study have significant clinical implications for the refinement and development of novel cell-based cartilage repair strategies. PMID:27781068

  14. Autologous Bone-Marrow-Derived-Mononuclear-Cells-Enriched Fat Transplantation in Breast Augmentation: Evaluation of Clinical Outcomes and Aesthetic Results in a 30-Year-Old Female

    Directory of Open Access Journals (Sweden)

    Dmitry Bulgin

    2013-01-01

    Full Text Available Autologous fat transfer (lipofilling is becoming an invaluable tool for breast augmentation as well as for breast reconstruction. Autologous lipofilling has several advantages, including biocompatibility, versatility, natural appearance, and low donor site morbidity. The main limitation is unpredictable fat graft resorption, which ranges from 25% to 80%, probably as a result of ischaemia and lack of neoangiogenesis. To obviate these disadvantages, several studies have searched for new ways of increasing the viability of the transplanted fat tissue. One promising approach is to enrich the fat graft with autologous bone-marrow-derived mononuclear cells (BMMNCs before transplantation. BMMNCs produce many angiogenic and antiapoptotic growth factors, and their secretion is significantly enhanced by hypoxia. All of these mechanisms of actions could be beneficial for the stimulation of angiogenesis in ischemic tissues by BMMNCs administration. In our aesthetic surgery practice, we use fat transplantation enriched with BMMNCs, which caused a significant improvement in survival of fat grafts, compared with that of traditional lipofilling. Our experience with freshly isolated autologous fat enriched with BMMNCs for breast augmentation procedures is presented. The concept of this surgical and tissue handling technique is based on ability of BMMNCs to stimulate blood vessel growth.

  15. A novel rat fibrosarcoma cell line from transformed bone marrow-derived mesenchymal stem cells with maintained in vitro and in vivo stemness properties.

    Science.gov (United States)

    Wang, Meng-Yu; Nestvold, Janne; Rekdal, Øystein; Kvalheim, Gunnar; Fodstad, Øystein

    2017-02-09

    Increasing evidence suggests a possible relationship between mesenchymal stem cells (MSCs) and sarcoma. MSCs are hypothesized to be the cells initiating sarcomagenesis, and cancer stem cells (CSCs) sharing features of MSCs have been identified in sarcomas. Here, we report on the characteristics of a bone marrow-derived rat mesenchymal stem cell line that spontaneously transformed in long-term culture. The rat transformed mesenchymal stem cells (rTMSCs) produced soft-tissue fibrosarcomas in immunocompromised mice and immunocompetent rats. In vitro, the rTMSCs displayed increased proliferation capacity compared to the untransformed cell line. The transformed MSCs maintained the mesenchymal phenotype by expression of the stem cell marker CD 90 and the lack of hematopoietic and endothelial markers. Cytogenetic analysis detected trisomy 6 in the rTMSCs. Side population (SP) isolation and tumorsphere cultivation of the transformed cells confirmed the presence of CSCs among the rTMSCs. Importantly, the rTMSCs retained their differentiation capacity towards osteogenic and adipogenic lineages. This transformed MSC-based cell line may be valuable in examining the balance in a mixed cell population between cancer stem cell properties and the ability to differentiate to specific non-transformed cell populations. Moreover, it may also be a useful tool to evaluate the efficacy of novel targeted immunotherapies in vivo.

  16. Peroxisome Proliferator-Activated Receptor Gamma Negatively Regulates the Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells Toward Myofibroblasts in Liver Fibrogenesis

    Directory of Open Access Journals (Sweden)

    Shuangshuang Jia

    2015-11-01

    Full Text Available Background/Aims: Bone marrow-derived mesenchymal stem cells (BMSCs have been confirmed to have capacity to differentiate toward hepatic myofibroblasts, which contribute to fibrogenesis in chronic liver diseases. Peroxisome proliferator-activated receptor gamma (PPARγ, a ligand-activated transcription factor, has gained a great deal of recent attention as it is involved in fibrosis and cell differentiation. However, whether it regulates the differentiation of BMSCs toward myofibroblasts remains to be defined. Methods: Carbon tetrachloride or bile duct ligation was used to induce mouse liver fibrosis. Expressions of PPARγ, α-smooth muscle actin, collagen α1 (I and collagen α1 (III were detected by real-time RT-PCR and Western blot or immunofluorescence assay. Results: PPARγ expression was decreased in mouse fibrotic liver. In addition, PPARγ was declined during the differentiation of BMSCs toward myofibroblasts induced by transforming growth factor β1. Activation of PPARγ stimulated by natural or synthetic ligands suppressed the differentiation of BMSCs. Additionally, knock down of PPARγ by siRNA contributed to BMSC differentiation toward myofibroblasts. Furthermore, PPARγ activation by natural ligand significantly inhibited the differentiation of BMSCs toward myofibroblasts in liver fibrogenesis and alleviated liver fibrosis. Conclusions: PPARγ negatively regulates the differentiation of BMSCs toward myofibroblasts, which highlights a further mechanism implicated in the BMSC differentiation.

  17. Acoustic-Frequency Vibratory Stimulation Regulates the Balance between Osteogenesis and Adipogenesis of Human Bone Marrow-Derived Mesenchymal Stem Cells

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    Xi Chen

    2015-01-01

    Full Text Available Osteoporosis can be associated with the disordered balance between osteogenesis and adipogenesis of bone marrow-derived mesenchymal stem cells (BM-MSCs. Although low-frequency mechanical vibration has been demonstrated to promote osteogenesis, little is known about the influence of acoustic-frequency vibratory stimulation (AFVS. BM-MSCs were subjected to AFVS at frequencies of 0, 30, 400, and 800 Hz and induced toward osteogenic or adipogenic-specific lineage. Extracellular matrix mineralization was determined by Alizarin Red S staining and lipid accumulation was assessed by Oil Red O staining. Transcript levels of osteogenic and adipogenic marker genes were evaluated by real-time reverse transcription-polymerase chain reaction. Cell proliferation of BM-MSCs was promoted following exposure to AFVS at 800 Hz. Vibration at 800 Hz induced the highest level of calcium deposition and significantly increased mRNA expression of COL1A1, ALP, RUNX2, and SPP1. The 800 Hz group downregulated lipid accumulation and levels of adipogenic genes, including FABP4, CEBPA, PPARG, and LEP, while vibration at 30 Hz supported adipogenesis. BM-MSCs showed a frequency-dependent response to acoustic vibration. AFVS at 800 Hz was the most favorable for osteogenic differentiation and simultaneously suppressed adipogenesis. Thus, acoustic vibration could potentially become a novel means to prevent and treat osteoporosis.

  18. Chromatin remodeling agent trichostatin A: a key-factor in the hepatic differentiation of human mesenchymal stem cells derived of adult bone marrow

    Directory of Open Access Journals (Sweden)

    Vinken Mathieu

    2007-04-01

    Full Text Available Abstract Background The capability of human mesenchymal stem cells (hMSC derived of adult bone marrow to undergo in vitro hepatic differentiation was investigated. Results Exposure of hMSC to a cocktail of hepatogenic factors [(fibroblast growth factor-4 (FGF-4, hepatocyte growth factor (HGF, insulin-transferrin-sodium-selenite (ITS and dexamethasone] failed to induce hepatic differentiation. Sequential exposure to these factors (FGF-4, followed by HGF, followed by HGF+ITS+dexamethasone, however, resembling the order of secretion during liver embryogenesis, induced both glycogen-storage and cytokeratin (CK18 expression. Additional exposure of the cells to trichostatin A (TSA considerably improved endodermal differentiation, as evidenced by acquisition of an epithelial morphology, chronological expression of hepatic proteins, including hepatocyte-nuclear factor (HNF-3β, alpha-fetoprotein (AFP, CK18, albumin (ALB, HNF1α, multidrug resistance-associated protein (MRP2 and CCAAT-enhancer binding protein (C/EBPα, and functional maturation, i.e. upregulated ALB secretion, urea production and inducible cytochrome P450 (CYP-dependent activity. Conclusion hMSC are able to undergo mesenchymal-to-epithelial transition. TSA is hereby essential to promote differentiation of hMSC towards functional hepatocyte-like cells.

  19. Clumping and Viability of Bone Marrow Derived Mesenchymal Stromal Cells under Different Preparation Procedures: A Flow Cytometry-Based In Vitro Study

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    Li-li Cui

    2016-01-01

    Full Text Available Complications of microocclusions have been reported after intra-arterial delivery of mesenchymal stromal cells. Hence, quantification and efficient limitation of cell clumps in suspension before transplantation is important to reduce the risk. We used a flow cytometry-based pulse-width assay to assess the effects of different cell suspension concentrations (0.2–2.0 × 106/mL, storage solutions (complete growth medium, Dulbecco’s phosphate-buffered saline, and normal saline, storage time in suspension (0–9 h, and freeze-thawing procedure on the clumping of rat bone marrow derived mesenchymal stromal cells (BMMSCs and also evaluated cell viability at the same time. Surprisingly, increasing the cell concentration did not result in more cell clumps in vitro. Freshly harvested (fresh cells in normal saline had significantly fewer cell clumps and also displayed high viability (>90%. A time-dependent reduction in viability was observed for cells in all three storage solutions, without any significant change in the clumping tendency except for cells in medium. Fresh cells were more viable than their frozen-thawed counterparts, and fresh cells in normal saline had fewer cell clumps. In conclusion, cell clumping and viability could be affected by different cell preparation procedures, and quantification of cell clumping can be conducted using the flow cytometry-based pulse-width assay before intra-arterial cell delivery.

  20. Biochemical properties of norepinephrine as a kind of neurotransmitter secreted by bone marrow-derived neural stem cells induced and differentiated in vitro

    Institute of Scientific and Technical Information of China (English)

    Jianrong Chen; Xiaodan Jiang; Ruxiang Xu; Peng Jin; Yuxi Zou; Lianshu Ding

    2006-01-01

    BACKGROUND: It has been proved by many experimental studies from the aspects of morphology and immunocytochemistry in recent years that bone marrow stromal cells (BMSCs) can in vitro induce and differentiate into the cells possessing the properties of nerve cells. But the functions of BMSCs-derived neural stem cells(NSCs) and the differentiated neuron-like cells are still unclear.OBJECTIVE: To observe whether bone marrow-derived NSCs can secrete norepinephrine (NE) under the condition of in vitro culture, induce and differentiation, and analyze the biochemical properties of BMSCs-derived NSCs.DESIGN: A non-randomized and controlled experimental observation.SETTING: Institute of Neuromedicine of Chinese PLA, Zhujiang Hospital, Southern Medical University.MATERIALS: This experiment was carried out in the Institute of Neuromedicine of Chinese PLA, Zhujiang Hospital, Southern Medical University. The bone marrow used in the experiment was collected from 1.5-month-old healthy New Zealand white rabbits.METHODS: This experiment was carried out in the Institute of Neuromedicine of Chinese PLA, Zhujiang Hospital, Southern Medical University. The bone marrow used in the experiment was collected from 1.5 month-old healthy New Zealand white rabbits. BMSCs of rabbits were isolated and performed in vitro culture, induce and differentiation with culture medium of NSCs and differentiation-inducing factor, then identified with immunocy-tochemical method. Experimental grouping: ①Negative control group: L-02 hepatic cell and RPMI1640 culture medium were used. ② Background culture group: Only culture medium of NSCs as culture solution was added into BMSCs to perform culture, and 0.1 volume fraction of imported fetal bovine serum was supplemented 72 hours later. ③Differentiation inducing factor group: After culture for 72 hours, retinoic acid and glial cell line-derived neurotrophic factors were added in the culture medium of BMSCs and NSCs as corresponding inducing factors. The

  1. Effects of magnetic nanoparticle-incorporated human bone marrow-derived mesenchymal stem cells exposed to pulsed electromagnetic fields on injured rat spinal cord.

    Science.gov (United States)

    Cho, Hyunjin; Choi, Yun-Kyong; Lee, Dong Heon; Park, Hee Jung; Seo, Young-Kwon; Jung, Hyun; Kim, Soo-Chan; Kim, Sung-Min; Park, Jung-Keug

    2013-01-01

    Transplanting mesenchymal stem cells into injured lesions is currently under study as a therapeutic approach for spinal cord injury. In this study, the effects of a pulsed electromagnetic field (PEMF) on injured rat spinal cord were investigated in magnetic nanoparticle (MNP)-incorporated human bone marrow-derived mesenchymal stem cells (hBM-MSCs). A histological analysis revealed significant differences in MNP-incorporated cell distribution near the injured site under the PEMF in comparison with that in the control group. We confirmed that MNP-incorporated cells were widely distributed in the lesions under PEMF. The results suggest that MNP-incorporated hBM-MSCs were guided by the PEMF near the injured site, and that PEMF exposure for 8 H per day over 4 weeks promoted behavioral recovery in spinal cord injured rats. The results show that rats with MNP-incorporated hBM-MSCs under a PEMF were more effective on the Basso, Beattie, and Bresnahan behavioral test and suggest that the PEMF enhanced the action of transplanted cells for recovery of the injured lesion.

  2. Human platelet lysate is an alternative to fetal bovine serum for large-scale expansion of bone marrow-derived mesenchymal stromal cells.

    Science.gov (United States)

    Gottipamula, Sanjay; Sharma, Archana; Krishnamurthy, Sagar; Majumdar, Anish Sen; Seetharam, Raviraja N

    2012-07-01

    Human platelet lysate (HPL) was evaluated as an alternative to fetal bovine serum (FBS) in large-scale culturing of bone marrow-derived mesenchymal stromal cells (BM-MSCs) for therapeutic applications. Dulbecco's modified Eagle medium (DMEM)of low glucose (LG) and Knock Out (KO) were used with human platelet lysate (HPL) as LG-HPL and KO-HPL, and with FBS as LG-FBS and KO-FBS to culture the BM-MSCs. HPL at 10 % (v/v) supported BM-MSCs growth and subsequent isolation efficiency generated >90 × 10(6) MSCs in LG-HPL. Population doublings (PDs) and population doubling times of LG-HPL and KO-HPL (PDT) were not significantly different but LG-HPL showed a significant clonogenic potential and HPL cultures had an average PDT of 36.5 ± 6.5 h and an average PDs of 5 ± 0.7/passage. BM-MSCs cultured with LG-HPL had significantly higher immunosuppression compared to LG-FBS, but KO-HPL and KO-FBS-grown cultures were not significantly different. HPL is therefore alternative to FBS for large-scale production of BM-MSCs for therapeutic applications.

