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Sample records for human marrow stromal

  1. CD146/MCAM defines functionality of human bone marrow stromal stem cell populations

    DEFF Research Database (Denmark)

    Harkness, Linda; Zaher, Walid; Ditzel, Nicholas

    2016-01-01

    BACKGROUND: Identification of surface markers for prospective isolation of functionally homogenous populations of human skeletal (stromal, mesenchymal) stem cells (hMSCs) is highly relevant for cell therapy protocols. Thus, we examined the possible use of CD146 to subtype a heterogeneous hMSC...... population. METHODS: Using flow cytometry and cell sorting, we isolated two distinct hMSC-CD146(+) and hMSC-CD146(-) cell populations from the telomerized human bone marrow-derived stromal cell line (hMSC-TERT). Cells were examined for differences in their size, shape and texture by using high...... and adipocytes on the basis of gene expression and protein production of lineage-specific markers. In vivo, hMSC-CD146(+) and hMSC-CD146(-) cells formed bone and bone marrow organ when implanted subcutaneously in immune-deficient mice. Bone was enriched in hMSC-CD146(-) cells (12.6 % versus 8.1 %) and bone...

  2. 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.

  3. Legumain Regulates Differentiation Fate of Human Bone Marrow Stromal Cells and Is Altered in Postmenopausal Osteoporosis

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    Abbas Jafari

    2017-02-01

    Full Text Available Secreted factors are a key component of stem cell niche and their dysregulation compromises stem cell function. Legumain is a secreted cysteine protease involved in diverse biological processes. Here, we demonstrate that legumain regulates lineage commitment of human bone marrow stromal cells and that its expression level and cellular localization are altered in postmenopausal osteoporotic patients. As shown by genetic and pharmacological manipulation, legumain inhibited osteoblast (OB differentiation and in vivo bone formation through degradation of the bone matrix protein fibronectin. In addition, genetic ablation or pharmacological inhibition of legumain activity led to precocious OB differentiation and increased vertebral mineralization in zebrafish. Finally, we show that localized increased expression of legumain in bone marrow adipocytes was inversely correlated with adjacent trabecular bone mass in a cohort of patients with postmenopausal osteoporosis. Our data suggest that altered proteolytic activity of legumain in the bone microenvironment contributes to decreased bone mass in postmenopausal osteoporosis.

  4. Multiple intracellular signaling pathways orchestrate adipocytic differentiation of human bone marrow stromal stem cells

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    Ayesh Hafez Ali, Dalia; Abuelreich, Sarah; Alkeraishan, Nora

    2018-01-01

    during adipocyte differentiation of human bone marrow stromal (mesenchymal) stem cells (hMSCs) and identified 2,589 up-regulated and 2,583 down-regulated mRNA transcripts. Pathway analysis on the up-regulated gene list untraveled enrichment in multiple signaling pathways including insulin receptor......Bone marrow adipocyte formation plays a role in bone homeostasis and whole body energy metabolism. However, the transcriptional landscape and signaling pathways associated with adipocyte lineage commitment and maturation are not fully delineated. Thus, we performed global gene expression profiling...... signaling, focal Adhesion, metapathway biotransformation, a number of metabolic pathways e.g. selenium metabolism, Benzo(a)pyrene metabolism, fatty acid, triacylglycerol, ketone body metabolism, tryptophan metabolism, and catalytic cycle of mammalian flavin-containing monooxygenase (FMOs). On the other hand...

  5. SERPINB2 is a novel TGFβ-responsive lineage fate determinant of human bone marrow stromal cells

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    Elsafadi, Mona; Manikandan, Muthurangan; Atteya, Muhammad

    2017-01-01

    TGF-β1, a multifunctional regulator of cell growth and differentiation, is the most abundant bone matrix growth factor. During differentiation of human bone stromal cells (hBMSCs), which constitute bone marrow osteoblast (OS) and adipocyte (AD) progenitor cells, continuous TGF-β1 (10 ng/ml) treat...

  6. 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.

  7. Pleiotrophin commits human bone marrow mesenchymal stromal cells towards hypertrophy during chondrogenesis.

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    Bouderlique, Thibault; Henault, Emilie; Lebouvier, Angelique; Frescaline, Guilhem; Bierling, Phillipe; Rouard, Helene; Courty, José; Albanese, Patricia; Chevallier, Nathalie

    2014-01-01

    Pleiotrophin (PTN) is a growth factor present in the extracellular matrix of the growth plate during bone development and in the callus during bone healing. Bone healing is a complicated process that recapitulates endochondral bone development and involves many cell types. Among those cells, mesenchymal stromal cells (MSC) are able to differentiate toward chondrogenic and osteoblastic lineages. We aimed to determine PTN effects on differentiation properties of human bone marrow stromal cells (hBMSC) under chondrogenic induction using histological analysis and quantitative reverse transcription polymerase chain reaction. PTN dramatically potentiated chondrogenic differentiation as indicated by a strong increase of collagen 2 protein, and cartilage-related gene expression. Moreover, PTN increased transcription of hypertrophic chondrocyte markers such as MMP13, collagen 10 and alkaline phosphatase and enhanced calcification and the content of collagen 10 protein. These effects are dependent on PTN receptors signaling and PI3 K pathway activation. These data suggest a new role of PTN in bone regeneration as an inducer of hypertrophy during chondrogenic differentiation of hBMSC.

  8. Pleiotrophin commits human bone marrow mesenchymal stromal cells towards hypertrophy during chondrogenesis.

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    Thibault Bouderlique

    Full Text Available Pleiotrophin (PTN is a growth factor present in the extracellular matrix of the growth plate during bone development and in the callus during bone healing. Bone healing is a complicated process that recapitulates endochondral bone development and involves many cell types. Among those cells, mesenchymal stromal cells (MSC are able to differentiate toward chondrogenic and osteoblastic lineages. We aimed to determine PTN effects on differentiation properties of human bone marrow stromal cells (hBMSC under chondrogenic induction using histological analysis and quantitative reverse transcription polymerase chain reaction. PTN dramatically potentiated chondrogenic differentiation as indicated by a strong increase of collagen 2 protein, and cartilage-related gene expression. Moreover, PTN increased transcription of hypertrophic chondrocyte markers such as MMP13, collagen 10 and alkaline phosphatase and enhanced calcification and the content of collagen 10 protein. These effects are dependent on PTN receptors signaling and PI3 K pathway activation. These data suggest a new role of PTN in bone regeneration as an inducer of hypertrophy during chondrogenic differentiation of hBMSC.

  9. Human Stromal (Mesenchymal) Stem Cells from Bone Marrow, Adipose Tissue and Skin Exhibit Differences in Molecular Phenotype and Differentiation Potential

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    Al-Nbaheen, May; Vishnubalaji, Radhakrishnan; Ali, Dalia

    2013-01-01

    Human stromal (mesenchymal) stem cells (hMSCs) are multipotent stem cells with ability to differentiate into mesoderm-type cells e.g. osteoblasts and adipocytes and thus they are being introduced into clinical trials for tissue regeneration. Traditionally, hMSCs have been isolated from bone marrow......, but the number of cells obtained is limited. Here, we compared the MSC-like cell populations, obtained from alternative sources for MSC: adipose tissue and skin, with the standard phenotype of human bone marrow MSC (BM-MSCs). MSC from human adipose tissue (human adipose stromal cells (hATSCs)) and human skin......, MSC populations obtained from different tissues exhibit significant differences in their proliferation, differentiation and molecular phenotype, which should be taken into consideration when planning their use in clinical protocols....

  10. Interleukin-1β modulates endochondral ossification by human adult bone marrow stromal cells

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    M Mumme

    2012-09-01

    Full Text Available Inflammatory cytokines present in the milieu of the fracture site are important modulators of bone healing. Here we investigated the effects of interleukin-1β (IL-1β on the main events of endochondral bone formation by human bone marrow mesenchymal stromal cells (BM-MSC, namely cell proliferation, differentiation and maturation/remodelling of the resulting hypertrophic cartilage. Low doses of IL-1β (50 pg/mL enhanced colony-forming units-fibroblastic (CFU-f and -osteoblastic (CFU-o number (up to 1.5-fold and size (1.2-fold in the absence of further supplements and glycosaminoglycan accumulation (1.4-fold upon BM-MSC chondrogenic induction. In osteogenically cultured BM-MSC, IL-1β enhanced calcium deposition (62.2-fold and BMP-2 mRNA expression by differential activation of NF-κB and ERK signalling. IL-1β-treatment of BM-MSC generated cartilage resulted in higher production of MMP-13 (14.0-fold in vitro, mirrored by an increased accumulation of the cryptic cleaved fragment of aggrecan, and more efficient cartilage remodelling/resorption after 5 weeks in vivo (i.e., more TRAP positive cells and bone marrow, less cartilaginous areas, resulting in the formation of mature bone and bone marrow after 12 weeks. In conclusion, IL-1β finely modulates early and late events of the endochondral bone formation by BM-MSC. Controlling the inflammatory environment could enhance the success of therapeutic approaches for the treatment of fractures by resident MSC and as well as improve the engineering of implantable tissues.

  11. Retrovirus-mediated gene transfer of a human c-fos cDNA into mouse bone marrow stromal cells.

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    Roux, P; Verrier, B; Klein, B; Niccolino, M; Marty, L; Alexandre, C; Piechaczyk, M

    1991-11-01

    A cDNA encoding a complete human c-fos protein was isolated and inserted into two different murine MoMuLV-derived recombinant retroviruses allowing expression of c-fos protein in different cell types. One c-fos-expressing retrovirus, chosen for its ability to express high levels of proteins in fibroblast-like cells, was shown to potentiate long-term cultures of mouse bone marrow stromal cells in vitro and therefore constitutes a potential tool for immortalizing such cells. Moreover, when tested in an in vitro differentiation assay, stromal cells constitutively expressing c-fos favor the granulocyte differentiation of hematopoietic precursors. Interestingly, retroviruses expressing v-src and v-abl oncogenes, included as controls in our experiments, do not produce any detectable effects, whereas those expressing polyoma virus middle T antigen facilitate long-term growth in vitro of stromal cells that favor the macrophage differentiation pathway of bone marrow stem cells. Our observation supports the idea that constitutive expression of some oncogenes, including c-fos and polyoma virus middle T antigen, may influence cytokine production by bone marrow stromal cells.

  12. Telomerase reverse transcriptase mediated immortalization of human bone marrow stromal cells

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    Yong Teng

    2014-02-01

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

  13. Nicotine-induced chondrogenic differentiation of human bone marrow stromal cells in vitro.

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    Ying, Xiaozhou; Zhang, Wei; Cheng, Shaowen; Nie, Pengfei; Cheng, Xiaojie; Shen, Yue; Wang, Wei; Xue, Enxing; Chen, Qingyu; Kou, Dongquan; Peng, Lei; Zhang, Yu; Lu, Chuanzhu

    2012-11-01

    Nicotine has been reported that it has a dose-dependent effect on matrix mineralization by human bone marrow cells. However, there is no relevant research concerning on chondrogenic differentiation potential of bone marrow stromal stem cells (BMSCs) treated with nicotine in vitro. The aims of the study were to examine the effects of nicotine (0, 10(-7), 10(-6) and 10(-5) M) on the proliferation and chondrogenic differentiation of BMSCs from three healthy donors in vitro. BMSCs proliferation was analyzed by CCK8 assay and real-time polymerase chain reaction was used to assay the expression of type II collagen, aggrecan, type I collagen and type X collagen. The proteoglycan content was stained by Alcian blue, and the sulfated glycosaminoglycan (sGAG) content of BMSCs was quantified spectrofluorometrically using dimethylmethylene blue. The cell viability was not significantly impaired until up to a concentration of 10(-5) M nicotine. Nicotine promoted the proliferation and enhanced the expression of type II collagen at the level up to 10(-6) M (P < 0.05). The expression of aggrecan was reduced at the concentration of 10(-5) M nicotine at day 14 (P < 0.05), and there was no significant difference in aggrecan gene expression at 10(-7) and 10(-6) M nicotine levels compared to control group (n.s.). Also the fibroblastic and hypertrophic gene expressions were down-regulated in the chondrogenic medium with 10(-7)-10(-5) M nicotine (P < 0.05). It was implied that local application of nicotine at an appropriate concentration may be a promising approach for enhancing chondrogenic differentiation capacity of BMSCs in cell-based cartilage tissue engineering. Also these results indicate that nicotine maybe a potentially useful drug for the treatment of Osteoarthritis.

  14. Effect of boron on osteogenic differentiation of human bone marrow stromal cells.

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    Ying, Xiaozhou; Cheng, Shaowen; Wang, Wei; Lin, Zhongqin; Chen, Qingyu; Zhang, Wei; Kou, Dongquan; Shen, Yue; Cheng, Xiaojie; Rompis, Ferdinand An; Peng, Lei; Zhu Lu, Chuan

    2011-12-01

    Bone marrow stromal cells (BMSCs) have been well established as an ideal source of cell-based therapy for bone tissue engineering applications. Boron (B) is a notable trace element in humans; so far, the effects of boron on the osteogenic differentiation of BMSCs have not been reported. The aim of this study was to evaluate the effects of boron (0, 1, 10,100, and 1,000 ng/ml) on osteogenic differentiation of human BMSCs. In this study, BMSCs proliferation was analyzed by cell counting kit-8 (CCK8) assay, and cell osteogenic differentiation was evaluated by alkaline phosphatase (ALP) activity assay, Von Kossa staining, and real-time PCR. The results indicated that the proliferation of BMSCs was no different from the control group when added with B at the concentration of 1, 10, and 100 ng/ml respectively (P > 0.05); in contrast, 1,000 ng/ml B inhibited the proliferation of BMSCs at days 4, 7, and 14 (P < 0.05). By ALP staining, we discovered that BMSCs treated with 10 and 100 ng/ml B presented a higher ALP activity compared with control (P < 0.05). By real-time PCR, we detected the messenger RNA expression of ALP, osteocalcin, collagen type I, and bone morphogenetic proteins 7 were also increased in 10 and 100 ng/ml B treatment groups (P < 0.05). The calcium depositions were increased in 1 and 10 ng/ml B treatment groups (P < 0.05). Taken all together, it was the first time to report that B could increase osteogenic effect by stimulating osteogenic differentiation-related marker gene synthesis during the proliferation and differentiation phase in human BMSCs and could be a promising approach for enhancing osteogenic capacity of cell-based construction in bone tissue engineering.

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

    International Nuclear Information System (INIS)

    Yang, Yingbin; Cai, Shaoxi; Yang, Li; Yu, Shuhui; Jiang, Jiahuan; Yan, Xiaoqing; Zhang, Haoxing; Liu, Lan; Liu, Qun; Du, Jun; Cai, Shaohui; Sung, K.L. Paul

    2010-01-01

    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.

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

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    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.

  17. 1,25-Dihydroxyvitamin D3 stimulates the production of insulin-like growth factor-binding proteins-2, -3 and -4 in human bone marrow stromal cells

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    Kveiborg, Marie; Flyvbjerg, Allan; Eriksen, E F

    2001-01-01

    1,25-Dihydroxyvitamin D3 (calcitriol) inhibits proliferation and stimulates differentiation of multiple cell types, including osteoblasts. Human (h) bone marrow stromal cells (MSCs) are a homogenous non-hematopoietic population of cells present in the bone marrow and exhibit a less differentiated...

  18. Ameliorating replicative senescence of human bone marrow stromal cells by PSMB5 overexpression

    International Nuclear Information System (INIS)

    Lu, Li; Song, Hui-Fang; Wei, Jiao-Long; Liu, Xue-Qin; Song, Wen-Hui; Yan, Ba-Yi; Yang, Gui-Jiao; Li, Ang; Yang, Wu-Lin

    2014-01-01

    Highlights: • PSMB5 overexpression restores the differentiation potential of aged hBMSCs. • PSMB5 overexpression enhances the proteasomal activity of late-stage hBMSCs. • PSMB5 overexpression inhibits replicative senescence and improved cell viability. • PSMB5 overexpression promotes cell growth by upregulating the Cyclin D1/CDK4 complex. - Abstract: Multipotent human bone marrow stromal cells (hBMSCs) potentially serve as a source for cell-based therapy in regenerative medicine. However, in vitro expansion was inescapably accompanied with cell senescence, characterized by inhibited proliferation and compromised pluripotency. We have previously demonstrated that this aging process is closely associated with reduced 20S proteasomal activity, with down-regulation of rate-limiting catalytic β-subunits particularly evident. In the present study, we confirmed that proteasomal activity directly contributes to senescence of hBMSCs, which could be reversed by overexpression of the β5-subunit (PSMB5). Knocking down PSMB5 led to decreased proteasomal activity concurrent with reduced cell proliferation in early-stage hBMSCs, which is similar to the senescent phenotype observed in late-stage cells. In contrast, overexpressing PSMB5 in late-stage cells efficiently restored the normal activity of 20S proteasomes and promoted cell growth, possibly via upregulating the Cyclin D1/CDK4 complex. Additionally, PSMB5 could enhance cell resistance to oxidative stress, as evidenced by the increased cell survival upon exposing senescent hBMSCs to hydrogen peroxide. Furthermore, PSMB5 overexpression retained the pluripotency of late-stage hBMSCs by facilitating their neural differentiation both in vitro and in vivo. Collectively, our work reveals a critical role of PSMB5 in 20S proteasome-mediated protection against replicative senescence, pointing to a possible strategy for maintaining the integrity of culture-expanded hBMSCs by manipulating the expression of PSMB5

  19. Ameliorating replicative senescence of human bone marrow stromal cells by PSMB5 overexpression

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    Lu, Li, E-mail: luli7300@126.com [Department of Anatomy, Shanxi Medical University, Taiyuan 030001 (China); Song, Hui-Fang; Wei, Jiao-Long; Liu, Xue-Qin [Department of Anatomy, Shanxi Medical University, Taiyuan 030001 (China); Song, Wen-Hui [Department of Orthopaedics, The Second Affiliated Hospital of Shanxi Medical University, Taiyuan 030001 (China); Yan, Ba-Yi; Yang, Gui-Jiao [Department of Anatomy, Shanxi Medical University, Taiyuan 030001 (China); Li, Ang [Department of Medicine, University of Hong Kong Faculty of Medicine, Hong Kong (Hong Kong); Department of Anatomy, University of Hong Kong Faculty of Medicine, Hong Kong (Hong Kong); Yang, Wu-Lin, E-mail: wulinyoung@163.com [School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei 230009 (China); Laboratory of Metabolic Medicine, Singapore Bioimaging Consortium (SBIC), Agency for Science, Technology and Research - A*STAR (Singapore)

    2014-01-24

    Highlights: • PSMB5 overexpression restores the differentiation potential of aged hBMSCs. • PSMB5 overexpression enhances the proteasomal activity of late-stage hBMSCs. • PSMB5 overexpression inhibits replicative senescence and improved cell viability. • PSMB5 overexpression promotes cell growth by upregulating the Cyclin D1/CDK4 complex. - Abstract: Multipotent human bone marrow stromal cells (hBMSCs) potentially serve as a source for cell-based therapy in regenerative medicine. However, in vitro expansion was inescapably accompanied with cell senescence, characterized by inhibited proliferation and compromised pluripotency. We have previously demonstrated that this aging process is closely associated with reduced 20S proteasomal activity, with down-regulation of rate-limiting catalytic β-subunits particularly evident. In the present study, we confirmed that proteasomal activity directly contributes to senescence of hBMSCs, which could be reversed by overexpression of the β5-subunit (PSMB5). Knocking down PSMB5 led to decreased proteasomal activity concurrent with reduced cell proliferation in early-stage hBMSCs, which is similar to the senescent phenotype observed in late-stage cells. In contrast, overexpressing PSMB5 in late-stage cells efficiently restored the normal activity of 20S proteasomes and promoted cell growth, possibly via upregulating the Cyclin D1/CDK4 complex. Additionally, PSMB5 could enhance cell resistance to oxidative stress, as evidenced by the increased cell survival upon exposing senescent hBMSCs to hydrogen peroxide. Furthermore, PSMB5 overexpression retained the pluripotency of late-stage hBMSCs by facilitating their neural differentiation both in vitro and in vivo. Collectively, our work reveals a critical role of PSMB5 in 20S proteasome-mediated protection against replicative senescence, pointing to a possible strategy for maintaining the integrity of culture-expanded hBMSCs by manipulating the expression of PSMB5.