  3. Full GMP-compliant validation of bone marrow-derived human CD133(+) cells as advanced therapy medicinal product for refractory ischemic cardiomyopathy.

    Science.gov (United States)

    Belotti, Daniela; Gaipa, Giuseppe; Bassetti, Beatrice; Cabiati, Benedetta; Spaltro, Gabriella; Biagi, Ettore; Parma, Matteo; Biondi, Andrea; Cavallotti, Laura; Gambini, Elisa; Pompilio, Giulio

    2015-01-01

    According to the European Medicine Agency (EMA) regulatory frameworks, Advanced Therapy Medicinal Products (ATMP) represent a new category of drugs in which the active ingredient consists of cells, genes, or tissues. ATMP-CD133 has been widely investigated in controlled clinical trials for cardiovascular diseases, making CD133(+) cells one of the most well characterized cell-derived drugs in this field. To ensure high quality and safety standards for clinical use, the manufacturing process must be accomplished in certified facilities following standard operative procedures (SOPs). In the present work, we report the fully compliant GMP-grade production of ATMP-CD133 which aims to address the treatment of chronic refractory ischemic heart failure. Starting from bone marrow (BM), ATMP-CD133 manufacturing output yielded a median of 6.66 × 10(6) of CD133(+) cells (range 2.85 × 10(6)-30.84 × 10(6)), with a viability ranged between 96,03% and 99,97% (median 99,87%) and a median purity of CD133(+) cells of 90,60% (range 81,40%-96,20%). Based on these results we defined our final release criteria for ATMP-CD133: purity ≥ 70%, viability ≥ 80%, cellularity between 1 and 12 × 10(6) cells, sterile, and endotoxin-free. The abovementioned criteria are currently applied in our Phase I clinical trial (RECARDIO Trial).

  4. The Effect of Bone Marrow-Derived Mesenchymal Stem Cells and Their Conditioned Media Topically Delivered in Fibrin Glue on Chronic Wound Healing in Rats.

    Science.gov (United States)

    Mehanna, Radwa A; Nabil, Iman; Attia, Noha; Bary, Amany A; Razek, Khalid A; Ahmed, Tamer A E; Elsayed, Fatma

    2015-01-01

    Bone marrow-derived mesenchymal stem cells (BM-MSCs) represent a modern approach for management of chronic skin injuries. In this work, we describe BM-MSCs application versus their conditioned media (CM) when delivered topically admixed with fibrin glue to enhance the healing of chronic excisional wounds in rats. Fifty-two adult male rats were classified into four groups after induction of large-sized full-thickness skin wound: control group (CG), fibrin only group (FG), fibrin + MSCs group (FG + SCs), and fibrin + CM group (FG + CM). Healing wounds were evaluated functionally and microscopically. Eight days after injury, number of CD68+ macrophages infiltrating granulation tissue was considerably higher in the latter two groups. Although--later--none of the groups depicted a substantially different healing rate, the quality of regenerated skin was significantly boosted by the application of either BM-MSCs or their CM both (1) structurally as demonstrated by the obviously increased mean area percent of collagen fibers in Masson's trichrome-stained skin biopsies and (2) functionally as supported by the interestingly improved epidermal barrier as well as dermal tensile strength. Thus, we conclude that topically applied BM-MSCs and their CM-via fibrin vehicle--could effectively improve the quality of healed skin in chronic excisional wounds in rats, albeit without true acceleration of wound closure.

  5. Stem Cell Ophthalmology Treatment Study (SCOTS):bone marrow-derived stem cells in the treatment of Leber’s hereditary optic neuropathy

    Institute of Scientific and Technical Information of China (English)

    Jeffrey N Weiss; Steven Levy; Susan C Benes

    2016-01-01

    hTe Stem Cell Ophthalmology Treatment Study (SCOTS) is currently the largest-scale stem cell ophthal-mology trial registered at ClinicalTrials.gov (identiifer: NCT01920867). SCOTS utilizes autologous bone marrow-derived stem cells (BMSCs) to treat optic nerve and retinal diseases. Treatment approaches include a combination of retrobulbar, subtenon, intravitreal, intra-optic nerve, subretinal, and intravenous injection of autologous BMSCs according to the nature of the disease, the degree of visual loss, and any risk factors related to the treatments. Patients with Leber’s hereditary optic neuropathy had visual acuity gains on the Early Treatment Diabetic Retinopathy Study (ETDRS) of up to 35 letters and Snellen acuity improvements from hand motion to 20/200 and from counting ifngers to 20/100. Visual ifeld improvements were noted. Macular and optic nerve head nerve ifber layer typically thickened. No serious complications were seen. hTe increases in visual acuity obtained in our study were encouraging and suggest that the use of autolo-gous BMSCs as provided in SCOTS for ophthalmologic mitochondrial diseases including Leber’s hereditary optic neuropathy may be a viable treatment option.

  6. Combined Effects of Mechanical Strain and Hydroxyapatite/Collagen Composite on Osteogenic Differentiation of Rat Bone Marrow Derived Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Yan Huang

    2013-01-01

    Full Text Available Mesenchymal stem cells (MSCs represent a promising source for bone repair and regeneration. Recent lines of evidence have shown that appropriate strain could regulate the osteogenic differentiation of MSCs. Our previous study demonstrated that hydroxyapatite/collagen (HA/Col composite also played an important role in the osteogenic differentiation of MSCs. The aim of this study is to investigate the effects of mechanical strain and HA/Col composite on the osteogenic differentiation of rat bone marrow derived MSCs (rBMSCs in vitro. rBMSCs were treated with cyclic strain generated by a self-designed stretching device with or without the presence of HA/Col composite. Osteogenic differentiation levels were evaluated using reverse transcription polymerase chain reaction (RT-PCR, alkaline phosphatase spectrophotometry, and western blotting. The results demonstrated that mechanical strain combined with HA/Col composite could obviously induce the differentiation of rBMSCs into osteoblasts, which had a better effect than only mechanical strain or HA/Col composite treatment. This provides a new avenue for mechanistic studies of stem cell differentiation and a novel approach to obtain more committed differentiated cells.

  7. Full GMP-Compliant Validation of Bone Marrow-Derived Human CD133+ Cells as Advanced Therapy Medicinal Product for Refractory Ischemic Cardiomyopathy

    Directory of Open Access Journals (Sweden)

    Daniela Belotti

    2015-01-01

    Full Text Available According to the European Medicine Agency (EMA regulatory frameworks, Advanced Therapy Medicinal Products (ATMP represent a new category of drugs in which the active ingredient consists of cells, genes, or tissues. ATMP-CD133 has been widely investigated in controlled clinical trials for cardiovascular diseases, making CD133+ cells one of the most well characterized cell-derived drugs in this field. To ensure high quality and safety standards for clinical use, the manufacturing process must be accomplished in certified facilities following standard operative procedures (SOPs. In the present work, we report the fully compliant GMP-grade production of ATMP-CD133 which aims to address the treatment of chronic refractory ischemic heart failure. Starting from bone marrow (BM, ATMP-CD133 manufacturing output yielded a median of 6.66 × 106 of CD133+ cells (range 2.85 × 106–30.84 × 106, with a viability ranged between 96,03% and 99,97% (median 99,87% and a median purity of CD133+ cells of 90,60% (range 81,40%–96,20%. Based on these results we defined our final release criteria for ATMP-CD133: purity ≥ 70%, viability ≥ 80%, cellularity between 1 and 12 × 106 cells, sterile, and endotoxin-free. The abovementioned criteria are currently applied in our Phase I clinical trial (RECARDIO Trial.

  8. Gene expression profiling suggests a pathological role of human bone marrow-derived mesenchymal stem cells in aging-related skeletal diseases.

    Science.gov (United States)

    Jiang, Shih Sheng; Chen, Chung-Hsing; Tseng, Kuo-Yun; Tsai, Fang-Yu; Wang, Ming Jen; Chang, I-Shou; Lin, Jiunn-Liang; Lin, Shankung

    2011-07-01

    Aging is associated with bone loss and degenerative joint diseases, in which the aging of bone marrow-derived mesenchymal stem cell (bmMSC)[1] may play an important role. In this study, we analyzed the gene expression profiles of bmMSC from 14 donors between 36 and 74 years old, and obtained age-associated genes (in the background of osteoarthritis) and osteoarthritis-associated genes (in the background of old age). Pathway analysis of these genes suggests that alterations in glycobiology might play an important role in the aging of human bmMSC. On the other hand, antigen presentation and signaling of immune cells were the top pathways enriched by osteoarthritis-associated genes, suggesting that alteration in immunology of bmMSC might be involved in the pathogenesis of osteoarthritis. Most intriguingly, we found significant age-associated differential expression of HEXA, HEXB, CTSK, SULF1, ADAMTS5, SPP1, COL8A2, GPNMB, TNFAIP6, and RPL29; those genes have been implicated in the bone loss and the pathology of osteoporosis and osteoarthritis in aging. Collectively, our results suggest a pathological role of bmMSC in aging-related skeletal diseases, and suggest the possibility that alteration in the immunology of bmMSC might also play an important role in the etiology of adult-onset osteoarthritis.

  9. Stem Cell Ophthalmology Treatment Study (SCOTS: bone marrow-derived stem cells in the treatment of Leber′s hereditary optic neuropathy

    Directory of Open Access Journals (Sweden)

    Jeffrey N Weiss

    2016-01-01

    Full Text Available The Stem Cell Ophthalmology Treatment Study (SCOTS is currently the largest-scale stem cell ophthalmology trial registered at ClinicalTrials.gov (identifier: NCT01920867. SCOTS utilizes autologous bone marrow-derived stem cells (BMSCs to treat optic nerve and retinal diseases. Treatment approaches include a combination of retrobulbar, subtenon, intravitreal, intra-optic nerve, subretinal, and intravenous injection of autologous BMSCs according to the nature of the disease, the degree of visual loss, and any risk factors related to the treatments. Patients with Leber′s hereditary optic neuropathy had visual acuity gains on the Early Treatment Diabetic Retinopathy Study (ETDRS of up to 35 letters and Snellen acuity improvements from hand motion to 20/200 and from counting fingers to 20/100. Visual field improvements were noted. Macular and optic nerve head nerve fiber layer typically thickened. No serious complications were seen. The increases in visual acuity obtained in our study were encouraging and suggest that the use of autologous BMSCs as provided in SCOTS for ophthalmologic mitochondrial diseases including Leber′s hereditary optic neuropathy may be a viable treatment option.

  10. The Effect of Bone Marrow-Derived Mesenchymal Stem Cells and Their Conditioned Media Topically Delivered in Fibrin Glue on Chronic Wound Healing in Rats

    Science.gov (United States)

    Mehanna, Radwa A.; Nabil, Iman; Attia, Noha; Bary, Amany A.; Razek, Khalid A.; Ahmed, Tamer A. E.; Elsayed, Fatma

    2015-01-01

    Bone marrow-derived mesenchymal stem cells (BM-MSCs) represent a modern approach for management of chronic skin injuries. In this work, we describe BM-MSCs application versus their conditioned media (CM) when delivered topically admixed with fibrin glue to enhance the healing of chronic excisional wounds in rats. Fifty-two adult male rats were classified into four groups after induction of large-sized full-thickness skin wound: control group (CG), fibrin only group (FG), fibrin + MSCs group (FG + SCs), and fibrin + CM group (FG + CM). Healing wounds were evaluated functionally and microscopically. Eight days after injury, number of CD68+ macrophages infiltrating granulation tissue was considerably higher in the latter two groups. Although—later—none of the groups depicted a substantially different healing rate, the quality of regenerated skin was significantly boosted by the application of either BM-MSCs or their CM both (1) structurally as demonstrated by the obviously increased mean area percent of collagen fibers in Masson's trichrome-stained skin biopsies and (2) functionally as supported by the interestingly improved epidermal barrier as well as dermal tensile strength. Thus, we conclude that topically applied BM-MSCs and their CM—via fibrin vehicle—could effectively improve the quality of healed skin in chronic excisional wounds in rats, albeit without true acceleration of wound closure. PMID:26236740

  11. The Effect of Bone Marrow-Derived Mesenchymal Stem Cells and Their Conditioned Media Topically Delivered in Fibrin Glue on Chronic Wound Healing in Rats

    Directory of Open Access Journals (Sweden)

    Radwa A. Mehanna

    2015-01-01

    Full Text Available Bone marrow-derived mesenchymal stem cells (BM-MSCs represent a modern approach for management of chronic skin injuries. In this work, we describe BM-MSCs application versus their conditioned media (CM when delivered topically admixed with fibrin glue to enhance the healing of chronic excisional wounds in rats. Fifty-two adult male rats were classified into four groups after induction of large-sized full-thickness skin wound: control group (CG, fibrin only group (FG, fibrin + MSCs group (FG + SCs, and fibrin + CM group (FG + CM. Healing wounds were evaluated functionally and microscopically. Eight days after injury, number of CD68+ macrophages infiltrating granulation tissue was considerably higher in the latter two groups. Although—later—none of the groups depicted a substantially different healing rate, the quality of regenerated skin was significantly boosted by the application of either BM-MSCs or their CM both (1 structurally as demonstrated by the obviously increased mean area percent of collagen fibers in Masson’s trichrome-stained skin biopsies and (2 functionally as supported by the interestingly improved epidermal barrier as well as dermal tensile strength. Thus, we conclude that topically applied BM-MSCs and their CM—via fibrin vehicle—could effectively improve the quality of healed skin in chronic excisional wounds in rats, albeit without true acceleration of wound closure.

  12. Activated microglia induce bone marrow mesenchymal stem cells to produce glial cell-derived neurotrophic factor and protect neurons against oxygen-glucose deprivation injury

    Directory of Open Access Journals (Sweden)

    Bingke Lv

    2016-12-01

    Full Text Available In this study, we investigated interactions among microglia (MG, bone marrow mesenchymal stem cells (BMSCs and neurons in cerebral ischemia and the potential mechanisms using an in vitro oxygen-glucose deprivation (OGD model. Rat BMSCs were incubated with conditioned medium (CM from in vitro cultures of OGD-activated rat MG and murine BV2 MG cells. Effects of glial cell-derived neurotrophic factor (GDNF on rat neuron viability, apoptosis, lactate dehydrogenase (LDH leakage and mitochondrial membrane potential (MMP were analyzed in this model. OGD-activated MG promoted GDNF production by BMSCs (P < 0.01. TNFα, but not IL6 or IL1β, promoted GDNF production by BMSCs (P < 0.001. GDNF or CM pre-treated BMSCs elevated neuronal viability and suppressed apoptosis (P < 0.05 or P < 0.01; these effects were inhibited by the RET antibody. GDNF activated MEK/ERK and PI3K/AKT signaling but not JNK/c-JUN. Furthermore, GDNF upregulated B cell lymphoma 2 (BCL2 and heat shock 60 kDa protein 1 (HSP60 levels, suppressed LDH leakage, and promoted MMP. Thus, activated MG produce TNFα to stimulate GDNF production by BMSCs, which prevents and repairs OGD-induced neuronal injury, possibly via regulating MEK/ERK and PI3K/AKT signaling. These findings will facilitate the prevention and treatment of neuronal injury by cerebral ischemia.

  13. p53/p21 Pathway involved in mediating cellular senescence of bone marrow-derived mesenchymal stem cells from systemic lupus erythematosus patients.

    Science.gov (United States)

    Gu, Zhifeng; Jiang, Jinxia; Tan, Wei; Xia, Yunfei; Cao, Haixia; Meng, Yan; Da, Zhanyun; Liu, Hong; Cheng, Chun

    2013-01-01

    Our and other groups have found that bone marrow-derived mesenchymal stem cells (BM-MSCs) from systemic lupus erythematosus (SLE) patients exhibited senescent behavior and are involved in the pathogenesis of SLE. Numerous studies have shown that activation of the p53/p21 pathway inhibits the proliferation of BM-MSCs. The aim of this study was to determine whether p53/p21 pathway is involved in regulating the aging of BM-MSCs from SLE patients and the underlying mechanisms. We further confirmed that BM-MSCs from SLE patients showed characteristics of senescence. The expressions of p53 and p21 were significantly increased, whereas levels of Cyclin E, cyclin-dependent kinase-2, and phosphorylation of retinoblastoma protein were decreased in the BM-MSCs from SLE patients and knockdown of p21 expression reversed the senescent features of BM-MSCs from SLE patients. Our results demonstrated that p53/p21 pathway played an important role in the senescence process of BM-MSCs from SLE.

  14. p53/p21 Pathway Involved in Mediating Cellular Senescence of Bone Marrow-Derived Mesenchymal Stem Cells from Systemic Lupus Erythematosus Patients

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    Zhifeng Gu

    2013-01-01

    Full Text Available Our and other groups have found that bone marrow-derived mesenchymal stem cells (BM-MSCs from systemic lupus erythematosus (SLE patients exhibited senescent behavior and are involved in the pathogenesis of SLE. Numerous studies have shown that activation of the p53/p21 pathway inhibits the proliferation of BM-MSCs. The aim of this study was to determine whether p53/p21 pathway is involved in regulating the aging of BM-MSCs from SLE patients and the underlying mechanisms. We further confirmed that BM-MSCs from SLE patients showed characteristics of senescence. The expressions of p53 and p21 were significantly increased, whereas levels of Cyclin E, cyclin-dependent kinase-2, and phosphorylation of retinoblastoma protein were decreased in the BM-MSCs from SLE patients and knockdown of p21 expression reversed the senescent features of BM-MSCs from SLE patients. Our results demonstrated that p53/p21 pathway played an important role in the senescence process of BM-MSCs from SLE.

  15. Bone Marrow Suppression by c-Kit Blockade Enhances Tumor Growth of Colorectal Metastases through the Action of Stromal Cell-Derived Factor-1

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    Kathrin Rupertus

    2012-01-01

    Full Text Available Background. Mobilization of c-Kit+ hematopoietic cells (HCs contributes to tumor vascularization. Whereas survival and proliferation of HCs are regulated by binding of the stem cell factor to its receptor c-Kit, migration of HCs is directed by stromal cell-derived factor (SDF-1. Therefore, targeting migration of HCs provides a promising new strategy of anti-tumor therapy. Methods. BALB/c mice (=16 were pretreated with an anti-c-Kit antibody followed by implantation of CT26.WT-GFP colorectal cancer cells into dorsal skinfold chambers. Animals (=8 additionally received a neutralizing anti-SDF-1 antibody. Animals (=8 treated with a control antibody served as controls. Investigations were performed using intravital fluorescence microscopy, immunohistochemistry, flow cytometry and western blot analysis. Results. Blockade of c-Kit significantly enhanced tumor cell engraftment compared to controls due to stimulation of tumor cell proliferation and invasion without markedly affecting tumor vascularization. C-Kit blockade significantly increased VEGF and CXCR4 expression within the growing tumors. Neutralization of SDF-1 completely antagonized this anti-c-Kit-associated tumor growth by suppression of tumor neovascularization, inhibition of tumor cell proliferation and reduction of muscular infiltration. Conclusion. Our study indicates that bone marrow suppression via anti-c-Kit pretreatment enhances tumor cell engraftment of colorectal metastases due to interaction with the SDF-1/CXCR4 pathway which is involved in HC-mediated tumor angiogenesis.

  16. The Expanding Family of Bone Marrow Homing Factors for Hematopoietic Stem Cells: Stromal Derived Factor 1 Is Not the Only Player in the Game

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    Mariusz Z. Ratajczak

    2012-01-01

    Full Text Available The α-chemokine stromal derived factor 1 (SDF-1, which binds to the CXCR4 and CXCR7 receptors, directs migration and homing of CXCR4+ hematopoietic stem/progenitor cells (HSPCs to bone marrow (BM and plays a crucial role in retention of these cells in stem cell niches. However, this unique role of SDF-1 has been recently challenged by several observations supporting SDF-1-CXCR4-independent BM homing. Specifically, it has been demonstrated that HSPCs respond robustly to some bioactive lipids, such as sphingosine-1-phosphate (S1P and ceramide-1-phosphate (C1P, and migrate in response to gradients of certain extracellular nucleotides, including uridine triphosphate (UTP and adenosine triphosphate (ATP. Moreover, the responsiveness of HSPCs to an SDF-1 gradient is enhanced by some elements of innate immunity (e.g., C3 complement cascade cleavage fragments and antimicrobial cationic peptides, such as cathelicidin/LL-37 or β2-defensin as well as prostaglandin E2 (PGE2. Since all these factors are upregulated in BM after myeloblative conditioning for transplantation, a more complex picture of homing emerges that involves several factors supporting, and in some situations even replacing, the SDF-1-CXCR4 axis.

  17. Biologic effect and immunoisolating behavior of BMP-2 gene-transfected bone marrow-derived mesenchymal stem cells in APA microcapsules.

    Science.gov (United States)

    Ding, H F; Liu, R; Li, B G; Lou, J R; Dai, K R; Tang, T T

    2007-11-03

    We investigated the encapsulation of BMP-2 gene-modified mesenchymal stem cells (MSCs) in alginate-poly-L-lysine (APA) microcapsules for the persistent delivery of bone morphogenic protein-2 (BMP-2) to induce bone formation. An electrostatic droplet generator was employed to produce APA microcapsules containing encapsulated beta-gal or BMP-2 gene-transfected bone marrow-derived MSCs. We found that X-gal staining was still positive 28 days after encapsulation. Encapsulated BMP-2 gene-transfected cells were capable of constitutive delivery of BMP-2 proteins for at least 30 days. The encapsulated BMP-2 gene-transfected MSCs or the encapsulated non-gene transfer MSCs (control group) were cocultured with the undifferentiated MSCs. The gene products from the encapsulated BMP-2 cells could induce the undifferentiated MSCs to become osteoblasts that had higher alkaline phosphatase (ALP) activity than those in the control group (pAPA microcapsules could inhibit the permeation of fluorescein isothiocyanate-conjuncted immunoglobulin G. Mixed lymphocyte reaction also indicates that the APA microcapsules could prevent the encapsulated BMP-2 gene-transfected MSCs from initiating the cellular immune response. These results demonstrated that the nonautologous BMP-2 gene-transfected stem cells are of potential utility for enhancement of bone repair and bone regeneration in vivo.

  18. Effect of Metformin on Viability, Morphology, and Ultrastructure of Mouse Bone Marrow-Derived Multipotent Mesenchymal Stromal Cells and Balb/3T3 Embryonic Fibroblast Cell Line

    Directory of Open Access Journals (Sweden)

    Agnieszka Śmieszek

    2015-01-01

    Full Text Available Metformin, a popular drug used to treat diabetes, has recently gained attention as a potentially useful therapeutic agent for treating cancer. In our research metformin was added to in vitro cultures of bone marrow-derived multipotent mesenchymal stromal cells (BMSCs and Balb/3T3 fibroblast at concentration of 1 mM, 5 mM, and 10 mM. Obtained results indicated that metformin negatively affected proliferation activity of investigated cells. The drug triggered the formation of autophagosomes and apoptotic bodies in all tested cultures. Additionally, we focused on determination of expression of genes involved in insulin-like growth factor 2 (IGF2 signaling pathway. The most striking finding was that the mRNA level of IGF2 was constant in both BMSCs and Balb/3T3. Further, the analysis of IGF2 concentration in cell supernatants showed that it decreased in BMSC cultures after 5 and 10 mM metformin treatments. In case of Balb/3T3 the concentration of IGF2 in culture supernatants decreased after 1 and 5 mM and increased after 10 mM of metformin. Our results suggest that metformin influences the cytophysiology of somatic cells in a dose- and time-dependent manner causing inhibition of proliferation and abnormalities of their morphology and ultrastructure.

  19. Salvianolic acid B improves bone marrow-derived mesenchymal stem cell differentiation into alveolar epithelial cells type I via Wnt signaling.

    Science.gov (United States)

    Gao, Peng; Yang, Jingxian; Gao, Xi; Xu, Dan; Niu, Dongge; Li, Jinglin; Wen, Qingping

    2015-08-01

    Acute lung injury (ALI) is among the most common causes of mortality in intensive care units. Previous studies have suggested that bone marrow-derived mesenchymal stem cells (BMSCs) may attenuate pulmonary edema. In addition, alveolar epithelial cells type I (ATI) are involved in reducing the alveolar edema in response to ALI. However, the mechanism involved in improving the efficiency of differentiation of MSCs into ATI remains to be elucidated. In the present study, the effect of salvianolic acid B (Sal B) on the differentiation of BMSCs into ATI and the activities of the Wnt signaling pathways were investigated. The BMSCs were supplemented with conditioned medium (CM). The groups were as follows: i) CM group: BMSCs were supplemented with CM; ii) lithium chloride (LiCl) group: BMSCs were supplemented with CM and 5 mM LiCl; iii) Sal B group: BMSCs were supplemented with CM and 10 mM Sal B. The samples were collected and assessed on days 7 and 14. It was revealed that aquaporin (AQP)-5 and T1α were expressed in BMSCs, and induction with LiCl or Sal B increased the expression of AQP-5 and T1α. Furthermore, the Wnt-1 and Wnt-3a signaling pathways were activated during the differentiation of BMSCs into ATI. In conclusion, it was suggested that the promotive effects of Sal B on the differentiation of BMSCs into ATI occurred through the activation of Wnt signaling pathways.

  20. The Chondrogenic Induction Potential for Bone Marrow-Derived Stem Cells between Autologous Platelet-Rich Plasma and Common Chondrogenic Induction Agents: A Preliminary Comparative Study

    Directory of Open Access Journals (Sweden)

    Shan-zheng Wang

    2015-01-01

    Full Text Available The interests in platelet-rich plasma (PRP and their application in stem cell therapy have contributed to a better understanding of the basic biology of the prochondrogenesis effect on bone marrow-derived stem cells (BMSCs. We aimed at comparing the effect of autologous PRP with common chondrogenic induction agents (CCIAs on the chondrogenic differentiation of BMSCs. Rabbit BMSCs were isolated and characterized by flow cytometry and differentiated towards adipocytes and osteoblasts. The chondrogenic response of BMSCs to autologous PRP and CCIAs which included transforming growth factor-β1 (TGF-β1, dexamethasone (DEX, and vitamin C (Vc was examined by cell pellet culture. The isolated BMSCs after two passages highly expressed CD29 and CD44 but minimally expressed CD45. The osteogenic and adipogenic differentiation potentials of the isolated BMSCs were also confirmed. Compared with common CCIAs, autologous PRP significantly upregulated the chondrogenic related gene expression, including Col-2, AGC, and Sox-9. Osteogenic related gene expression, including Col-1 and OCN, was not of statistical significance between these two groups. Thus, our data shows that, compared with common chondrogenic induction agents, autologous PRP can be more effective in promoting the chondrogenesis of BMSCs.

  1. Biological Response of Human Bone Marrow-Derived Mesenchymal Stem Cells to Commercial Tantalum Coatings with Microscale and Nanoscale Surface Topographies

    Science.gov (United States)

    Skoog, Shelby A.; Kumar, Girish; Goering, Peter L.; Williams, Brian; Stiglich, Jack; Narayan, Roger J.