  20. 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

  1. Comparison of clinical grade human platelet lysates for cultivation of mesenchymal stromal cells from bone marrow and adipose tissue

    DEFF Research Database (Denmark)

    Juhl, Morten; Tratwal, Josefine; Follin, Bjarke

    2016-01-01

    be devoid of any animal derived components. We have evaluated whether human Platelet Lysate (hPL) could be an attractive alternative to animal supplements. METHODS: MSCs from bone marrow (BMSCs) and adipose tissue-derived stromal cells (ASCs) obtained from three donors were culture expanded in three...... culture conditions with 10% fetal bovine serum (FBS). Cell morphology, proliferation, phenotype, genomic stability, and differentiation potential were analyzed. RESULTS: Regardless of manufacturer, BMSCs and ASCs cultured in hPL media showed a significant increase in proliferation capacity compared to FBS...

  2. In vitro induction of alkaline phosphatase levels predicts in vivo bone forming capacity of human bone marrow stromal cells

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    Henk-Jan Prins

    2014-03-01

    Full Text Available One of the applications of bone marrow stromal cells (BMSCs that are produced by ex vivo expansion is for use in in vivo bone tissue engineering. Cultured stromal cells are a mixture of cells at different stages of commitment and expansion capability, leading to a heterogeneous cell population that each time can differ in the potential to form in vivo bone. A parameter that predicts for in vivo bone forming capacity is thus far lacking. We employed single colony-derived BMSC cultures to identify such predictive parameters. Using limiting dilution, we have produced sixteen single CFU-F derived BMSC cultures from human bone marrow and found that only five of these formed bone in vivo. The single colony-derived BMSC strains were tested for proliferation, osteogenic-, adipogenic- and chondrogenic differentiation capacity and the expression of a variety of associated markers. The only robust predictors of in vivo bone forming capacity were the induction of alkaline phosphatase, (ALP mRNA levels and ALP activity during in vitro osteogenic differentiation. The predictive value of in vitro ALP induction was confirmed by analyzing “bulk-cultured” BMSCs from various bone marrow biopsies. Our findings show that in BMSCs, the additional increase in ALP levels over basal levels during in vitro osteogenic differentiation is predictive of in vivo performance.

  3. Maintenance of osteoblastic and adipocytic differentiation potential with age and osteoporosis in human marrow stromal cell cultures

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    Justesen, J; Dokkedahl, Karin Stenderup; Eriksen, E F

    2002-01-01

    Osteoblasts and adipocytes share a common precursor cell in the bone marrow stroma, termed marrow stromal cell (MSC). As the volume of bone adipose tissue increases in vivo with age, we hypothesized that decreased bone formation observed during aging and in patients with osteoporosis (OP) is the ...

  4. Enhancing proliferation and optimizing the culture condition for human bone marrow stromal cells using hypoxia and fibroblast growth factor-2

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    Jung-Seok Lee

    2018-04-01

    Full Text Available This study aimed to determine the cellular characteristics and behaviors of human bone marrow stromal cells (hBMSCs expanded in media in a hypoxic or normoxic condition and with or without fibroblast growth factor-2 (FGF-2 treatment. hBMSCs isolated from the vertebral body and expanded in these four groups were evaluated for cellular proliferation/migration, colony-forming units, cell-surface characterization, in vitro differentiation, in vivo transplantation, and gene expression. Culturing hBMSCs using a particular environmental factor (hypoxia and with the addition of FGF-2 increased the cellular proliferation rate while enhancing the regenerative potential, modulated the multipotency-related processes (enhanced chondrogenesis-related processes/osteogenesis, but reduced adipogenesis, and increased cellular migration and collagen formation. The gene expression levels in the experimental samples showed activation of the hypoxia-inducible factor-1 pathway and glycolysis in the hypoxic condition, with this not being affected by the addition of FGF-2. The concurrent application of hypoxia and FGF-2 could provide a favorable condition for culturing hBMSCs to be used in clinical applications associated with bone tissue engineering, due to the enhancement of cellular proliferation and regenerative potential. Keywords: Bone marrow stromal cells, Hypoxia, Fibroblast growth factor, Tissue regeneration, Microenvironment interactions

  5. Survival of human mesenchymal stromal cells from bone marrow and adipose tissue after xenogenic transplantation in immunocompetent mice

    DEFF Research Database (Denmark)

    Niemeyer, P; Vohrer, J; Schmal, H

    2008-01-01

    of the current paper was to evaluate the survival of undifferentiated and osteogenically induced human MSC from different origins after transplantation in immunocompetent mice. METHODS: Human MSC were isolated from bone marrow (BMSC) and adipose tissue (ASC). After cultivation on mineralized collagen, MSC were......INTRODUCTION: Mesenchymal stromal cells (MSC) represent an attractive cell population for tissue engineering purposes. As MSC are described as immunoprivileged, non-autologous applications seem possible. A basic requirement is the survival of MSC after transplantation in the host. The purpose...... transplanted subcutaneously into immunocompetent mice (n=12). Undifferentiated MSC (group A) were compared with osteogenic-induced MSC (group B). Human-specific in situ hybridization and anti-vimentin staining was used to follow MSC after transplantation. Quantitative evaluation of lymphocytes and macrophages...

  6. Telomerase expression extends the proliferative life-span and maintains the osteogenic potential of human bone marrow stromal cells

    DEFF Research Database (Denmark)

    Simonsen, Janne Lytoft; Rosada, Cecilia; Serakinci, Nedime

    2002-01-01

    Human bone marrow stromal cells (hMSCs) were stably transduced by a retroviral vector containing the gene for the catalytic subunit of human telomerase (hTERT). Transduced cells (hMSC-TERTs) had telomerase activity, and the mean telomere length was increased as compared with that of control cells....... The transduced cells have now undergone more than 260 population doublings (PD) and continue to proliferate, whereas control cells underwent senescence-associated proliferation arrest after 26 PD. The cells maintained production of osteoblastic markers and differentiation potential during continuous subculturing......, did not form tumors, and had a normal karyotype. When implanted subcutaneously in immunodeficient mice, the transduced cells formed more bone than did normal cells. These results suggest that ectopic expression of telomerase in hMSCs prevents senescence-associated impairment of osteoblast functions....

  7. Activation of non-canonical Wnt/JNK pathway by Wnt3a is associated with differentiation fate determination of human bone marrow stromal (mesenchymal) stem cells

    DEFF Research Database (Denmark)

    Qiu, Weimin; Chen, Li; Kassem, Moustapha

    2011-01-01

    The canonical Wnt signaling pathway can determine human bone marrow stromal (mesenchymal) stem cell (hMSC) differentiation fate into osteoblast or adipocyte lineages. However, its downstream targets in MSC are not well characterized. Thus, using DNA microarrays, we compared global gene expression...

  8. Low/Negative Expression of PDGFR-α Identifies the Candidate Primary Mesenchymal Stromal Cells in Adult Human Bone Marrow

    DEFF Research Database (Denmark)

    Li, Hongzhe; Ghazanfari, Roshanak; Zacharaki, Dimitra

    2014-01-01

    Human bone marrow (BM) contains a rare population of nonhematopoietic mesenchymal stromal cells (MSCs), which are of central importance for the hematopoietic microenvironment. However, the precise phenotypic definition of these cells in adult BM has not yet been reported. In this study, we show...... exhibited high levels of genes associated with mesenchymal lineages and HSC supportive function. Moreover, lin(-)/CD45(-)/CD271(+)/CD140a(low/-) cells effectively mediated the ex vivo expansion of transplantable CD34(+) hematopoietic stem cells. Taken together, these data indicate that CD140a is a key...... that low/negative expression of CD140a (PDGFR-α) on lin(-)/CD45(-)/CD271(+) BM cells identified a cell population with very high MSC activity, measured as fibroblastic colony-forming unit frequency and typical in vitro and in vivo stroma formation and differentiation capacities. Furthermore, these cells...

  9. Association between in vivo bone formation and ex vivo migratory capacity of human bone marrow stromal cells

    DEFF Research Database (Denmark)

    Andersen, Rikke K.; Zaher, Walid; Larsen, Kenneth Hauberg

    2015-01-01

    INTRODUCTION: There is a clinical need for developing systemic transplantation protocols for use of human skeletal stem cells (also known bone marrow stromal stem cells) (hBMSC) in tissue regeneration. In systemic transplantation studies, only a limited number of hBMSC home to injured tissues...... populations derived from telomerized hBMSC (hBMSC-TERT) with variable ability to form heterotopic bone when implanted subcutaneously in immune deficient mice. In vitro transwell migration assay was used and the in vivo homing ability of transplanted hBMSC to bone fractures in mice was visualized...... suggesting that only a subpopulation of hBMSC possesses "homing" capacity. Thus, we tested the hypothesis that a subpopulation of hBMSC defined by ability to form heterotopic bone in vivo, is capable of homing to injured bone. METHODS: We tested ex vivo and in vivo homing capacity of a number of clonal cell...