    2016-06-01

    Tantalum is a promising orthopaedic implant coating material due to its robust mechanical properties, corrosion resistance, and excellent biocompatibility. Previous studies have demonstrated improved biocompatibility and tissue integration of surface-treated tantalum coatings compared to untreated tantalum. Surface modification of tantalum coatings with biologically inspired microscale and nanoscale features may be used to evoke optimal tissue responses. The goal of this study was to evaluate commercial tantalum coatings with nanoscale, sub-microscale, and microscale surface topographies for orthopaedic and dental applications using human bone marrow-derived mesenchymal stem cells (hBMSCs). Tantalum coatings with different microscale and nanoscale surface topographies were fabricated using a diffusion process or chemical vapor deposition. Biological evaluation of the tantalum coatings using hBMSCs showed that tantalum coatings promote cellular adhesion and growth. Furthermore, hBMSC adhesion to the tantalum coatings was dependent on surface feature characteristics, with enhanced cell adhesion on sub-micrometer- and micrometer-sized surface topographies compared to hybrid nano-/microstructures. Nanostructured and microstructured tantalum coatings should be further evaluated to optimize the surface coating features to promote osteogenesis and enhance osseointegration of tantalum-based orthopaedic implants.

  2. Cellular behaviour of hepatocyte-like cells from nude mouse bone marrow-derived mesenchymal stem cells on galactosylated poly(D,L-lactic-co-glycolic acid).

    Science.gov (United States)

    Roh, Hyun; Yang, Dae Hyeok; Chun, Heung Jae; Khang, Gilson

    2015-07-01

    Previously, the galactosylation of poly(d,l-lactic-co-glycolic acid) (PLGA) surface was accomplished by grafting allylamine (AA), using inductively coupled plasma-assisted chemical vapour deposition (ICP-CVD) and conjugating lactobionic acid (LA) with AA via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS) activation for hepatic tissue-engineering purposes. As a continuation study, the cellular behaviour of hepatocyte-like cells (HLCs) on the surface of the galactosylated PLGA were investigated. Nude mouse bone marrow-derived mesenchymal stem cells (MSCs) were cultured under hepatogenic conditions and the differentiated cells were characterized by reverse-transcription polymerase chain reaction (RT-PCR), immunofluorescence and periodic acid-Schiff (PAS) staining. Galactosylated PLGA enhanced the proliferation rate of HLCs compared to the control; HLCs on the surface of the sample became aggregated and formed spheroids after 3 days of culture. A large number of cells on the surface of the sample exhibited increased liver-specific functional activities, such as albumin and urea secretions. In addition, multicellular spheroids in the sample strongly expressed phospholyated focal adhesion kinase (pFAK) (cell-matrix interactions), E-cadherin (cell-cell interactions) and connexin 32 (Cox32; gap junction).

  3. SF20/IL-25, a novel bone marrow stroma-derived growth factor that binds to mouse thymic shared antigen-1 and supports lymphoid cell proliferation.

    Science.gov (United States)

    Tulin, E E; Onoda, N; Nakata, Y; Maeda, M; Hasegawa, M; Nomura, H; Kitamura, T

    2001-12-01

    Using a forward genetic approach and phenotype-based complementation screening to search for factors that stimulate cell proliferation, we have isolated a novel secreted bone marrow stroma-derived growth factor, which we termed SF20/IL-25. This protein signals cells to proliferate via its receptor, which we have identified as mouse thymic shared Ag-1 (TSA-1). Enforced expression of TSA-1 in IL-3-dependent Ba/F3 cells that do not express endogenous TSA-1 rendered cells to proliferate in a dose-dependent manner when stimulated with SF20/IL-25. FDCP2, a factor-dependent hemopoietic cell line that expresses endogenous TSA-1, could also be stimulated to proliferate with SF20/IL-25. Binding of SF20 to TSA-1 was blocked by anti-TSA-1 Ab and SF20-induced proliferation of TSA-1-expressing cells was inhibited by anti-TSA-1. In vitro assay revealed that SF20/IL-25 has no detectable myelopoietic activity but supports proliferation of cells in the lymphoid lineage.

  4. Human Amnion-Derived Mesenchymal Stem Cells Promote Osteogenic Differentiation in Human Bone Marrow Mesenchymal Stem Cells by Influencing the ERK1/2 Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Yuli Wang

    2016-01-01

    Full Text Available Human amnion-derived mesenchymal stem cells (HAMSCs are considered to be an important resource in the field of tissue engineering because of their anti-inflammatory properties and fewer ethical issues associated with their use compared with other sources of stem cells. HAMSCs can be obtained from human amniotic membranes, a readily available and abundant tissue. However, the potential of HAMSCs as seed cells for treating bone deficiency is unknown. In this study, HAMSCs were used to promote proliferation and osteoblastic differentiation in human bone marrow mesenchymal stem cells (HBMSCs in a Transwell coculture system. Proliferation levels were investigated by flow cytometry and immunofluorescence staining of 5-ethynyl-2′-deoxyuridine (EdU. Osteoblastic differentiation and mineralization were evaluated in chromogenic alkaline phosphatase (ALP activity substrate assays, Alizarin red S staining, and RT-PCR analysis of early HBMSCs osteogenic marker expression. We demonstrated that HAMSCs stimulated increased alkaline phosphatase (ALP activity, mRNA expression of osteogenic marker genes, and mineralized matrix deposition. Moreover, the effect of HAMSCs was significantly inhibited by U0126, a highly selective inhibitor of extracellular signaling-regulated kinase 1/2 (ERK1/2 signaling. We demonstrate that HAMSCs promote osteogenic differentiation in HBMSCs by influencing the ERK1/2 signaling pathway. These observations confirm the potential of HAMSCs as a seed cell for the treatment of bone deficiency.

  5. Lack of galectin-3 increases Jagged1/Notch activation in bone marrow-derived dendritic cells and promotes dysregulation of T helper cell polarization.

    Science.gov (United States)

    Fermino, Marise L; Dylon, L Sebastian D; Cecílio, Nerry T; Santos, Sofia N; Toscano, Marta A; Dias-Baruffi, Marcelo; Roque-Barreira, Maria C; Rabinovich, Gabriel A; Bernardes, Emerson S

    2016-08-01

    Galectin-3, an endogenous glycan-binding protein, is abundantly expressed at sites of inflammation and immune cell activation. Although this lectin has been implicated in the control of T helper (Th) polarization, the mechanisms underlying this effect are not well understood. Here, we investigated the role of endogenous galectin-3 during the course of experimental Leishmania major infection using galectin-3-deficient (Lgals3(-/-)) mice in a BALB/c background and the involvement of Notch signaling pathway in this process. Lgals3(-/-) mice displayed an augmented, although mixed Th1/Th2 responses compared with wild-type (WT) mice. Concomitantly, lymph node and footpad lesion cells from infected Lgals3(-/-) mice showed enhanced levels of Notch signaling components (Notch-1, Jagged1, Jagged2 and Notch target gene Hes-1). Bone marrow-derived dendritic cells (BMDCs) from uninfected Lgals3(-/-) mice also displayed increased expression of the Notch ligands Delta-like-4 and Jagged1 and pro-inflammatory cytokines. In addition, activation of Notch signaling in BMDCs upon stimulation with Jagged1 was more pronounced in Lgals3(-/-) BMDCs compared to WT BMDCs; this condition resulted in increased production of IL-6 by Lgals3(-/-) BMDCs. Finally, addition of exogenous galectin-3 to Lgals3(-/-) BMDCs partially reverted the increased sensitivity to Jagged1 stimulation. Our results suggest that endogenous galectin-3 regulates Notch signaling activation in BMDCs and influences polarization of T helper responses, thus increasing susceptibility to L. major infection.

  6. Bone marrow cell derived arginase I is the major source of allergen-induced lung arginase but is not required for airway hyperresponsiveness, remodeling and lung inflammatory responses in mice

    Directory of Open Access Journals (Sweden)

    Rothenberg Marc E

    2009-06-01

    Full Text Available Abstract Background Arginase is significantly upregulated in the lungs in murine models of asthma, as well as in human asthma, but its role in allergic airway inflammation has not been fully elucidated in mice. Results In order to test the hypothesis that arginase has a role in allergic airway inflammation we generated arginase I-deficient bone marrow (BM chimeric mice. Following transfer of arginase I-deficient BM into irradiated recipient mice, arginase I expression was not required for hematopoietic reconstitution and baseline immunity. Arginase I deficiency in bone marrow-derived cells decreased allergen-induced lung arginase by 85.8 ± 5.6%. In contrast, arginase II-deficient mice had increased lung arginase activity following allergen challenge to a similar level to wild type mice. BM-derived arginase I was not required for allergen-elicited sensitization, recruitment of inflammatory cells in the lung, and proliferation of cells. Furthermore, allergen-induced airway hyperresponsiveness and collagen deposition were similar in arginase-deficient and wild type mice. Additionally, arginase II-deficient mice respond similarly to their control wild type mice with allergen-induced inflammation, airway hyperresponsiveness, proliferation and collagen deposition. Conclusion Bone marrow cell derived arginase I is the predominant source of allergen-induced lung arginase but is not required for allergen-induced inflammation, airway hyperresponsiveness or collagen deposition.

  7. Resveratrol protects bone marrow mesenchymal stem cell derived chondrocytes cultured on chitosan-gelatin scaffolds from the inhibitory effect of interleukin-1β

    Institute of Scientific and Technical Information of China (English)

    Ming LEI; Shi-qing LIU; Yu-lan LIU

    2008-01-01

    Aim: To investigate the effects of resveratrol on interleukin-lbeta (IL-1β) induced catabolism in bone marrow mesenchymal stem cell (MSC) derived chon-drocytes cultured on chitosan-gelatin scaffolds (CGS). Methods: The chondro-genesis of alginate-encapsulated MSCs was evaluated by toluidine blue staining, RT-PCR, and immunostaing. MSC-derived chondrocyte morphology cultured on CGS was evaluated by a scanning electron microscope (SEM) and a laser confocal microscope (LCM). When these cells on CGS were pre-stimulated with IL-1β or co-treated with IL-1β and resveratrol in the absence and presence of the specific β1-integrin blocking antibody, collagen type Ⅱ, aggrecan, matrix metalloproteinase-13 (MMP-13) expression, and the translocation of nuclear factor kappaB (NF-κB) were analyzed by Western blot analysis. Results: Transforming growth factor beta 3 (TGF-β3) combined with insulin-like growth factor Ⅰ (IGF-Ⅰ) induced the cartilage-specific collagen type Ⅱ, aggrecan expression and extracellular matrix (ECM) accumulation at the end of a 3-week culture. CGS supported those differentiated chondrocytes' attachment, proliferation, migration, and ECM formation. When those cells cultured on CGS were stimulated with IL-1β alone, collagen type Ⅱ and aggrecan expression was inhibited. However, MMP-13 expression increased. Resveratrol reversed the catabolic effects by reducing the translocation of NF-κB. A specific β1-integrin blocking antibody abrogated the effects of resveratrol on IL-1β stimulated MSC-derived chondrocytes. Conclusion: These results indicated that resveratrol acta as a NF-κB inhibitor to protect MSC-derived chondrocytes on the CGS from the IL-1β catabolism and these effects are mediated by β1-integrin.

  8. Organotins Are Potent Activators of PPARγ and Adipocyte Differentiation in Bone Marrow Multipotent Mesenchymal Stromal Cells

    OpenAIRE

    2011-01-01

    Adipocyte differentiation in bone marrow is potentially deleterious to both bone integrity and lymphopoiesis. Here, we examine the hypothesis that organotins, common environmental contaminants that are dual ligands for peroxisome proliferator–activated receptor (PPAR) γ and its heterodimerization partner retinoid X receptor (RXR), are potent activators of bone marrow adipogenesis. A C57Bl/6-derived bone marrow multipotent mesenchymal stromal cell (MSC) line, BMS2, was treated with rosiglitazo...

  9. The effects of dynamic compression on the development of cartilage grafts engineered using bone marrow and infrapatellar fat pad derived stem cells.

    Science.gov (United States)

    Luo, Lu; Thorpe, Stephen D; Buckley, Conor T; Kelly, Daniel J

    2015-09-21

    Bioreactors that subject cell seeded scaffolds or hydrogels to biophysical stimulation have been used to improve the functionality of tissue engineered cartilage and to explore how such constructs might respond to the application of joint specific mechanical loading. Whether a particular cell type responds appropriately to physiological levels of biophysical stimulation could be considered a key determinant of its suitability for cartilage tissue engineering applications. The objective of this study was to determine the effects of dynamic compression on chondrogenesis of stem cells isolated from different tissue sources. Porcine bone marrow (BM) and infrapatellar fat pad (FP) derived stem cells were encapsulated in agarose hydrogels and cultured in a chondrogenic medium in free swelling (FS) conditions for 21 d, after which samples were subjected to dynamic compression (DC) of 10% strain (1 Hz, 1 h d(-1)) for a further 21 d. Both BM derived stem cells (BMSCs) and FP derived stem cells (FPSCs) were capable of generating cartilaginous tissues with near native levels of sulfated glycosaminoglycan (sGAG) content, although the spatial development of the engineered grafts strongly depended on the stem cell source. The mechanical properties of cartilage grafts generated from both stem cell sources also approached that observed in skeletally immature animals. Depending on the stem cell source and the donor, the application of DC either enhanced or had no significant effect on the functional development of cartilaginous grafts engineered using either BMSCs or FPSCs. BMSC seeded constructs subjected to DC stained less intensely for collagen type I. Furthermore, histological and micro-computed tomography analysis showed mineral deposition within BMSC seeded constructs was suppressed by the application of DC. Therefore, while the application of DC in vitro may only lead to modest improvements in the mechanical functionality of cartilaginous grafts, it may play an important

  10. Morphological, molecular and functional differences of adult bone marrow- and adipose-derived stem cells isolated from rats of different ages

    Energy Technology Data Exchange (ETDEWEB)

    Mantovani, Cristina [Blond McIndoe Laboratories, School of Biomedicine, The University of Manchester, Room 3,106 Stopford Building, Oxford Road, Manchester M13 9PT, Academic Health Science Centre, Faculty of Medicine and Human Sciences (United Kingdom); Department of Integrative Medical Biology and Surgical and Perioperative Science, Umea University, Umea (Sweden); Department of Surgical and Perioperative Science, Umea University, Umea (Sweden); Raimondo, Stefania [Dipartimento di Scienze Cliniche e Biologiche, University of Turin (Italy); Haneef, Maryam S. [Blond McIndoe Laboratories, School of Biomedicine, The University of Manchester, Room 3,106 Stopford Building, Oxford Road, Manchester M13 9PT, Academic Health Science Centre, Faculty of Medicine and Human Sciences (United Kingdom); Geuna, Stefano [Dipartimento di Scienze Cliniche e Biologiche, University of Turin (Italy); Terenghi, Giorgio [Blond McIndoe Laboratories, School of Biomedicine, The University of Manchester, Room 3,106 Stopford Building, Oxford Road, Manchester M13 9PT, Academic Health Science Centre, Faculty of Medicine and Human Sciences (United Kingdom); Shawcross, Susan G., E-mail: sue.shawcross@manchester.ac.uk [Blond McIndoe Laboratories, School of Biomedicine, The University of Manchester, Room 3,106 Stopford Building, Oxford Road, Manchester M13 9PT, Academic Health Science Centre, Faculty of Medicine and Human Sciences (United Kingdom); Wiberg, Mikael [Department of Integrative Medical Biology and Surgical and Perioperative Science, Umea University, Umea (Sweden); Department of Surgical and Perioperative Science, Umea University, Umea (Sweden)

    2012-10-01

    Adult mesenchymal stem cells have self-renewal and multiple differentiation potentials, and play important roles in regenerative medicine. However, their use may be limited by senescence or age of the donor, leading to changes in stem cell functionality. We investigated morphological, molecular and functional differences between bone marrow-derived (MSC) and adipose-derived (ASC) stem cells isolated from neonatal, young and old rats compared to Schwann cells from the same animals. Immunocytochemistry, RT-PCR, proliferation assays, western blotting and transmission electron microscopy were used to investigate expression of senescence markers. Undifferentiated and differentiated ASC and MSC from animals of different ages expressed Notch-2 at similar levels; protein-38 and protein-53 were present in all groups of cells with a trend towards increased levels in cells from older animals compared to those from neonatal and young rats. Following co-culture with adult neuronal cells, dMSC and dASC from animals of all ages elicited robust neurite outgrowth. Mitotracker{sup Registered-Sign} staining was consistent with ultrastructural changes seen in the mitochondria of cells from old rats, indicative of senescence. In conclusion, this study showed that although the cells from aged animals expressed markers of senescence, aged MSC and ASC differentiated into SC-like cells still retain potential to support axon regeneration. -- Highlights: Black-Right-Pointing-Pointer Aged MSC and ASC differentiated into Schwann-like cells support axon regeneration. Black-Right-Pointing-Pointer p53 expression does not appreciably influence the biology of Schwann or stem cells. Black-Right-Pointing-Pointer Notch 2 expression was similar in cells derived from animals of different ages. Black-Right-Pointing-Pointer Proliferation rates of dMSC varied little over time or with animal age.

  11. CXCR5-transduced bone marrow-derived dendritic cells traffic to B cell zones of lymph nodes and modify antigen-specific immune responses.

    Science.gov (United States)

    Wu, Meng-Tse; Hwang, Sam T

    2002-05-15

    Skin-derived migratory dendritic cells (DC), in contrast to bone marrow-derived DC (BMDC), express CXCR5, respond to the chemokine CXC ligand 13 (CXCL13) in vitro, and are capable of migrating to B cell zones (BCZ) in lymph nodes (LN) in vivo. Herein, we analyzed the surface phenotype of skin-derived migratory DC and found that 15-35% of MHC class II(high) cells showed high levels of expression of CXCR5 but expressed low levels of DEC205, a suggested characteristic of dermal-type DC in mice. To study the effects of CXCR5 on the trafficking dynamics of DC, we stably expressed CXCR5 in BMDC by retroviral gene transduction. CXCR5 was detected by flow cytometry on transduced cells, which responded to CXCL13 in vitro in chemotaxis assays (3-fold over nontransduced BMDC, p BMDC were observed in BCZ of draining LN. Mice were vaccinated with CXCR5- and vector-BMDC that were pulsed with keyhole limpet hemocyanin (KLH) to induce Ag-specific cellular and humoral immune responses. Mice injected with CXCR5-BMDC (vs vector-BMDC) demonstrated marginally less footpad swelling in response to intradermal injection of KLH. Interestingly, significantly higher levels of KLH-specific IgG (p BMDC compared with mice immunized with vector-transduced BMDC. Thus, CXCR5 is predominantly expressed by dermal-type DC. Moreover, CXCR5 directs BMDC to BCZ of LN in vivo and modifies Ag-specific immune responses induced by BMDC vaccination.

  12. Bone Marrow Transplants: "Another Possibility at Life"

    Science.gov (United States)

    ... page please turn Javascript on. Feature: Bone Marrow Transplants “Another Possibility at Life” Past Issues / Summer 2011 ... for 16,000 of them, a bone marrow transplant is the best treatment option, notes Susan F. ...

  13. Direct Comparison of Wharton's Jelly and Bone Marrow-Derived Mesenchymal Stromal Cells to Enhance Engraftment of Cord Blood CD34+ Transplants

    Science.gov (United States)

    van der Garde, Mark; van Pel, Melissa; Millán Rivero, Jose Eduardo; de Graaf-Dijkstra, Alice; Slot, Manon C.; Kleinveld, Yoshiko; Watt, Suzanne M.; Roelofs, Helene

    2015-01-01

    Cotransplantation of CD34+ hematopoietic stem and progenitor cells (HSPCs) with mesenchymal stromal cells (MSCs) enhances HSPC engraftment. For these applications, MSCs are mostly obtained from bone marrow (BM). However, MSCs can also be isolated from the Wharton's jelly (WJ) of the human umbilical cord. This source, regarded to be a waste product, enables a relatively low-cost MSC acquisition without any burden to the donor. In this study, we evaluated the ability of WJ MSCs to enhance HSPC engraftment. First, we compared cultured human WJ MSCs with human BM-derived MSCs (BM MSCs) for in vitro marker expression, immunomodulatory capacity, and differentiation into three mesenchymal lineages. Although we confirmed that WJ MSCs have a more restricted differentiation capacity, both WJ MSCs and BM MSCs expressed similar levels of surface markers and exhibited similar immune inhibitory capacities. Most importantly, cotransplantation of either WJ MSCs or BM MSCs with CB CD34+ cells into NOD SCID mice showed similar enhanced recovery of human platelets and CD45+ cells in the peripheral blood and a 3-fold higher engraftment in the BM, blood, and spleen 6 weeks after transplantation when compared to transplantation of CD34+ cells alone. Upon coincubation, both MSC sources increased the expression of adhesion molecules on CD34+ cells, although stromal cell-derived factor-1 (SDF-1)-induced migration of CD34+ cells remained unaltered. Interestingly, there was an increase in CFU-GEMM when CB CD34+ cells were cultured on monolayers of WJ MSCs in the presence of exogenous thrombopoietin, and an increase in BFU-E when BM MSCs replaced WJ MSCs in such cultures. Our results suggest that WJ MSC is likely to be a practical alternative for BM MSC to enhance CB CD34+ cell engraftment. PMID:26414086

  14. Xeno-free culture condition for human bone marrow and umbilical cord matrix-derived mesenchymal stem/stromal cells using human umbilical cord blood serum

    Science.gov (United States)

    Esmaeli, Azadeh; Moshrefi, Mojgan; Shamsara, Ali; Eftekhar-vaghefi, Seyed Hasan; Nematollahi-mahani, Seyed Noureddin

    2016-01-01

    Background: Fetal bovine serum (FBS) is widely used in cell culture laboratories, risk of zoonotic infections and allergic side effects create obstacles for its use in clinical trials. Therefore, an alternative supplement with proper inherent growth-promoting activities is demanded. Objective: To find FBS substitute, we tested human umbilical cord blood serum (hUCS) for proliferation of human umbilical cord matrix derived mesenchymal stem cells (hUC-MSCs) and human bone marrow-derived mesenchymal cells (hBM-MSCs). Materials and Methods: Umbilical cord blood of healthy neonates, delivered by Caesarian section, was collected and the serum was separated. hUC-MSCs and hBM-MSCs were isolated and characterized by assessment of cell surface antigens by flow cytometry, alkaline phosphatase activity and osteogenic/adipogenic differentiation potential. The cells were then cultured in Iscove's Modified Dulbecco's Medium (IMDM) by conventional methods in three preparations: 1- with hUCS, 2- with FBS, and 3- without serum supplements. Cell proliferation was measured using WST-1 assay, and cell viability was assessed by trypan blue staining. Results: The cells cultured in hUCS and FBS exhibited similar morphology and mesenchymal stem cells properties. WST-1 proliferation assay data showed no significant difference between the proliferation rate of either cells following hUCS and FBS supplementation. Trypan blue exclusion dye test also revealed no significant difference for viability between hUCS and FBS groups. A significant difference was detected between the proliferation rate of stem cells cultured in serum-supplemented medium compared with serum-free medium. Conclusion: Our results indicate that human umbilical cord serum can effectively support proliferation of hBM-MSCS and hUC-MSCs in vitro and can be used as an appropriate substitute for FBS, especially in clinical studies. PMID:27738658

  15. Derivation of male germ cells from ram bone marrow mesenchymal stem cells by three different methods and evaluation of their fate after transplantation into the testis.

    Science.gov (United States)

    Ghasemzadeh-Hasankolaei, Mohammad; Eslaminejad, Mohammadreza Baghaban; Sedighi-Gilani, Mohammadali

    2016-01-01

    Mesenchymal stem cells (MSCs) have the capacity to differentiate into germ cells (GCs). This research, for the first time, has evaluated the fate of in vitro MSC-derived GCs generated by three different induction methods and compared them after transplantation into the testes of rams. Passage-3 ram bone marrow (BM)-MSCs were divided into three treatment groups: (1) 14-d treatment with 10 μM retinoic acid (RA; RA14), (2) 21-d treatment with 10 μM RA (RA21), and (3) 21-d treatment with 10 ng/ml transforming growth factor beta-1 (TGFb1). After confirmation of the existence of germ-like cells in the culture, the treated cells were labeled and transplanted into the testes of ram lambs. After 2 mo, we conducted histological evaluations of the rams' testes. Results showed that in vitro-derived GCs from all treatment groups survived in the testes. Some of these GCs homed at the basement membrane of seminiferous tubules and formed colonies. The homed cells and cell colonies were similar to testicular native spermatogonia and expressed PGP9.5. TGFb1 exhibited the highest efficiency for in vitro production of GCs as well as the highest capability for homing and colony formation in the testes. RA21 was less efficient than TGFb1, particularly in colony formation. RA14 was the weakest group. No further differentiation of the transplanted GCs was observed. From our results, it could be concluded that a 21-d treatment period of BM-MSCs with TGFb1 is the most efficient method for in vitro generation of spermatogonia-like cells that survive, home, and form colonies in the testes.

  16. Validity of T2 mapping in characterization of the regeneration tissue by bone marrow derived cell transplantation in osteochondral lesions of the ankle

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    Battaglia, M., E-mail: milva.battaglia@ior.it [Service of Ecography and Radiology, Rizzoli Orthopaedic Institute, via Pupilli n. 1, 40136 Bologna (Italy); Rimondi, E. [Service of Ecography and Radiology, Rizzoli Orthopaedic Institute, via Pupilli n. 1, 40136 Bologna (Italy); Monti, C. [Service of CT and MRI, Casa di Cura Madre Fortunata Toniolo, Bologna (Italy); Guaraldi, F. [Department of Pathology, The Johns Hopkins University, School of Medicine, Baltimore, MD (United States); Sant' Andrea, A. [Service of CT and MRI, Casa di Cura Madre Fortunata Toniolo, Bologna (Italy); Buda, R.; Cavallo, M.; Giannini, S.; Vannini, F. [Clinical Orthopaedic and Traumatology Unit II, Rizzoli Orthopaedic Institute, Bologna (Italy)

    2011-11-15

    Objective: Bone marrow derived cell transplantation (BMDCT) has been recently suggested as a possible surgical technique to repair osteochondral lesions. To date, no qualitative MRI studies have evaluated its efficacy. The aim of our study is to investigate the validity of MRI T2-mapping sequence in characterizing the reparative tissue obtained and its ability to correlate with clinical results. Methods and materials: 20 patients with an osteochondral lesion of the talus underwent BMDCT and were evaluated at 2 years follow up using MRI T2-mapping sequence. 20 healthy volunteers were recruited as controls. MRI images were acquired using a protocol suggested by the International Cartilage Repair Society, MOCART scoring system and T2 mapping. Results were then correlated with AOFAS clinical score. Results: AOFAS score increased from 66.8 {+-} 14.5 pre-operatively to 91.2 {+-} 8.3 (p < 0.0005) at 2 years follow-up. T2-relaxation time value of 35-45 ms was derived from healthy ankles evaluation and assumed as normal hyaline cartilage value and used as a control. Regenerated tissue with a T2-relaxation time value comparable to hyaline cartilage was found in all the cases treated, covering a mean of 78% of the repaired lesion area. A high clinical score was related directly to isointense signal in DPFSE fat sat (p = 0.05), and percentage of regenerated hyaline cartilage (p = 0.05), inversely to the percentage of regenerated fibrocartilage. Lesion's depth negatively related to the integrity of the repaired tissue's surface (tau = -0.523, p = 0.007), and to the percentage of regenerated hyaline cartilage (rho = -0.546, p = 0.013). Conclusions: Because of its ability to detect cartilage's quality and to correlate to the clinical score, MRI T2-mapping sequence integrated with Mocart score represent a valid, non-invasive technique for qualitative cartilage assessment after regenerative surgical procedures.