  10. Direct Reprogramming of Human Bone Marrow Stromal Cells into Functional Renal Cells Using Cell-free Extracts

    Directory of Open Access Journals (Sweden)

    Evangelia Papadimou

    2015-04-01

    Full Text Available The application of cell-based therapies in regenerative medicine is gaining recognition. Here, we show that human bone marrow stromal cells (BMSCs, also known as bone-marrow-derived mesenchymal cells, can be reprogrammed into renal proximal tubular-like epithelial cells using cell-free extracts. Streptolysin-O-permeabilized BMSCs exposed to HK2-cell extracts underwent morphological changes—formation of “domes” and tubule-like structures—and acquired epithelial functional properties such as transepithelial-resistance, albumin-binding, and uptake and specific markers E-cadherin and aquaporin-1. Transmission electron microscopy revealed the presence of brush border microvilli and tight intercellular contacts. RNA sequencing showed tubular epithelial transcript abundance and revealed the upregulation of components of the EGFR pathway. Reprogrammed BMSCs integrated into self-forming kidney tissue and formed tubular structures. Reprogrammed BMSCs infused in immunodeficient mice with cisplatin-induced acute kidney injury engrafted into proximal tubuli, reduced renal injury and improved function. Thus, reprogrammed BMSCs are a promising cell resource for future cell therapy.

  11. The Bone Marrow-Derived Stromal Cells

    DEFF Research Database (Denmark)

    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...

  12. Comparative study of the chondrogenic potential of human bone marrow stromal cells, neonatal chondrocytes and adult chondrocytes

    International Nuclear Information System (INIS)

    Saha, Sushmita; Kirkham, Jennifer; Wood, David; Curran, Stephen; Yang, Xuebin

    2010-01-01

    Research highlights: → This study has characterised three different cell types under conditions similar to those used for autologous chondrocyte implantation (ACI) for applications in cartilage repair/regeneration. → Compared for the first time the chondrogenic potential of neonatal chondrocytes with human bone marrow stromal cells (HBMSCs) and adult chondrocytes. → Demonstrated that adult chondrocytes hold greatest potential for use in ACI based on their higher proliferation rates, lower alkaline phosphatise activity and enhanced expression of chondrogenic genes. → Demonstrated the need for chondroinduction as a necessary pre-requisite to efficient chondrogenesis in vitro and, by extrapolation, for cell based therapy (e.g. ACI or cartilage tissue engineering). -- Abstract: Cartilage tissue engineering is still a major clinical challenge with optimisation of a suitable source of cells for cartilage repair/regeneration not yet fully addressed. The aims of this study were to compare and contrast the differences in chondrogenic behaviour between human bone marrow stromal cells (HBMSCs), human neonatal and adult chondrocytes to further our understanding of chondroinduction relative to cell maturity and to identify factors that promote chondrogenesis and maintain functional homoeostasis. Cells were cultured in monolayer in either chondrogenic or basal medium, recapitulating procedures used in existing clinical procedures for cell-based therapies. Cell doubling time, morphology and alkaline phosphatase specific activity (ALPSA) were determined at different time points. Expression of chondrogenic markers (SOX9, ACAN and COL2A1) was compared via real time polymerase chain reaction. Amongst the three cell types studied, HBMSCs had the highest ALPSA in basal culture and lowest ALPSA in chondrogenic media. Neonatal chondrocytes were the most proliferative and adult chondrocytes had the lowest ALPSA in basal media. Gene expression analysis revealed a difference in the

  13. Comparative study of the chondrogenic potential of human bone marrow stromal cells, neonatal chondrocytes and adult chondrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Saha, Sushmita [Biomaterials and Tissue Engineering Group, Leeds Dental Institute, University of Leeds, LS29LU (United Kingdom); Kirkham, Jennifer [Biomineralisation Group, Leeds Dental Institute, University of Leeds, LS29LU (United Kingdom); NIHR Leeds Musculoskeletal Biomedical Research Unit, University of Leeds, Chapel Allerton Hospital, Leeds LS74SA (United Kingdom); Wood, David [Biomaterials and Tissue Engineering Group, Leeds Dental Institute, University of Leeds, LS29LU (United Kingdom); Curran, Stephen [Smith and Nephew Research Centre, YO105DF (United Kingdom); Yang, Xuebin, E-mail: X.B.Yang@leeds.ac.uk [Biomaterials and Tissue Engineering Group, Leeds Dental Institute, University of Leeds, LS29LU (United Kingdom); NIHR Leeds Musculoskeletal Biomedical Research Unit, University of Leeds, Chapel Allerton Hospital, Leeds LS74SA (United Kingdom)

    2010-10-22

    Research highlights: {yields} This study has characterised three different cell types under conditions similar to those used for autologous chondrocyte implantation (ACI) for applications in cartilage repair/regeneration. {yields} Compared for the first time the chondrogenic potential of neonatal chondrocytes with human bone marrow stromal cells (HBMSCs) and adult chondrocytes. {yields} Demonstrated that adult chondrocytes hold greatest potential for use in ACI based on their higher proliferation rates, lower alkaline phosphatise activity and enhanced expression of chondrogenic genes. {yields} Demonstrated the need for chondroinduction as a necessary pre-requisite to efficient chondrogenesis in vitro and, by extrapolation, for cell based therapy (e.g. ACI or cartilage tissue engineering). -- Abstract: Cartilage tissue engineering is still a major clinical challenge with optimisation of a suitable source of cells for cartilage repair/regeneration not yet fully addressed. The aims of this study were to compare and contrast the differences in chondrogenic behaviour between human bone marrow stromal cells (HBMSCs), human neonatal and adult chondrocytes to further our understanding of chondroinduction relative to cell maturity and to identify factors that promote chondrogenesis and maintain functional homoeostasis. Cells were cultured in monolayer in either chondrogenic or basal medium, recapitulating procedures used in existing clinical procedures for cell-based therapies. Cell doubling time, morphology and alkaline phosphatase specific activity (ALPSA) were determined at different time points. Expression of chondrogenic markers (SOX9, ACAN and COL2A1) was compared via real time polymerase chain reaction. Amongst the three cell types studied, HBMSCs had the highest ALPSA in basal culture and lowest ALPSA in chondrogenic media. Neonatal chondrocytes were the most proliferative and adult chondrocytes had the lowest ALPSA in basal media. Gene expression analysis revealed

  14. An in vivo model to assess magnesium alloys and their biological effect on human bone marrow stromal cells.

    Science.gov (United States)

    Yoshizawa, Sayuri; Chaya, Amy; Verdelis, Kostas; Bilodeau, Elizabeth A; Sfeir, Charles

    2015-12-01

    Magnesium (Mg) alloys have many unique qualities which make them ideal candidates for bone fixation devices, including biocompatibility and degradation in vivo. Despite a rise in Mg alloy production and research, there remains no standardized system to assess their degradation or biological effect on human stem cells in vivo. In this study, we developed a novel in vivo model to assess Mg alloys for craniofacial and orthopedic applications. Our model consists of a collagen sponge seeded with human bone marrow stromal cells (hBMSCs) around a central Mg alloy rod. These scaffolds were implanted subcutaneously in mice and analyzed after eight weeks. Alloy degradation and biological effect were determined by microcomputed tomography (microCT), histological staining, and immunohistochemistry (IHC). MicroCT showed greater volume loss for pure Mg compared to AZ31 after eight weeks in vivo. Histological analysis showed that hBMSCs were retained around the Mg implants after 8 weeks. Furthermore, immunohistochemistry showed the expression of dentin matrix protein 1 and osteopontin around both pure Mg and AZ31 with implanted hBMSCs. In addition, histological sections showed a thin mineral layer around all degrading alloys at the alloy-tissue interface. In conclusion, our data show that degrading pure Mg and AZ31 implants are cytocompatible and do not inhibit the osteogenic property of hBMSCs in vivo. These results demonstrate that this model can be used to efficiently assess the biological effect of corroding Mg alloys in vivo. Importantly, this model may be modified to accommodate additional cell types and clinical applications. Magnesium (Mg) alloys have been investigated as ideal candidates for bone fixation devices due to high biocompatibility and degradation in vivo, and there is a growing need of establishing an efficient in vivo material screening system. In this study, we assessed degradation rate and biological effect of Mg alloys by transplanting Mg alloy rod with

  15. Low/Negative Expression of PDGFR-α Identifies the Candidate Primary Mesenchymal Stromal Cells in Adult Human Bone Marrow

    Directory of Open Access Journals (Sweden)

    Hongzhe Li

    2014-12-01

    Full Text Available Human bone marrow (BM contains a rare population of nonhematopoietic mesenchymal stromal cells (MSCs, which are of central importance for the hematopoietic microenvironment. However, the precise phenotypic definition of these cells in adult BM has not yet been reported. In this study, we show that low/negative expression of CD140a (PDGFR-α on lin−/CD45−/CD271+ BM cells identified a cell population with very high MSC activity, measured as fibroblastic colony-forming unit frequency and typical in vitro and in vivo stroma formation and differentiation capacities. Furthermore, these cells exhibited high levels of genes associated with mesenchymal lineages and HSC supportive function. Moreover, lin−/CD45−/CD271+/CD140alow/− cells effectively mediated the ex vivo expansion of transplantable CD34+ hematopoietic stem cells. Taken together, these data indicate that CD140a is a key negative selection marker for adult human BM-MSCs, which enables to prospectively isolate a close to pure population of candidate human adult stroma stem/progenitor cells with potent hematopoiesis-supporting capacity.