  17. Effect of Chromatin-Remodeling Agents in Hepatic Differentiation of Rat Bone Marrow-Derived Mesenchymal Stem Cells In Vitro and In Vivo

    Directory of Open Access Journals (Sweden)

    Danna Ye

    2016-01-01

    Full Text Available Epigenetic events, including covalent histone modifications and DNA methylation, play fundamental roles in the determination of lineage-specific gene expression and cell fates. The aim of this study was to determine whether the DNA methyltransferase inhibitor (DNMTi 5-aza-2′-deoxycytidine (5-aza-dC and the histone deacetylase inhibitor (HDACi trichostatin A (TSA promote the hepatic differentiation of rat bone marrow-derived mesenchymal stem cells (rBM-MSCs and their therapeutic effect on liver damage. 1 μM TSA and 20 μM 5-aza-dC were added to standard hepatogenic medium especially at differentiation and maturation steps and their potential function on hepatic differentiation in vitro and in vivo was determined. Exposure of rBM-MSCs to 1 μM TSA at both the differentiation and maturation steps considerably improved hepatic differentiation. TSA enhanced the development of the hepatocyte shape, promoted the chronological expression of hepatocyte-specific markers, and improved hepatic functions. In contrast, treatment of rBM-MSCs with 20 μM 5-aza-dC alone or in combination with TSA was ineffective in improving hepatic differentiation in vitro. TSA and/or 5-aza-dC derived hepatocytes-like cells failed to improve the therapeutic potential in liver damage. We conclude that HDACis enhance hepatic differentiation in a time-dependent manner, while DNMTis do not induce the hepatic differentiation of rBM-MSCs in vitro. Their in vivo function needs further investigation.

  18. Transplantation of bone marrow-derived mesenchymal stem cells rescues partially rachitic phenotypes induced by 1,25-Dihydroxyvitamin D deficiency in mice

    Science.gov (United States)

    Zhang, Zengli; Yin, Shaomeng; Xue, Xian; Ji, Ji; Tong, Jian; Goltzman, David; Miao, Dengshun

    2016-01-01

    To determine whether the transplantation of bone marrow-derived mesenchymal stem cells (BM-MSCs) can improve the 1,25(OH)2D deficiency-induced rachitic phenotype, 2×106 BM-MSCs from wild-type mice or vehicle were transplanted by tail vein injection into mice deficient in 1,25(OH)2D due to targeted deletion of 1α(OH)ase (1α(OH)ase-/-). Our results show that 1α(OH)ase mRNA was expressed in the BM-MSCs derived from wild-type mice, and was detected in long bone, kidney and intestine from BM-MSC-transplanted 1α(OH)ase-/- recipients. Serum calcium, 1,25(OH)2D3 levels and body weight were significantly increased in BM-MSC-transplanted 1α(OH)ase-/- recipients compared to vehicle-treated 1α(OH)ase-/- mice. Skeletal mineralization improved in 1α(OH)ase-/- recipients as demonstrated by BMD measurement, micro-CT analysis and von Kossa staining of undecalcified sections. Expression levels of type I collagen, osteocalcin, bone sialoprotein and vitronectin and the size of calcified nodules were decreased in BM-MSC cultures from 1α(OH)ase-/- mice compared with those from wild-type mice, however, these parameters were increased in those from BM-MSCs-transplanted 1α(OH)ase-/- recipients compared with those from vehicle-treated 1α(OH)ase-/- mice. This study indicates that donor BM-MSCs cells can relocate to multiple tissues where they synthesize 1α(OH)ase and produce 1,25(OH)2D that contributes to the improvement of serum calcium and skeletal mineralization. Results from this study suggest that BM-MSC transplantation may provide a therapeutic approach to treatment of pseudovitamin D-deficiency rickets. PMID:27830022

  19. Inhibition of tubular cell proliferation by neutralizing endogenous HGF leads to renal hypoxia and bone marrow-derived cell engraftment in acute renal failure.

    Science.gov (United States)

    Ohnishi, Hiroyuki; Mizuno, Shinya; Nakamura, Toshikazu

    2008-02-01

    During the progression of acute renal failure (ARF), the renal tubular S3 segment is sensitive to ischemic stresses. For reversing tubular damage, resident tubular cells proliferate, and bone marrow-derived cells (BMDC) can be engrafted into injured tubules. However, how resident epithelium or BMDC are involved in tubular repair remains unknown. Using a mouse model of ARF, we examined whether hepatocyte growth factor (HGF) regulates a balance of resident cell proliferation and BMDC recruitment. Within 48 h post-renal ischemia, tubular destruction became evident, followed by two-waved regenerative events: 1) tubular cell proliferation between 2 and 4 days, along with an increase in blood HGF; and 2) appearance of BMDC in the tubules from 6 days postischemia. When anti-HGF IgG was injected in the earlier stage, tubular cell proliferation was inhibited, leading to an increase in BMDC in renal tubules. Under the HGF-neutralized state, stromal cell-derived factor-1 (SDF1) levels increased in renal tubules, associated with the enhanced hypoxia. Administrations of anti-SDF1 receptor IgG into ARF mice reduced the number of BMDC in interstitium and tubules. Thus possible cascades include 1) inhibition of tubular cell proliferation by neutralizing HGF leads to renal hypoxia and SDF1 upregulation; and 2) BMDC are eventually engrafted in tubules through SDF1-mediated chemotaxis. Inversely, administration of recombinant HGF suppressed the renal hypoxia, SDF1 upregulation, and BMDC engraftment in ARF mice by enhancing resident tubular cell proliferation. Thus we conclude that HGF is a positive regulator for eliciting resident tubular cell proliferation, and SDF1 for BMDC engraftment during the repair process of ARF.

  20. CO2 laser increases the regenerative capacity of human adipose-derived stem cells by a mechanism involving the redox state and enhanced secretion of pro-angiogenic molecules.

    Science.gov (United States)

    Constantin, Alina; Dumitrescu, Madalina; Mihai Corotchi, Maria Cristina; Jianu, Dana; Simionescu, Maya

    2017-01-01

    CO2 laser has a beneficial effect on stem cells by mechanisms that are not clearly elucidated. We hypothesize that the effect of fractional CO2 laser on human adipose-derived stem cells (ADSC) could be due to changes in redox homeostasis and secretion of factors contributing to cellular proliferation and angiogenic potential. ADSC incubated in medium containing 0.5 or 10 % FBS were exposed to a single irradiation of a 10,600-nm fractional CO2 laser; non-irradiated ADSC were used as control. Viability/proliferation of ADSC was assessed by MTT assay; the intracellular reactive oxygen species (ROS) levels and the mitochondrial membrane potential (∆Ψm) were determined with DCFH-DA and JC-1 fluorescent probes, respectively. Molecules secreted by ADSC in the medium were determined by ELISA assay, and their capacity to support endothelial tube-like formation by the Matrigel assay. The results showed that compared to controls, ADSC kept in low FBS medium and irradiated with CO2 laser at 9 W exhibited: (a) increased proliferation (∼20 %), (b) transient increase of mitochondrial ROS and the capacity to restore Δψm after rotenone induced depolarization, and (c) augmented secretion in the conditioned medium of MMP-2 (twofold), MMP-9 (eightfold), VEGF (twofold), and adiponectin (∼50 %) that have the capacity to support angiogenesis of endothelial progenitor cells. In conclusion, the mechanisms underlying the benefic effect of CO2 laser on ADSC are the activation of the redox pathways which increases cell proliferation and enhances secretion of angiogenic molecules. These results explain, in part, the mechanisms involved in the increased regenerative potential of CO2 laser-exposed ADSC that could be exploited for clinical applications.

  1. A stepwise procedure for isolation of murine bone marrow and generation of dendritic cells

    Directory of Open Access Journals (Sweden)

    Alka Madaan

    2014-02-01

    Full Text Available Bone marrow derived Dendritic cells (BMDCs are routinely employed in cell based assays to evaluate immunomodulatory and anti-inflammatory activities. Hence, simplified, stepwise, defined and standardized methods are required for isolation of bone marrow cells from mice, propagating them in presence of growth factors and obtaining high and reproducible yields of BMDCs. Here, we describe a detailed, stepwise protocol with pictorial representation to isolate bone marrow from mouse femur and development of dendritic cells. Mouse bone marrow cells are cultured in presence of granulocyte-macrophage colony stimulating factor (GM-CSF for 6 days to generate BMDCs.

  2. Protective effect of bone marrow-derived mesenchymal stem cells on dopaminergic neurons against 1-methyl-4-phenylpyridinium ion-induced neurotoxicity in rat brain slices

    Institute of Scientific and Technical Information of China (English)

    Lirong Jin; Zhen Hong; Chunjiu Zhong; Yang Wang

    2009-01-01

    BACKGROUND: To date, the use of bone marrow-derived mesenchymal stem cells (MSCs) for the treatment of Parkinson's disease have solely focused on in vivo animal models. Because of the number of influencing factors, it has been difficult to determine a consistent outcome. OBJECTIVE: To establish an injury model in brain slices of substantia nigra and striatum using 1-methyl-4-phenylpytidinium ion (MPP+), and to investigate the effect of MSCs on dopaminergic neurons following MPP+ induced damage.DESIGN, TIME AND SETTING: An in vitro, randomized, controlled, animal experiment using immunohistochemistry was performed at the Laboratory of the Department of Anatomy, Fudan University between January 2004 and December 2006.MATERIALS: Primary MSC cultures were obtained from femurs and tibias of adult Sprague Dawley rats. Organotypic brain slices were isolated from substantia nigra and striatum of 1-day-old Sprague Dawley rat pups. Monoclonal antibodies for tyrosine hydroxylase (TH, 1:5 000) were from Santa Cruz (USA); goat anti-rabbit IgG antibodies labeled with FITC were from Boster Company (China).METHODS: Organotypic brain slices were cultured for 5 days in whole culture medium supplemented with 50% DMEM, 25% equine serum, and 25% Tyrode's balanced salt solution. The medium was supplemented with 5 μg/mL Ara-C, and the culture was continued for an additional 5 days. The undergrowth of brain slices was discarded at day 10. Eugonic brain slices were cultured with basal media for an additional 7 days. The brain slices were divided into three groups: control, MPP+ exposure, and co-culture. For the MPP+ group, MPP+ (30 μmol/L) was added to the media at day 17 and brain slices were cultured for 4 days, followed by control media. For the co-culture group, the MPP+ injured brain slices were placed over MSCs in the well and were further cultured for 7 days.MAIN OUTCOME MEASURES: After 28 days in culture, neurite outgrowth was examined in the brain slices under phase

  3. 肌肉源性和骨髓源性干细胞成骨活性的比较研究%Osteogenic activity of muscle derived stem cells versus bone marrow derived stem cells:a ;comparative study

    Institute of Scientific and Technical Information of China (English)

    邹俊; 袁晨曦; 吴春屾; 曹成; 施勤; 杨惠林

    2015-01-01

    目的:本研究旨在探讨大鼠肌肉源性干细胞分离培养方法,及向成骨细胞分化的能力,并与传统的骨髓间充质干细胞相比较。方法从大鼠双下肢处获取骨骼肌肉组织,胶原酶消化分离,培养出肌肉源性干细胞。抽取骨髓组织,经过分离,培养出骨髓间充质干细胞。两种干细胞经基础培养基传至第二代时改换诱导培养基,向成骨细胞分化,采用CCK⁃8法检测两种细胞的增殖能力,矿化结节染色及RT⁃PCR检测比较向成骨细胞分化能力。结果两种细胞经过诱导后,均可以向成骨细胞方向分化。CCK⁃8检测显示肌肉源性干细胞细胞活性更强,矿化结节染色也有更多的结节形成。RT⁃PCR检测显示两种细胞ALP、Osteocalcin和Osteopontin均有表达,但肌肉源性干细胞表达量更高。结论大鼠骨骼肌肉组织可以成功分离培养出肌肉源性干细胞,通过诱导能够较好地向成骨细胞方向分化,有希望成为组织工程理想的种子细胞来源,成为骨髓间充质干细胞一种可供选择的替代。%Objective To investigate the isolation and culture ,and osteogenic differentiation of rat muscle derived stem cells ,and to compare those with the conventional bone marrow mesenchymal stem cells. Methods The skeletal muscle tissues of rat lower limbs were obtained and isolated by collagenase digestion to culture muscle derived stem cells. The bone marrow tissues were obtained and isolated to culture bone marrow mesenchymal stem cells. After transformed to the second generation,the two kinds of stem cells were cultured from basic medium to induction medium,and were differentiated to osteoblasts. The proliferation of the two stem cells was determined by CCK⁃8 method. Mineralized nodules staining and RT⁃PCR test were used to compare the osteogenic differentiation of the two stem cells. Results Both of the muscle derived stem cells and the conventional bone

  4. Adipocyte differentiation of human bone marrow-derived stromal cells is modulated by microRNA-155, microRNA-221, and microRNA-222.

    Science.gov (United States)

    Skårn, Magne; Namløs, Heidi M; Noordhuis, Paul; Wang, Meng-Yu; Meza-Zepeda, Leonardo A; Myklebost, Ola

    2012-04-10

    Human mesenchymal stromal cells (hMSCs) are capable of limited self-renewal and multilineage differentiation in vitro. Several studies have demonstrated that microRNAs (miRNAs, miRs), post-transcriptional modifiers of mRNA stability and protein translation, play crucial roles in the regulation of these complex processes. To gain knowledge regarding the role of miRNAs in human adipocyte differentiation, we examined the miRNA expression profile of the immortalized human bone marrow-derived stromal cell line hMSC-Tert20. Such a model system has the advantage of a reproducible and consistent phenotype while maintaining important properties of the primary donor cells, including the potential to differentiate to adipocytes, osteoblasts, and chondrocytes. We identified 12 miRNAs that were differentially expressed during adipogenesis, of which several have been previously shown to play important roles in adipocyte biology. Among these, the expression of miRNA-155, miRNA-221, and miRNA-222 decreased during the adipogenic program of both immortalized and primary hMSCs, suggesting that they act as negative regulators of differentiation. Interestingly, ectopic expression of the miRNAs significantly inhibited adipogenesis and repressed induction of the master regulators PPARγ and CCAAT/enhancer-binding protein alpha. Our study provides the first experimental evidence that miRNA-155, miRNA-221, and miRNA-222 have an important function in human adipocyte differentiation, and that their downregulation is necessary to relieve the repression of genes crucial for this process.