  16. Modified Titanium Surface-Mediated Effects on Human Bone Marrow Stromal Cell Response

    Directory of Open Access Journals (Sweden)

    Amol Chaudhari

    2013-11-01

    Full Text Available Surface modification of titanium implants is used to enhance osseointegration. The study objective was to evaluate five modified titanium surfaces in terms of cytocompatibility and pro-osteogenic/pro-angiogenic properties for human mesenchymal stromal cells: amorphous microporous silica (AMS, bone morphogenetic protein-2 immobilized on AMS (AMS + BMP, bio-active glass (BAG and two titanium coatings with different porosity (T1; T2. Four surfaces served as controls: uncoated Ti (Ti, Ti functionalized with BMP-2 (Ti + BMP, Ti surface with a thickened titanium oxide layer (TiO2 and a tissue culture polystyrene surface (TCPS. The proliferation of eGFP-fLuc (enhanced green fluorescence protein-firefly luciferase transfected cells was tracked non-invasively by fluorescence microscopy and bio-luminescence imaging. The implant surface-mediated effects on cell differentiation potential was tracked by determination of osteogenic and angiogenic parameters [alkaline phosphatase (ALP; osteocalcin (OC; osteoprotegerin (OPG; vascular endothelial growth factor-A (VEGF-A]. Unrestrained cell proliferation was observed on (unfunctionalized Ti and AMS surfaces, whereas BAG and porous titanium coatings T1 and T2 did not support cell proliferation. An important pro-osteogenic and pro-angiogenic potential of the AMS + BMP surface was observed. In contrast, coating the Ti surface with BMP did not affect the osteogenic differentiation of the progenitor cells. A significantly slower BMP-2 release from AMS compared to Ti supports these findings. In the unfunctionalized state, Ti was found to be superior to AMS in terms of OPG and VEGF-A production. AMS is suggested to be a promising implant coating material for bioactive agents delivery.

  17. Apatite formation on bioactive calcium-silicate cements for dentistry affects surface topography and human marrow stromal cells proliferation.

    Science.gov (United States)

    Gandolfi, Maria Giovanna; Ciapetti, Gabriela; Taddei, Paola; Perut, Francesca; Tinti, Anna; Cardoso, Marcio Vivan; Van Meerbeek, Bart; Prati, Carlo

    2010-10-01

    The effect of ageing in phosphate-containing solution of bioactive calcium-silicate cements on the chemistry, morphology and topography of the surface, as well as on in vitro human marrow stromal cells viability and proliferation was investigated. A calcium-silicate cement (wTC) mainly based on dicalcium-silicate and tricalcium-silicate was prepared. Alpha-TCP was added to wTC to obtain wTC-TCP. Bismuth oxide was inserted in wTC to prepare a radiopaque cement (wTC-Bi). A commercial calcium-silicate cement (ProRoot MTA) was tested as control. Cement disks were aged in DPBS for 5 h ('fresh samples'), 14 and 28 days, and analyzed by ESEM/EDX, SEM/EDX, ATR-FTIR, micro-Raman techniques and scanning white-light interferometry. Proliferation, LDH release, ALP activity and collagen production of human marrow stromal cells (MSC) seeded for 1-28 days on the cements were evaluated. Fresh samples exposed a surface mainly composed of calcium-silicate hydrates CSH (from the hydration of belite and alite), calcium hydroxide, calcium carbonate, and ettringite. Apatite nano-spherulites rapidly precipitated on cement surfaces within 5 h. On wTC-TCP the Ca-P deposits appeared thicker than on the other cements. Aged cements showed an irregular porous calcium-phosphate (Ca-P) coating, formed by aggregated apatite spherulites with interspersed calcite crystals. All the experimental cements exerted no acute toxicity in the cell assay system and allowed cell growth. Using biochemical results, the scores were: fresh cements>aged cements for cell proliferation and ALP activity (except for wTC-Bi), whereas fresh cements

  18. Bone Marrow Derived Mesenchymal Stromal Cells Harness Purinergenic Signaling to Tolerize Human Th1 Cells In Vivo

    Science.gov (United States)

    Amarnath, Shoba; Foley, Jason E.; Farthing, Don E.; Gress, Ronald E.; Laurence, Arian; Eckhaus, Michael A.; Métais, Jean-Yves; Rose, Jeremy J.; Hakim, Frances T.; Felizardo, Tania C.; Cheng, Austin V.; Robey, Pamela G.; Stroncek, David E.; Sabatino, Marianna; Battiwalla, Minoo; Ito, Sawa; Fowler, Daniel H.; Barrett, Austin J.

    2014-01-01

    The use of bone marrow derived mesenchymal stromal cells (BMSC) in the treatment of alloimmune and autoimmune conditions has generated much interest, yet an understanding of the therapeutic mechanism remains elusive. We therefore explored immune modulation by a clinical-grade BMSC product in a model of human-into-mouse xenogeneic GVHD (x-GVHD) mediated by human CD4+ Th1 cells. BMSC reversed established, lethal x-GVHD through marked inhibition of Th1 cell effector function. Gene marking studies indicated BMSC engraftment was limited to the lung; further, there was no increase in regulatory T cells, thereby suggesting a paracrine mechanism of BMSC action. BMSC recipients had increased serum CD73 expressing exosomes that promoted adenosine accumulation ex vivo. Importantly, immune modulation mediated by BMSC was fully abrogated by pharmacologic therapy with an adenosine A2A receptor antagonist. To investigate the potential clinical relevance of these mechanistic findings, patient serum samples collected pre- and post-BMSC treatment were studied for exosome content: CD73 expressing exosomes promoting adenosine accumulation were detected in post-BMSC samples. In conclusion, BMSC effectively modulate experimental GVHD through a paracrine mechanism that promotes adenosine-based immune suppression. PMID:25532725

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

    International Nuclear Information System (INIS)

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

    2014-01-01

    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

  20. Response of Primary Human Bone Marrow Mesenchymal Stromal Cells and Dermal Keratinocytes to Thermal Printer Materials In Vitro.

    Science.gov (United States)

    Schmelzer, Eva; Over, Patrick; Gridelli, Bruno; Gerlach, Jörg C

    Advancement in thermal three-dimensional printing techniques has greatly increased the possible applications of various materials in medical applications and tissue engineering. Yet, potential toxic effects on primary human cells have been rarely investigated. Therefore, we compared four materials commonly used in thermal printing for bioengineering, namely thermally printed acrylonitrile butadiene styrene, MED610, polycarbonate, and polylactic acid, and investigated their effects on primary human adult skin epidermal keratinocytes and bone marrow mesenchymal stromal cells (BM-MSCs) in vitro. We investigated indirect effects on both cell types caused by potential liberation of soluble substances from the materials, and also analyzed BM-MSCs in direct contact with the materials. We found that even in culture without direct contact with the materials, the culture with MED610 (and to a lesser extent acrylonitrile butadiene styrene) significantly affected keratinocytes, reducing cell numbers and proliferation marker Ki67 expression, and increasing glucose consumption, lactate secretion, and expression of differentiation-associated genes. BM-MSCs had decreased metabolic activity, and exhibited increased cell death in direct culture on the materials. MED610 and acrylonitrile butadiene styrene induced the strongest expression of genes associated to differentiation and estrogen receptor activation. In conclusion, we found strong cell-type-specific effects of the materials, suggesting that materials for applications in regenerative medicine should be carefully selected not only based on their mechanical properties but also based on their cell-type-specific biological effects.

  1. Novel strontium-doped bioactive glass nanoparticles enhance proliferation and osteogenic differentiation of human bone marrow stromal cells

    Science.gov (United States)

    Strobel, L. A.; Hild, N.; Mohn, D.; Stark, W. J.; Hoppe, A.; Gbureck, U.; Horch, R. E.; Kneser, U.; Boccaccini, A. R.

    2013-07-01

    The present study investigates a new family of bioactive glass nanoparticles with and without Sr-doping focusing on the influence of the nanoparticles on human bone marrow stromal cells (hBMSCs) in vitro. The bioactive glass nanoparticles were fabricated by flame spray synthesis and a particle diameter of 30-35 nm was achieved. Glass nanoparticles were undoped (BG 13-93-0Sr) or doped with 5 wt% strontium (Sr) (BG 13-93-5Sr) and used at concentrations of 10 and 100 μg/cm² (particles per culture plate area), respectively. Cells were cultured for 14 days after which the samples were analysed regarding metabolic activity and expression of various bone-specific genes. Cell growth and morphology indicated the high cytocompatibility of the nanoparticulate bioactive glass. The presence of the nanoparticles enhanced cell growth compared to the plain polystyrene control group. At a concentration of 100 μg/cm², Sr-doped particles led to significantly enhanced gene expression of osteocalcin, collagen type 1 and vascular endothelial growth factor. Thus, Sr-doped nanoparticles showing a dose-dependent increase of osteogenic differentiation in hBMSCs are a promising biomaterial for bone regeneration purposes.

  2. Evaluation of gene delivery strategies to efficiently overexpress functional HLA-G on human bone marrow stromal cells

    Directory of Open Access Journals (Sweden)

    Joana S Boura

    2014-01-01

    Full Text Available Mesenchymal stromal cells (MSC constitutively express low levels of human leukocyte antigen-G (HLA-G, which has been shown to contribute to their immunomodulatory and anti-inflammatory properties. Here, we hypothesized that overexpression of HLA-G on bone marrow-derived MSC would improve their immunomodulatory function, thus increasing their therapeutic potential. Therefore, we investigated which gene transfer system is best suited for delivering this molecule while maintaining its immunomodulatory effects. We performed a side-by-side comparison between three nonviral plasmid-based platforms (pmax-HLA-G1; MC-HLA-G1; pEP-HLA-G1 and a viral system (Lv-HLA-G1 using gene transfer parameters that yielded similar levels of HLA-G1-expressing MSC. Natural killer (NK cell–mediated lysis assays and T cell proliferation assays showed that MSC modified with the HLA-G1 expressing viral vector had significantly lower susceptibility to NK-lysis and significantly reduced T cell proliferation when compared to nonmodified cells or MSC modified with plasmid. We also show that, in plasmid-modified MSC, an increase in Toll-like receptor (TLR9 expression is the mechanism responsible for the abrogation of HLA-G1's immunomodulatory effect. Although MSC can be efficiently modified to overexpress HLA-G1 using viral and nonviral strategies, only viral-based delivery of HLA-G1 is suitable for improvement of MSC's immunomodulatory properties.