  5. Preferential magnetic nanoparticle uptake by bone marrow derived macrophages sub-populations: effect of surface coating on polarization, toxicity, and in vivo MRI detection

    Energy Technology Data Exchange (ETDEWEB)

    Al Faraj, Achraf, E-mail: aalfaraj@ksu.edu.sa [College of Applied Medical Sciences, King Saud University, Molecular and Cellular Imaging Lab, Department of Radiological Sciences (Saudi Arabia)

    2013-07-15

    Noninvasive imaging of macrophages activity has raised increasing interest for diagnosis of different diseases, which make them attractive vehicles to deliver contrast agents or drugs for diagnostic or therapeutic purposes. In this study, the effect of polyethylene glycol functionalization of magnetic iron oxide nanoparticles and their further surface modification with carboxylic groups on bone marrow-derived M1 and M2 macrophages phenotype, labeling efficiency, uptake mechanism, biocompatibility, and their in vivo MR detection was assessed. An enhanced labeling efficiency was observed for carboxylic surface-modified superparamagnetic iron oxide (SPIO) compared to PEGylated SPIO and to a higher extent to plain SPIO along with a higher uptake by M2 subsets. Magnetic nanoparticles were found located in the periphery of the vesicles dispersed in the cytoplasm in TEM. Investigation of the labeling mechanism by inhibiting different uptake pathways revealed that endocytosis via scavenger receptor A, a process known to be clathrin mediated, plays a central role in the cellular uptake kinetics of both macrophages subpopulations. Biocompatibility evaluation showed no variation in cell viability and mitochondrial membrane potential with a low release of ROS. Flow cytometry and measurement of iNOS and Arginase 1 activity as marker of M1 and M2 macrophages polarization confirmed that magnetic labeling of macrophages subsets did not affect their polarization. In addition, no variation was observed in the biodistribution of magnetic iron oxide-labeled M1 and M2 macrophages subsets when monitored using noninvasive magnetic resonance imaging with a better detection for the enhanced SPIO-PEG-COOH-labeled cells.

  6. Bone marrow-derived and peritoneal macrophages have different inflammatory response to oxLDL and M1/M2 marker expression – implications for atherosclerosis research

    Science.gov (United States)

    Bisgaard, Line S.; Mogensen, Christina K.; Rosendahl, Alexander; Cucak, Helena; Nielsen, Lars Bo; Rasmussen, Salka E.; Pedersen, Tanja X.

    2016-01-01

    Macrophages are heterogeneous and can polarize into specific subsets, e.g. pro-inflammatory M1-like and re-modelling M2-like macrophages. To determine if peritoneal macrophages (PEMs) or bone marrow derived macrophages (BMDMs) resembled aortic macrophages from ApoE−/− mice, their M1/M2 phenotype, inflammatory status, and lipid metabolism signatures were compared. oxLDL accumulation was similar in PEMs and BMDMs. On protein expression level, BMDMs showed an M2-like CD206highCD11clow profile, while cholesterol loading led to enhanced CD11c expression and reduced MCP-1 secretion. In contrast, PEMs expressed low levels of CD206 and CD11c, and responded to cholesterol loading by increasing CD11c expression and MCP-1 secretion. mRNA expression of M1/M2 markers was higher in PEMS than BMDMs, while lipid metabolism genes were similarly expressed. Whole aorta flow cytometry showed an accumulation of M2-like CD206highCD11clow macrophages in advanced versus early atherosclerotic disease in ApoE−/− mice. In isolated lesions, mRNA levels of the M2 markers Socs2, CD206, Retnla, and IL4 were downregulated with increasing disease severity. Likewise, mRNA expression of lipid metabolism genes (SREBP2, ACSL1, SRB1, DGAT1, and cpt1a) was decreased in advanced versus early lesions. In conclusion, PEMs and BMDMs are phenotypically distinct and differ from macrophages in lesions with respect to expression of M1/M2 markers and lipid metabolism genes. PMID:27734926

  7. Endocannabinoid System Contributes to Liver Injury and Inflammation by Activation of Bone Marrow-Derived Monocytes/Macrophages in a CB1-Dependent Manner.

    Science.gov (United States)

    Mai, Ping; Yang, Le; Tian, Lei; Wang, Lin; Jia, Shuangshuang; Zhang, Yuanyuan; Liu, Xin; Yang, Lin; Li, Liying

    2015-10-01

    Hepatic injury undergoes significant increases in endocannabinoidsand infiltrations of macrophages, yet the concrete mechanisms of changes in endocannabinoids and the functions of macrophage-expressed cannabinoid receptors (CBs) are unclear. Biosynthetic and degradative enzymes of endocannabinoids revealed a significant change in human fibrotic liver. Meanwhile, we showed dynamic changes of these enzymes and CBs (CB1 and CB2) from 1 to 56 d in carbon tetrachloride-induced murine liver injury. Biosynthetic enzymes (N-acylphosphatidyl-ethanolamine selective phospholipase D and diacylglycerol lipase-α) and CBs were markedly increased, whereas degradative enzymes (fatty acid amidohydrolase and monoacylglycerol lipase) were downregulated. Moreover, these enzymes intimately correlated with the fibrosis parameter [procollagen α1(III)]. Bone marrow-derived monocytes/macrophages (BMM) expressed CBs. Interestingly, CB1 but not CB2 mediated BMM migration through a Boyden chambers assay, and the effect depended on the G(α)i/o/RhoA/ROCK signaling pathway. ICR mice were lethally irradiated and received BM transplants from enhanced GFP transgenic mice. Four weeks later, mice of BM reconstruction were subjected to carbon tetrachloride-induced liver injury. In the chimeric murine model, we found that blockade of CB1 by administration of a CB1 antagonist inhibited the recruitment of BMM into injured liver using immunofluorescence staining and FACS, but it did not have effects on migration of T cells and dendritic cells without CB1 expression. Furthermore, activation of CB1 enhanced cytokine expression of BMM. In vivo, inhibition of CB1 attenuated the inflammatory cytokine level through real-time RT-PCR and cytometric bead array, ameliorating hepatic inflammation and fibrosis. In this study, we identify inactivation of BMM-expressed CB1 as a therapeutic strategy for reducing hepatic inflammation and fibrosis.

  8. Neuron-like differentiation of adult rat bone marrow stromal cells induced by transforming growth factor-beta and brain-derived neurotrophic factor

    Institute of Scientific and Technical Information of China (English)

    Chang Liu; Xifan Mei; Gang Lü; Yansong Wang; Quanshuang Li; Zhanpeng Guo

    2009-01-01

    BACKGROUND: It has been demonstrated that transforming growth factor-β (TGF-β) and brain-derived neurotrophic factor (BDNF) can induce stem cell differentiation into neuron-like cells.OBJECTIVE: To investigate the efficacy of TGF-β and BDNF at inducing the differentiation of adult rat bone marrow stromal cells (BMSCs) into neuron-like cells, both in combination or alone.DESIGN, TIME AND SETTING: A comparative observation experiment was performed at the Department of Orthopedics, First Affiliated Hospital of Liaoning Medical University between October 2007 and January 2008.MATERIALS: TGF-βand BDNF were purchased from Sigma, USA; mouse anti-rat neuron specific enolase, neurofilament and glial fibrillary acidic protein were purchased from Beijing HMHL Biochem Ltd., China.METHODS: BMSCs were isolated from rats aged 4 weeks and incubated with TGF-β(1μg/L) and/or BDNF (50μg/mL).MAIN OUTCOME MEASURES: Expression of neuron-specific enolase, neurofilament and glial fibrillary acidic protein were determined by immunocytochemistry.RESULTS: BMSCs differentiated into neuron-like cells following induction of TGF-β and BDNF, and expressed both neuron-specific enolase and neurofilament. The percent of positive cells was significantly greater in the combination group than those induced with TGF-β or BDNF alone (P<0.01).CONCLUSION: Treatment of BMSCs with a combination of TGF-β and BDNF induced differentiation into neuron-like cells, with the induction being significantly greater than with TGF-β or BDNF alone.

  9. Cell source-dependent in vivo immunosuppressive properties of mesenchymal stem cells derived from the bone marrow and synovial fluid of minipigs

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Won-Jae [College of Veterinary Medicine, Gyeongsang National University, Jinju 660-701, Gyeongnam (Korea, Republic of); Hah, Young-Sool [Biomedical Research Institute, Gyeongsang National University Hospital, Jinju (Korea, Republic of); Ock, Sun-A. [Animal Biotechnology Division, National Institute of Animal Science, RDA, Suwon 441-706, Gyeonggi (Korea, Republic of); Lee, Jae-Hoon; Jeon, Ryong-Hoon; Park, Ji-Sung [College of Veterinary Medicine, Gyeongsang National University, Jinju 660-701, Gyeongnam (Korea, Republic of); Lee, Sang-Il [Department of Internal Medicine and Institute of Health Sciences, Gyeongsang National University, Jinju (Korea, Republic of); Rho, Na-Young [Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada N1G 4S7 (Canada); Rho, Gyu-Jin [College of Veterinary Medicine, Gyeongsang National University, Jinju 660-701, Gyeongnam (Korea, Republic of); Research Institute of Life Sciences, Gyeongsang National University, Jinju 660-701, Gyeongnam (Korea, Republic of); Lee, Sung-Lim, E-mail: sllee@gnu.ac.kr [College of Veterinary Medicine, Gyeongsang National University, Jinju 660-701, Gyeongnam (Korea, Republic of); Research Institute of Life Sciences, Gyeongsang National University, Jinju 660-701, Gyeongnam (Korea, Republic of)

    2015-05-01

    The in vitro differentiation and immunosuppressive capacity of mesenchymal stem cells (MSCs) derived from synovial fluid (SF-MSCs) and bone marrow extract (BM-MSCs) in an isogenic background of minipigs were comparatively analyzed in a collagen-induced arthritis (CIA) mouse model of rheumatoid arthritis (RA). The proliferation capacity and expression of pluripotent transcription factors (Oct3/4 and Sox2) were significantly (P<0.05) higher in SF-MSCs than in BM-MSCs. The differentiation capacity of SF-MSCs into adipocytes, osteocytes and neurocytes was significantly (P<0.05) lower than that of BM-MSCs, and the differentiation capacity of SF-MSCs into chondrocytes was significantly (P<0.05) higher than that of BM-MSCs. Systemic injection of BM- and SF-MSCs significantly (P<0.05) ameliorated the clinical symptoms of CIA mice, with SF-MSCs having significantly (P<0.05) higher clinical and histopathological recovery scores than BM-MSCs. Furthermore, the immunosuppressive properties of SF-MSCs in CIA mice were associated with increased levels of the anti-inflammatory cytokine interleukin (IL)-10, and decreased levels of the pro-inflammatory cytokine IL-1β and osteoclast-related sRANKL. In conclusion, SF-MSCs exhibited eminent pluripotency and differentiation capacity into chondrocytes, addition to substantial in vivo immunosuppressive capacity by elevating IL-10 and reducing IL-1β levels in CIA mice. - Highlights: • Immunosuppressive capacity of BM-, SM-, and SF-MSCs was evaluated in an RA model. • Proliferation, pluripotency and chondrogenic differentiation capacity were higher in SF-MSCs. • SF-MSCs exhibited improved therapeutic effects than BM-MSCs. • SF-MSCs may have applications as immunosuppressive therapy in autoimmune diseases.

  10. Brain-derived neurotrophic factor gene delivery in an animal model of multiple sclerosis using bone marrow stem cells as a vehicle.

    Science.gov (United States)

    Makar, Tapas K; Bever, Christopher T; Singh, Ishwar S; Royal, Walter; Sahu, Surasri Nandan; Sura, Tushar P; Sultana, Shireen; Sura, Karna T; Patel, Niraj; Dhib-Jalbut, Suhayl; Trisler, David

    2009-05-29

    Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, is neuroprotective in animal models of neurodegenerative diseases. However, BDNF has a short half-life and its efficacy in the central nervous system (CNS), when delivered peripherally, is limited due to the blood-brain barrier (BBB). We have developed a means of delivering BDNF into the CNS using genetically engineered bone marrow stem cells (BMSCs) as a vehicle, and have explored the clinical effects of BDNF on outcomes in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). BDNF-engineered-BMSCs were transplanted (i.v.) into irradiated 2-week-old SJL/J female mice. Eight weeks after transplantation, mice were immunized with a peptide of proteolipid protein (PLP(139-151)). Mice, which had received BDNFengineered BMSCs, showed a significant delay in EAE onset and a reduction in overall clinical severity compared to mice receiving BMSC transfected with an empty vector lacking the BDNF gene. In addition, pathological examination showed that BDNF delivery reduced demyelination and increased remyelination. Inhibition of pro-inflammatory cytokines TNF-alpha and IFN-gamma and enhanced expression of the antiinflammatory cytokines IL-4, IL-10, and IL-11 were found in the CNS tissues of the BDNF transplanted group. These results support the use of BMSCs as vehicles to deliver BDNF into the CNS of EAE animals. This is a potentially novel therapeutic approach that might be used to deliver BDNF gene or genes for other therapeutic proteins into the CNS in MS or in other diseases of the CNS in which accessibility of therapeutic proteins is limited due to the BBB.