  3. Novel strontium-doped bioactive glass nanoparticles enhance proliferation and osteogenic differentiation of human bone marrow stromal cells

    Energy Technology Data Exchange (ETDEWEB)

    Strobel, L. A. [University of Erlangen-Nuremberg Medical Center, Department of Plastic and Hand Surgery (Germany); Hild, N.; Mohn, D.; Stark, W. J. [ETH Zurich, Department of Chemistry and Applied Biosciences, Institute for Chemical and Bioengineering (Switzerland); Hoppe, A. [University of Erlangen-Nuremberg, Department of Materials Science and Engineering, Institute of Biomaterials (Germany); Gbureck, U. [University of Wuerzburg, Department for Functional Materials in Medicine and Dentistry (Germany); Horch, R. E.; Kneser, U. [University of Erlangen-Nuremberg Medical Center, Department of Plastic and Hand Surgery (Germany); Boccaccini, A. R., E-mail: aldo.boccaccini@ww.uni-erlangen.de [University of Erlangen-Nuremberg, Department of Materials Science and Engineering, Institute of Biomaterials (Germany)

    2013-07-15

    The present study investigates a new family of bioactive glass nanoparticles with and without Sr-doping focusing on the influence of the nanoparticles on human bone marrow stromal cells (hBMSCs) in vitro. The bioactive glass nanoparticles were fabricated by flame spray synthesis and a particle diameter of 30-35 nm was achieved. Glass nanoparticles were undoped (BG 13-93-0Sr) or doped with 5 wt% strontium (Sr) (BG 13-93-5Sr) and used at concentrations of 10 and 100 {mu}g/cm Superscript-Two (particles per culture plate area), respectively. Cells were cultured for 14 days after which the samples were analysed regarding metabolic activity and expression of various bone-specific genes. Cell growth and morphology indicated the high cytocompatibility of the nanoparticulate bioactive glass. The presence of the nanoparticles enhanced cell growth compared to the plain polystyrene control group. At a concentration of 100 {mu}g/cm Superscript-Two , Sr-doped particles led to significantly enhanced gene expression of osteocalcin, collagen type 1 and vascular endothelial growth factor. Thus, Sr-doped nanoparticles showing a dose-dependent increase of osteogenic differentiation in hBMSCs are a promising biomaterial for bone regeneration purposes.

  4. Novel strontium-doped bioactive glass nanoparticles enhance proliferation and osteogenic differentiation of human bone marrow stromal cells

    International Nuclear Information System (INIS)

    Strobel, L. A.; Hild, N.; Mohn, D.; Stark, W. J.; Hoppe, A.; Gbureck, U.; Horch, R. E.; Kneser, U.; Boccaccini, A. R.

    2013-01-01

    The present study investigates a new family of bioactive glass nanoparticles with and without Sr-doping focusing on the influence of the nanoparticles on human bone marrow stromal cells (hBMSCs) in vitro. The bioactive glass nanoparticles were fabricated by flame spray synthesis and a particle diameter of 30–35 nm was achieved. Glass nanoparticles were undoped (BG 13-93-0Sr) or doped with 5 wt% strontium (Sr) (BG 13-93-5Sr) and used at concentrations of 10 and 100 μg/cm² (particles per culture plate area), respectively. Cells were cultured for 14 days after which the samples were analysed regarding metabolic activity and expression of various bone-specific genes. Cell growth and morphology indicated the high cytocompatibility of the nanoparticulate bioactive glass. The presence of the nanoparticles enhanced cell growth compared to the plain polystyrene control group. At a concentration of 100 μg/cm², Sr-doped particles led to significantly enhanced gene expression of osteocalcin, collagen type 1 and vascular endothelial growth factor. Thus, Sr-doped nanoparticles showing a dose-dependent increase of osteogenic differentiation in hBMSCs are a promising biomaterial for bone regeneration purposes

  5. Endogenously produced Indian Hedgehog regulates TGFβ-driven chondrogenesis of human bone marrow stromal/stem cells.

    Science.gov (United States)

    Handorf, Andrew M; Chamberlain, Connie S; Li, Wan-Ju

    2015-04-15

    Human bone marrow stromal/stem cells (hBMSCs) have an inherent tendency to undergo hypertrophy when induced into the chondrogenic lineage using transforming growth factor-beta 1 (TGFβ) in vitro, reminiscent of what occurs during endochondral ossification. Surprisingly, Indian Hedgehog (IHH) has received little attention for its role during hBMSC chondrogenesis despite being considered a master regulator of endochondral ossification. In this study, we investigated the role that endogenously produced IHH plays during hBMSC chondrogenesis. We began by analyzing the expression of IHH throughout differentiation using quantitative polymerase chain reaction and found that IHH expression was upregulated dramatically upon chondrogenic induction and peaked from days 9 to 12 of differentiation, which coincided with a concomitant increase in the expression of chondrogenesis- and hypertrophy-related markers, suggesting a potential role for endogenously produced IHH in driving hBMSC chondrogenesis. More importantly, pharmacological inhibition of Hedgehog signaling with cyclopamine or knockdown of IHH almost completely blocked TGFβ1-induced chondrogenesis in hBMSCs, demonstrating that endogenously produced IHH is necessary for hBMSC chondrogenesis. Furthermore, overexpression of IHH was sufficient to drive chondrogenic differentiation, even when TGFβ signaling was inhibited. Finally, stimulation with TGFβ1 induced a significant and sustained upregulation of IHH expression within 3 h that preceded an upregulation in all cartilage-related genes analyzed, and knockdown of IHH blocked the effects of TGFβ1 entirely, suggesting that the effects of TGFβ1 are being mediated through endogenously produced IHH. Together, our findings demonstrate that endogenously produced IHH is playing a critical role in regulating hBMSC chondrogenesis.

  6. The Src inhibitor dasatinib accelerates the differentiation of human bone marrow-derived mesenchymal stromal cells into osteoblasts

    International Nuclear Information System (INIS)

    Id Boufker, Hichame; Lagneaux, Laurence; Najar, Mehdi; Piccart, Martine; Ghanem, Ghanem; Body, Jean-Jacques; Journé, Fabrice

    2010-01-01

    The proto-oncogene Src is an important non-receptor protein tyrosine kinase involved in signaling pathways that control cell adhesion, growth, migration and differentiation. It negatively regulates osteoblast activity, and, as such, its inhibition is a potential means to prevent bone loss. Dasatinib is a new dual Src/Bcr-Abl tyrosine kinase inhibitor initially developed for the treatment of chronic myeloid leukemia. It has also shown promising results in preclinical studies in various solid tumors. However, its effects on the differentiation of human osteoblasts have never been examined. We evaluated the effects of dasatinib on bone marrow-derived mesenchymal stromal cells (MSC) differentiation into osteoblasts, in the presence or absence of a mixture of dexamethasone, ascorbic acid and β-glycerophosphate (DAG) for up to 21 days. The differentiation kinetics was assessed by evaluating mineralization of the extracellular matrix, alkaline phosphatase (ALP) activity, and expression of osteoblastic markers (receptor activator of nuclear factor kappa B ligand [RANKL], bone sialoprotein [BSP], osteopontin [OPN]). Dasatinib significantly increased the activity of ALP and the level of calcium deposition in MSC cultured with DAG after, respectively, 7 and 14 days; it upregulated the expression of BSP and OPN genes independently of DAG; and it markedly downregulated the expression of RANKL gene and protein (decrease in RANKL/OPG ratio), the key factor that stimulates osteoclast differentiation and activity. Our results suggest a dual role for dasatinib in both (i) stimulating osteoblast differentiation leading to a direct increase in bone formation, and (ii) downregulating RANKL synthesis by osteoblasts leading to an indirect inhibition of osteoclastogenesis. Thus, dasatinib is a potentially interesting candidate drug for the treatment of osteolysis through its dual effect on bone metabolism

  7. Tissue-engineered bone formation using human bone marrow stromal cells and novel β-tricalcium phosphate

    International Nuclear Information System (INIS)

    Liu Guangpeng; Zhao Li; Cui Lei; Liu Wei; Cao Yilin

    2007-01-01

    In this study we investigated not only the cellular proliferation and osteogenic differentiation of human bone marrow stromal cells (hBMSCs) on the novel β-tricalcium phosphate (β-TCP) scaffolds in vitro but also bone formation by ectopic implantation in athymic mice in vivo. The interconnected porous β-TCP scaffolds with pores of 300-500 μm in size were prepared by the polymeric sponge method. β-TCP scaffolds with the dimension of 3 mm x 3 mm x 3 mm were combined with hBMSCs, and incubated with (+) or without (-) osteogenic medium in vitro. Cell proliferation and osteogenic differentiation on the scaffolds were evaluated by scanning electron microscopy (SEM) observation, MTT assay, alkaline phosphatase (ALP) activity and osteocalcin (OCN) content measurement. SEM observation showed that hBMSCs attached well on the scaffolds and proliferated rapidly. No significant difference in the MTT assay could be detected between the two groups, but the ALP activity and OCN content of scaffolds (+) were much higher than those of the scaffolds (-) (p < 0.05). These results indicated that the novel porous β-TCP scaffolds can support the proliferation and subsequent osteogenic differentiation of hBMSCs in vitro. After being cultured in vitro for 14 days, the scaffolds (+) and (-) were implanted into subcutaneous sites of athymic mice. In β-TCP scaffolds (+), woven bone formed after 4 weeks of implantation and osteogenesis progressed with time. Furthermore, tissue-engineered bone could be found at 8 weeks, and remodeled lamellar bone was also observed at 12 weeks. However, no bone formation could be found in β-TCP scaffolds (-) at each time point checked. The above findings illustrate that the novel porous β-TCP scaffolds developed in this work have prominent osteoconductive activity and the potential for applications in bone tissue engineering

  8. Tissue-engineered bone formation using human bone marrow stromal cells and novel {beta}-tricalcium phosphate

    Energy Technology Data Exchange (ETDEWEB)

    Liu Guangpeng [National Tissue Engineering Research and Development Center, Shanghai 200235 (China); Zhao Li [National Tissue Engineering Research and Development Center, Shanghai 200235 (China); Cui Lei [National Tissue Engineering Research and Development Center, Shanghai 200235 (China); Liu Wei [National Tissue Engineering Research and Development Center, Shanghai 200235 (China); Cao Yilin [National Tissue Engineering Research and Development Center, Shanghai 200235 (China)

    2007-06-01

    In this study we investigated not only the cellular proliferation and osteogenic differentiation of human bone marrow stromal cells (hBMSCs) on the novel {beta}-tricalcium phosphate ({beta}-TCP) scaffolds in vitro but also bone formation by ectopic implantation in athymic mice in vivo. The interconnected porous {beta}-TCP scaffolds with pores of 300-500 {mu}m in size were prepared by the polymeric sponge method. {beta}-TCP scaffolds with the dimension of 3 mm x 3 mm x 3 mm were combined with hBMSCs, and incubated with (+) or without (-) osteogenic medium in vitro. Cell proliferation and osteogenic differentiation on the scaffolds were evaluated by scanning electron microscopy (SEM) observation, MTT assay, alkaline phosphatase (ALP) activity and osteocalcin (OCN) content measurement. SEM observation showed that hBMSCs attached well on the scaffolds and proliferated rapidly. No significant difference in the MTT assay could be detected between the two groups, but the ALP activity and OCN content of scaffolds (+) were much higher than those of the scaffolds (-) (p < 0.05). These results indicated that the novel porous {beta}-TCP scaffolds can support the proliferation and subsequent osteogenic differentiation of hBMSCs in vitro. After being cultured in vitro for 14 days, the scaffolds (+) and (-) were implanted into subcutaneous sites of athymic mice. In {beta}-TCP scaffolds (+), woven bone formed after 4 weeks of implantation and osteogenesis progressed with time. Furthermore, tissue-engineered bone could be found at 8 weeks, and remodeled lamellar bone was also observed at 12 weeks. However, no bone formation could be found in {beta}-TCP scaffolds (-) at each time point checked. The above findings illustrate that the novel porous {beta}-TCP scaffolds developed in this work have prominent osteoconductive activity and the potential for applications in bone tissue engineering.

  9. MicroRNA-4739 regulates osteogenic and adipocytic differentiation of immortalized human bone marrow stromal cells via targeting LRP3

    Directory of Open Access Journals (Sweden)

    Mona Elsafadi

    2017-04-01

    Full Text Available Understanding the regulatory networks underlying lineage differentiation and fate determination of human bone marrow stromal cells (hBMSC is a prerequisite for their therapeutic use. The goal of the current study was to unravel the novel role of the low-density lipoprotein receptor-related protein 3 (LRP3 in regulating the osteogenic and adipogenic differentiation of immortalized hBMSCs. Gene expression profiling revealed significantly higher LRP3 levels in the highly osteogenic hBMSC clone imCL1 than in the less osteogenic clone imCL2, as well as a significant upregulation of LRP3 during the osteogenic induction of the imCL1 clone. Data from functional and gene expression assays demonstrated the role of LRP3 as a molecular switch promoting hBMSC lineage differentiation into osteoblasts and inhibiting differentiation into adipocytes. Interestingly, microRNA (miRNA expression profiling identified miR-4739 as the most under-represented miRNA (−36.11 fold in imCL1 compared to imCL2. The TargetScan prediction algorithm, combined with functional and biochemical assays, identified LRP3 mRNA as a novel target of miR-4739, with a single potential binding site for miR-4739 located in the LRP3 3′ UTR. Regulation of LRP3 expression by miR-4739 was subsequently confirmed by qRT-PCR, western blotting, and luciferase assays. Over-expression of miR-4739 mimicked the effects of LRP3 knockdown on promoting adipogenic and suppressing osteogenic differentiation of hBMSCs. Hence, we report for the first time a novel biological role for the LRP3/hsa-miR-4739 axis in balancing osteogenic and adipocytic differentiation of hBMSCs. Our data support the potential utilization of miRNA-based therapies in regenerative medicine.

  10. 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

  11. Effect of human bone marrow mesenchymal stromal cells on cytokine production by peripheral blood naive, memory, and effector T cells.

    Science.gov (United States)

    Laranjeira, Paula; Pedrosa, Monia; Pedreiro, Susana; Gomes, Joana; Martinho, Antonio; Antunes, Brigida; Ribeiro, Tania; Santos, Francisco; Trindade, Helder; Paiva, Artur

    2015-01-05

    The different distribution of T cells among activation/differentiation stages in immune disorders may condition the outcome of mesenchymal stromal cell (MSC)-based therapies. Indeed, the effect of MSCs in the different functional compartments of T cells is not completely elucidated. We investigated the effect of human bone marrow MSCs on naturally occurring peripheral blood functional compartments of CD4(+) and CD8(+) T cells: naive, central memory, effector memory, and effector compartments. For that, mononuclear cells (MNCs) stimulated with phorbol myristate acetate (PMA) plus ionomycin were cultured in the absence/presence of MSCs. The percentage of cells expressing tumor necrosis factor-alpha (TNF-α), interferon gamma (IFNγ), and interleukin-2 (IL-2), IL-17, IL-9, and IL-6 and the amount of cytokine produced were assessed by flow cytometry. mRNA levels of IL-4, IL-10, transforming growth factor-beta (TGF-β), and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) in purified CD4(+) and CD8(+) T cells, and phenotypic and mRNA expression changes induced by PMA + ionomycin stimulation in MSCs, were also evaluated. MSCs induced the reduction of the percentage of CD4(+) and CD8(+) T cells producing TNF-α, IFNγ, and IL-2 in all functional compartments, except for naive IFNγ(+)CD4(+) T cells. This inhibitory effect differentially affected CD4(+) and CD8(+) T cells as well as the T-cell functional compartments; remarkably, different cytokines showed distinct patterns of inhibition regarding both the percentage of producing cells and the amount of cytokine produced. Likewise, the percentages of IL-17(+), IL-17(+)TNF-α(+), and IL-9(+) within CD4(+) and CD8(+) T cells and of IL-6(+)CD4(+) T cells were decreased in MNC-MSC co-cultures. MSCs decreased IL-10 and increased IL-4 mRNA expression in stimulated CD4(+) and CD8(+) T cells, whereas TGF-β was reduced in CD8(+) and augmented in CD4(+) T cells, with no changes for CTLA4. Finally, PMA

  12. Fibroblast Growth Factor-2 Enhances Expansion of Human Bone Marrow-Derived Mesenchymal Stromal Cells without Diminishing Their Immunosuppressive Potential

    OpenAIRE

    Auletta, Jeffery J.; Zale, Elizabeth A.; Welter, Jean F.; Solchaga, Luis A.

    2011-01-01

    Allogeneic hematopoietic stem cell transplantation is the main curative therapy for many hematologic malignancies. Its potential relies on graft-versus-tumor effects which associate with graft-versus-host disease. Mesenchymal stromal cells (MSCs) possess immunomodulatory properties that make them attractive therapeutic alternatives. We evaluated the in vitro immunosuppressive activity of medium conditioned by human MSCs from 5 donors expanded 13 passages with or without FGF-2. FGF-2 supplemen...

  13. Immunoregulatory effects on T lymphocytes by human mesenchymal stromal cells isolated from bone marrow, amniotic fluid, and placenta.

    Science.gov (United States)

    Mareschi, Katia; Castiglia, Sara; Sanavio, Fiorella; Rustichelli, Deborah; Muraro, Michela; Defedele, Davide; Bergallo, Massimiliano; Fagioli, Franca

    2016-02-01

    Mesenchymal stromal cells (MSCs) are a promising tool in cell therapies because of their multipotent, bystander, and immunomodulatory properties. Although bone marrow represents the main source of MSCs, there remains a need to identify a stem cell source that is safe and easily accessible and yields large numbers of cells without provoking debates over ethics. In this study, MSCs isolated from amniotic fluid and placenta were compared with bone marrow MSCs. Their immunomodulatory properties were studied in total activated T cells (peripheral blood mononuclear cells) stimulated with phytohemagglutinin (PHA-PBMCs). In particular, an in vitro co-culture system was established to study: (i) the effect on T-lymphocyte proliferation; (ii) the presence of T regulatory lymphocytes (Treg); (iii) the immunophenotype of various T subsets (Th1 and Th2 naïve, memory, effector lymphocytes); (iv) cytokine release and master gene expression to verify Th1, Th2, and Th17 polarization; and (v) IDO production. Under all co-culture conditions with PHA-PBMCs and MSCs (independently of tissue origin), data revealed: (i) T proliferation inhibition; (ii) increase in naïve T and decrease in memory T cells; (iii) increase in T regulatory lymphocytes; (iv) strong Th2 polarization associated with increased interleukin-10 and interleukin-4 levels, Th1 inhibition (significant decreases in interleukin-2, tumor necrosis factor-α, interferon-γ, and interleukin-12) and Th17 induction (production of high concentrations of interleukins-6 and -17); (v) indoleamine-2,3-dioxygenase mRNA induction in MSCs co-cultured with PHA-PBMCs. AF-MSCs had a more potent immunomodulatory effect on T cells than BM-MSCs, only slightly higher than that of placenta MSCs. This study indicates that MSCs isolated from fetal tissues may be considered a good alternative to BM-MSCs for clinical applications. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights

  14. Human stromal (mesenchymal) stem cells

    DEFF Research Database (Denmark)

    Aldahmash, Abdullah; Zaher, Walid; Al-Nbaheen, May

    2012-01-01

    Human stromal (mesenchymal) stem cells (hMSC) represent a group of non-hematopoietic stem cells present in the bone marrow stroma and the stroma of other organs including subcutaneous adipose tissue, placenta, and muscles. They exhibit the characteristics of somatic stem cells of self......-renewal and multi-lineage differentiation into mesoderm-type of cells, e.g., to osteoblasts, adipocytes, chondrocytes and possibly other cell types including hepatocytes and astrocytes. Due to their ease of culture and multipotentiality, hMSC are increasingly employed as a source for cells suitable for a number...

  15. 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

  16. Production of human platelet lysate by use of ultrasound for ex vivo expansion of human bone marrow-derived mesenchymal stromal cells.