  11. Neural Differentiation of Mouse Bone Marrow-Derived Mesenchymal Stem Cells Treated with Sex Steroid Hormones and Basic Fibroblast Growth Factor

    Directory of Open Access Journals (Sweden)

    Kazem Parivar

    2015-04-01

    Full Text Available Objective: There are several factors, like environmental agents, neurotrophic factors, serotonin and some hormones such as estrogen, affecting neurogenesis and neural differentiation. Regarding to importance of proliferation and regeneration in central nervous system, and a progressive increase in neurodegenerative diseases, cell therapy is an attractive approach in neuroscience. The aim of the present study was to investigate the effects of sex steroid hormones and basic fibroblast growth factor (bFGF on neuronal differentiation of mouse bone marrow-derived mesenchymal stem cells (BM-MSCs. Materials and Methods: This experimental study was established in Kharazmi University. BM was isolated from the bones of femur and tibia of 4-6-week old Naval Medical Research Institute (NMRI mice, and the cells were cultured. The cells were divided into following 4 groups based on the applied treatments: I. control (no treatment, II. steroid hormones (β-estradiol, progesterone and testosterone, III. bFGF and IV. combination of steroid hormones and bFGF. Immunocytochemistry and flow cytometery analyses were applied for beta III-tubulin (β-III tubulin and microtubule-associated proteins-2 (MAP-2 in 4 days of treatment for all groups. Results: The cells treated with combination of bFGF and steroid hormones represented more expressions of neural markers as compared to control and to other two groups treated with either bFGF or steroid hormones. Conclusion: This study showed that BM-MSCs can express specific neural markers after receiving bFGF pretreatment that was followed by sex steroid hormones treatment. More investigations are necessary to specify whether steroid hormones and bFGF can be considered for treatment of CNS diseases and disorders.

  12. Sphingosine-1-phosphate/S1P receptors signaling modulates cell migration in human bone marrow-derived mesenchymal stem cells.

    Science.gov (United States)

    Kong, Yaxian; Wang, Hong; Lin, Tao; Wang, Shuling

    2014-01-01

    The recruitment of bone marrow-derived mesenchymal stem cells (BMSCs) to damaged tissues and sites of inflammation is an essential step for clinical therapy. However, the signals regulating the motility of these cells are still not fully understood. Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, is known to have a variety of biological effects on various cells. Here, we investigated the roles of S1P and S1P receptors (S1PRs) in migration of human BMSCs. We found that S1P exerted a powerful migratory action on human BMSCs. Moreover, by employing RNA interference technology and pharmacological tools, we demonstrated that S1PR1 and S1PR3 are responsible for S1P-induced migration of human BMSCs. In contrast, S1PR2 mediates the inhibition of migration. Additionally, we explored the downstream signaling pathway of the S1P/S1PRs axis and found that activation of S1PR1 or S1PR3 increased migration of human BMSCs through a G i /extracellular regulated protein kinases 1/2- (ERK1/2-) dependent pathway, whereas activation of S1PR2 decreased migration through the Rho/Rho-associated protein kinase (ROCK) pathway. In conclusion, we reveal that the S1P/S1PRs signaling axis regulates the migration of human BMSCs via a dual-directional mechanism. Thus, selective modulation of S1PR's activity on human BMSCs may provide an effective approach to immunotherapy or tissue regeneration.

  13. Heparin improves BMSC cell therapy: Anticoagulant treatment by heparin improves the safety and therapeutic effect of bone marrow-derived mesenchymal stem cell cytotherapy

    Science.gov (United States)

    Liao, Li; Shi, Bingzheng; Chang, Heran; Su, Xiaoxia; Zhang, Lichao; Bi, Chunsheng; Shuai, Yi; Du, Xiaoyan; Deng, Zhihong; Jin, Yan

    2017-01-01

    Systemic infusion of bone marrow-derived mesenchymal stem cells (BMSCs) has become a promising strategy for disease treatment and tissue regeneration. Strategies to enhance the efficiency of BMSC cell therapy are crucial to promote its clinical application. Here, we aimed to improve BMSC cell therapy by inhibiting the BMSC-induced coagulation reaction. Intravenous injection of gradient BMSCs into mice showed that BMSCs were not fully compatible with blood. Large doses of BMSCs induced a series of symptoms of respiratory failure and heart failure. Histological and homeostasis analysis confirmed that large doses of BMSCs induced disseminated intravascular thrombosis, exhaustion of platelets and coagulation factors, and prolonged prothrombin time (PT) and activated partial thromboplastin time (APTT). Similar to mouse BMSCs, goat and human BMSCs also induced coagulation reactions in vitro and in vivo. The coagulation was induced mostly by tissue factor, the overexpression of which enhanced the procoagulant activity of BMSCs during in vitro culture. Notably, clinical doses of BMSCs in cell therapy also induced mild and reversible coagulation, which increased BMSC lung embolism and clearance. Anticoagulation treatment by heparin (400 U/kg) prevented BMSC-induced coagulation and the acute adverse effects of large-dose BMSCs infusion efficiently. Importantly, heparin treatment led to decreased BMSC lung embolism and enhanced migration and maintenance of BMSCs to target organs in cell therapy. Based on an experimental colitis model, we confirmed that heparin treatment enhanced the effect of BMSC therapy efficiently to reduce mortality, prevent weight loss, suppress inflammation reaction and alleviate tissue injury. In conclusion, BMSCs possess procoagulant activity that could induce disseminated coagulation and thrombosis in recipients. Anticoagulation treatment by heparin is a practical strategy to improve both the safety and therapeutic effect of BMSC therapy. PMID

  14. Mast cell growth-enhancing activity (MEA) stimulates interleukin 6 production in a mouse bone marrow-derived mast cell line and a malignant subline.

    Science.gov (United States)

    Hültner, L; Moeller, J

    1990-09-01

    A novel mast cell growth-enhancing activity (MEA/P40/interleukin 9 [IL-9]) purified from the conditioned medium of a murine interleukin 2 (IL-2)-dependent Mlsa-specific T-cell line (MLS4.2) was tested for its capacity to induce interleukin 6 (IL-6) production in a mouse bone marrow-derived factor-dependent mast cell line (L138.8A). This interleukin 3 (IL-3)/interleukin 4 (IL-4)/MEA-responsive cell line was demonstrated recently to express IL-6 mRNA and to secrete IL-6 when cultured with IL-3/IL-4. Now we were able to show that conditioned medium from L138.8A mast cells stimulated with MEA alone contained growth factor activity for the IL-6-dependent mouse hybridoma cell line 7TD1 that was completely blocked by the monoclonal anti-IL-6 antibody 6B4. A dose-response study including IL-3, IL-4, and MEA tested either alone or in different combinations revealed that among these growth factors MEA was the most potent inducer of IL-6 in L138.8A cells. Moreover, IL-4 but not IL-3 had a strong synergistic effect on MEA-induced IL-6 production. The autonomous malignant mast cell subline L138Cauto also showed enhanced IL-6 production when stimulated with MEA. Our findings indicate that MEA (IL-9) not only provides a proliferation signal, but also leads to a marked functional activation of responsive mast cells.

  15. TNF-α Inhibits FoxO1 by Upregulating miR-705 to Aggravate Oxidative Damage in Bone Marrow-Derived Mesenchymal Stem Cells during Osteoporosis.

    Science.gov (United States)

    Liao, Li; Su, Xiaoxia; Yang, Xiaohong; Hu, Chenghu; Li, Bei; Lv, Yajie; Shuai, Yi; Jing, Huan; Deng, Zhihong; Jin, Yan

    2016-04-01

    Decline of antioxidant defense after estrogen deficiency leads to oxidative damage in bone marrow-derived mesenchymal stem cells (BMMSCs), resulting a defect of bone formation in osteoporosis. Forkhead box O1 (FoxO1) protein is crucial for defending physiological oxidative damage in bone. But whether FoxO1 is involved in the oxidative damage during osteoporosis is largely unknown. In this study, we found that FoxO1 protein accumulation was decreased in BMMSCs of ovariectomized mice. The decrease of FoxO1 resulted in the suppression of manganese superoxide dismutase (Sod2) and catalase (Cat) expression and accumulation of reactive oxygen species (ROS), inhibiting the osteogenic differentiation of BMMSCs. The decline of FoxO1 protein was caused by tumor necrosis factor-alpha (TNF-α) accumulated after estrogen deficiency. Mechanistically, TNF-α activated NF-κB pathway to promote microRNA-705 expression, which function as a repressor of FoxO1 through post-transcriptional regulation. Inhibition of NF-κB pathway or knockdown of miR-705 largely prevented the decline of FoxO1-mediated antioxidant defense caused by TNF-α and ameliorated the oxidative damage in osteoporotic BMMSCs. Moreover, the accumulated ROS further activated NF-κB pathway with TNF-α, which formed a feed-forward loop to persistently inhibiting FoxO1 protein accumulation in BMMSCs. In conclusion, our study revealed that the decline of FoxO1 is an important etiology factor of osteoporosis and unclosed a novel mechanism of FoxO1 regulation by TNF-α. These findings suggested a close correlation between inflammation and oxidative stress in stem cell dysfunction during degenerative bone diseases.

  16. Comparison of the behavior of fibroblast and bone marrow-derived mesenchymal stem cell on nitrogen plasma-treated gelatin films

    Energy Technology Data Exchange (ETDEWEB)

    Prasertsung, I. [Chemical Engineering Program, Department of Industrial Engineering, Faculty of Engineering, Naresuan University, Phitsanulok 65000 (Thailand); Research Unit on Functionalized Material for Chemical, Biochemical and Biomedical Technology, Faculty of Engineering, Naresuan University, Phitsanulok 65000 (Thailand); Kanokpanont, S. [Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Bangkok 10330 (Thailand); Mongkolnavin, R. [Department of Physics, Faculty of Science, Chulalongkorn University, Bangkok 10330 (Thailand); Plasma Technology and Nuclear Fusion Research Unit, Chulalongkorn University, Bangkok 10330 (Thailand); Wong, C.S. [Plasma Technology Research Centre, Department of Physics, Faculty of Science, University of Malaya, 50603 Kuala Lumpur (Malaysia); Panpranot, J. [Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Bangkok 10330 (Thailand); Damrongsakkul, S., E-mail: siriporn.d@chula.ac.th [Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Bangkok 10330 (Thailand); Plasma Technology and Nuclear Fusion Research Unit, Chulalongkorn University, Bangkok 10330 (Thailand)

    2013-10-01

    The attachment and growth behavior of mouse fibroblast (L929) and rat bone marrow-derived mesenchymal stem cell (MSC) on nitrogen plasma-treated and untreated gelatin films was investigated and compared. The gelatin films were prepared by solution casting (0.05% w/v) and crosslinked using dehydrothermal treatment. The crosslinked gelatin films were treated with nitrogen alternating current (AC) 50 Hz plasma systems at various treatment time. The results on the attachment and growth of two cells; L929 and MSC, on plasma-treated gelatin film showed that the number of attached and proliferated cells on plasma-treated gelatin films was significantly increased compared to untreated samples. However, no significant difference between the number of attached L929 and MSC on plasma-treated gelatin was observed. The shorter population doubling time and higher growth rate of cells cultured on plasma-treated film indicated the greater growth of cells, compared to ones on untreated films. The greatest enhancement of cell attachment and growth were noticed when the film was treated with nitrogen plasma for 9 to 15 s. This suggested that the greater attachment and growth of both cells on gelatin films resulted from the change of surface properties, i.e. hydrophilicity, surface energy, and chemistry. The suitable water contact angle and oxygen/nitrogen ratio (O/N) of gelatin film for best L929 and MSC attachment were observed at 27–32° and 1.4, respectively. These conditions also provided the best proliferation of cells on plasma-treated gelatin films. - Highlights: • We compared the attachment and growth behavior of L929 and MSC. • The attachment of two cells on plasma-treated gelatin was significantly increased. • The shorter population doubling time and higher growth rate of cells were observed. • L929 fibroblast exhibited the greater proliferation, compared to MSC.

  17. Choose your models wisely: how different murine bone marrow-derived dendritic cell protocols influence the success of nanoparticulate vaccines in vitro.

    Science.gov (United States)

    Dewitte, Heleen; Verbeke, Rein; Breckpot, Karine; Vandenbroucke, Roosmarijn E; Libert, Claude; De Smedt, Stefaan C; Lentacker, Ine

    2014-12-10

    Dendritic cell (DC)-based cancer vaccination has shown great potential in cancer immunotherapy. As a result, novel nanoparticles aiming to load DCs with tumor antigens are being developed and evaluated in vitro. For this, murine bone marrow-derived DCs (BM-DCs) are most commonly used as model DCs. However, many different protocols exist to generate these cells. Therefore, we investigated to what extent different BM-DC culture protocols impact on the immunobiology of the cells, as well as their response to particulate antigens. We evaluated 4 different BM-DC protocols with 2 main variables: bovine serum and cytokine combinations. Our results show distinct differences in yield, phenotypical maturation status and the production of immune stimulatory and immune suppressive cytokines by the different BM-DCs. Importantly, we demonstrate that the antigen-loading of these different BM-DCs via transfection with mRNA lipoplexes results in large differences in transfection efficiency as well as in the capacity of mRNA-transfected BM-DCs to stimulate antigen-specific T cells. Thus, it is clear that the BM-DC model can have significant confounding effects on the evaluation of novel nanoparticulate vaccines. To take this into account when testing novel particulate antigen-delivery systems in BM-DC models, we propose to (1) perform a thorough immunological characterization of the BM-DCs and to (2) not only judge a particle's potential for cancer vaccination based on transfection efficiency, but also to include an evaluation of functional end-points such as T cell activation.

  18. Topographical and biological evidence revealed FTY720-mediated anergy-polarization of mouse bone marrow-derived dendritic cells in vitro.

    Science.gov (United States)

    Zeng, Xiangfeng; Wang, Tong; Zhu, Cairong; Xing, Xiaobo; Ye, Yanxia; Lai, Xinqiang; Song, Bing; Zeng, Yaoying

    2012-01-01

    Abnormal inflammations are central therapeutic targets in numerous infectious and autoimmune diseases. Dendritic cells (DCs) are involved in these inflammations, serving as both antigen presenters and proinflammatory cytokine providers. As an immuno-suppressor applied to the therapies of multiple sclerosis and allograft transplantation, fingolimod (FTY720) was shown to affect DC migration and its crosstalk with T cells. We posit FTY720 can induce an anergy-polarized phenotype switch on DCs in vitro, especially upon endotoxic activation. A lipopolysaccharide (LPS)-induced mouse bone marrow-derived dendritic cell (BMDC) activation model was employed to test FTY720-induced phenotypic changes on immature and mature DCs. Specifically, methods for morphology, nanostructure, cytokine production, phagocytosis, endocytosis and specific antigen presentation studies