    Science.gov (United States)

    Bernardi, Martina; Albiero, Elena; Alghisi, Alberta; Chieregato, Katia; Lievore, Chiara; Madeo, Domenico; Rodeghiero, Francesco; Astori, Giuseppe

    2013-08-01

    A medium supplemented with fetal bovine serum (FBS) is of common use for the expansion of human mesenchymal stromal cells (MSCs). However, its use is discouraged by regulatory authorities because of the risk of zoonoses and immune reactions. Human platelet lysate (PL) obtained by freezing/thawing disruption of platelets has been proposed as a possible substitute of FBS. The process is time-consuming and not well standardized. A new method for obtaining PL that is based on the use of ultrasound is proposed. Platelet sonication was performed by submerging platelet-containing plastic bags in an ultrasonic bath. To evaluate platelet lysis we measured platelet-derived growth factor-AB release. PL efficiency was tested by expanding bone marrow (BM)-MSCs, measuring population doubling time, differentiation capacity and immunogenic properties. Safety was evaluated by karyotyping expanded cells. After 30 minutes of sonication, 74% of platelet derived growth factor-AB was released. PL enhanced BM-MSC proliferation rate compared with FBS. The mean cumulative population doubling (cPD) of cells growth in PL at 10%, 7.5% and 5% was better compared with cPD obtained with 10% FBS. PD time (hours) of MSCs with PL obtained by sonication was shorter than for cPD with PL obtained by freezing/thawing (18.9 versus 17.4, P < 0.01). BM mononucleated cells expressed MSC markers and were able to differentiate into adipogenic, osteogenic and chondrogenic lineages. When BM-MSCs and T cells were co-cultured in close contact, immunosuppressive activity of BM-MSCs was maintained. Cell karyotype showed no genetic alterations. The proposed method for the production of PL by sonication could be a safe, efficient and fast substitute of FBS, without the potential risks of FBS. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  17. Chondrogenesis of human bone marrow mesenchymal stromal cells in highly porous alginate-foams supplemented with chondroitin sulfate

    International Nuclear Information System (INIS)

    Huang, Zhao; Nooeaid, Patcharakamon; Kohl, Benjamin; Roether, Judith A.; Schubert, Dirk W.; Meier, Carola; Boccaccini, Aldo R.; Godkin, Owen; Ertel, Wolfgang; Arens, Stephan; Schulze-Tanzil, Gundula

    2015-01-01

    To overcome the limited intrinsic cartilage repair, autologous chondrocyte or bone-marrow-derived mesenchymal stromal cell (BM-MSC) was implanted into cartilage defects. For this purpose suitable biocompatible scaffolds are needed to provide cell retention, chondrogenesis and initial mechanical stability. The present study should indicate whether a recently developed highly porous alginate (Alg) foam scaffold supplemented with chondroitin sulfate (CS) allows the attachment, survival and chondrogenesis of BM-MSCs and articular chondrocytes. The foams were prepared using a freeze-drying method; some of them were supplemented with CS and subsequently characterized for porosity, biodegradation and mechanical profile. BM-MSCs were cultured for 1–2 weeks on the scaffold either under chondrogenic or maintenance conditions. Cell vitality assays, histology, glycosaminoglycan (sGAG) assay, and type II and I collagen immunolabelings were performed to monitor cell growth and extracellular matrix (ECM) synthesis in the scaffolds. Scaffolds had a high porosity ~ 93–95% with a mean pore sizes of 237 ± 48 μm (Alg) and 197 ± 61 μm (Alg/CS). Incorporation of CS increased mechanical strength of the foams providing gradually CS release over 7 days. Most of the cells survived in the scaffolds. BM-MSCs and articular chondrocytes formed rounded clusters within the scaffold pores. The BM-MSCs, irrespective of whether cultured under non/chondrogenic conditions and chondrocytes produced an ECM containing sGAGs, and types II and I collagen. Total collagen and sGAG contents were higher in differentiated BM-MSC cultures supplemented with CS than in CS-free foams after 14 days. The cell cluster formation induced by the scaffolds might stimulate chondrogenesis via initial intense cell–cell contacts. - Highlights: • Alginate foam scaffolds revealed a high porosity and mean pore size of 197–237 μm. • Chondroitin sulfate was released over 14 days by the scaffolds. • Chondrocytes

  18. Chondrogenesis of human bone marrow mesenchymal stromal cells in highly porous alginate-foams supplemented with chondroitin sulfate

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Zhao [Department of Orthopaedic, Trauma and Reconstructive Surgery, Charité-Universitätsmedizin-Berlin Campus Benjamin Franklin, Berlin (Germany); Nooeaid, Patcharakamon [Institute of Biomaterials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg (Germany); Kohl, Benjamin [Department of Orthopaedic, Trauma and Reconstructive Surgery, Charité-Universitätsmedizin-Berlin Campus Benjamin Franklin, Berlin (Germany); Roether, Judith A.; Schubert, Dirk W. [Institute of Polymer Materials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg (Germany); Meier, Carola [Department of Orthopaedic, Trauma and Reconstructive Surgery, Charité-Universitätsmedizin-Berlin Campus Benjamin Franklin, Berlin (Germany); Boccaccini, Aldo R. [Institute of Biomaterials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg (Germany); Godkin, Owen; Ertel, Wolfgang; Arens, Stephan [Department of Orthopaedic, Trauma and Reconstructive Surgery, Charité-Universitätsmedizin-Berlin Campus Benjamin Franklin, Berlin (Germany); Schulze-Tanzil, Gundula, E-mail: gundula.schulze@pmu.ac.at [Department of Orthopaedic, Trauma and Reconstructive Surgery, Charité-Universitätsmedizin-Berlin Campus Benjamin Franklin, Berlin (Germany); Institute of Anatomy, Paracelsus Medical University, Nuremberg (Germany)

    2015-05-01

    To overcome the limited intrinsic cartilage repair, autologous chondrocyte or bone-marrow-derived mesenchymal stromal cell (BM-MSC) was implanted into cartilage defects. For this purpose suitable biocompatible scaffolds are needed to provide cell retention, chondrogenesis and initial mechanical stability. The present study should indicate whether a recently developed highly porous alginate (Alg) foam scaffold supplemented with chondroitin sulfate (CS) allows the attachment, survival and chondrogenesis of BM-MSCs and articular chondrocytes. The foams were prepared using a freeze-drying method; some of them were supplemented with CS and subsequently characterized for porosity, biodegradation and mechanical profile. BM-MSCs were cultured for 1–2 weeks on the scaffold either under chondrogenic or maintenance conditions. Cell vitality assays, histology, glycosaminoglycan (sGAG) assay, and type II and I collagen immunolabelings were performed to monitor cell growth and extracellular matrix (ECM) synthesis in the scaffolds. Scaffolds had a high porosity ~ 93–95% with a mean pore sizes of 237 ± 48 μm (Alg) and 197 ± 61 μm (Alg/CS). Incorporation of CS increased mechanical strength of the foams providing gradually CS release over 7 days. Most of the cells survived in the scaffolds. BM-MSCs and articular chondrocytes formed rounded clusters within the scaffold pores. The BM-MSCs, irrespective of whether cultured under non/chondrogenic conditions and chondrocytes produced an ECM containing sGAGs, and types II and I collagen. Total collagen and sGAG contents were higher in differentiated BM-MSC cultures supplemented with CS than in CS-free foams after 14 days. The cell cluster formation induced by the scaffolds might stimulate chondrogenesis via initial intense cell–cell contacts. - Highlights: • Alginate foam scaffolds revealed a high porosity and mean pore size of 197–237 μm. • Chondroitin sulfate was released over 14 days by the scaffolds. • Chondrocytes

  19. NOTCH-Mediated Maintenance and Expansion of Human Bone Marrow Stromal/Stem Cells: A Technology Designed for Orthopedic Regenerative Medicine.

    Science.gov (United States)

    Dong, Yufeng; Long, Teng; Wang, Cuicui; Mirando, Anthony J; Chen, Jianquan; O'Keefe, Regis J; Hilton, Matthew J

    2014-12-01

    Human bone marrow-derived stromal/stem cells (BMSCs) have great therapeutic potential for treating skeletal disease and facilitating skeletal repair, although maintaining their multipotency and expanding these cells ex vivo have proven difficult. Because most stem cell-based applications to skeletal regeneration and repair in the clinic would require large numbers of functional BMSCs, recent research has focused on methods for the appropriate selection, expansion, and maintenance of BMSC populations during long-term culture. We describe here a novel biological method that entails selection of human BMSCs based on NOTCH2 expression and activation of the NOTCH signaling pathway in cultured BMSCs via a tissue culture plate coated with recombinant human JAGGED1 (JAG1) ligand. We demonstrate that transient JAG1-mediated NOTCH signaling promotes human BMSC maintenance and expansion while increasing their skeletogenic differentiation capacity, both ex vivo and in vivo. This study is the first of its kind to describe a NOTCH-mediated methodology for the maintenance and expansion of human BMSCs and will serve as a platform for future clinical or translational studies aimed at skeletal regeneration and repair. ©AlphaMed Press.

  20. Standardization of Good Manufacturing Practice-compliant production of bone marrow-derived human mesenchymal stromal cells for immunotherapeutic applications.

    Science.gov (United States)

    Wuchter, Patrick; Bieback, Karen; Schrezenmeier, Hubert; Bornhäuser, Martin; Müller, Lutz P; Bönig, Halvard; Wagner, Wolfgang; Meisel, Roland; Pavel, Petra; Tonn, Torsten; Lang, Peter; Müller, Ingo; Renner, Matthias; Malcherek, Georg; Saffrich, Rainer; Buss, Eike C; Horn, Patrick; Rojewski, Markus; Schmitt, Anita; Ho, Anthony D; Sanzenbacher, Ralf; Schmitt, Michael

    2015-02-01

    Human mesenchymal stem or stromal cells (MSCs) represent a potential resource not only for regenerative medicine but also for immunomodulatory cell therapies. The application of different MSC culture protocols has significantly hampered the comparability of experimental and clinical data from different laboratories and has posed a major obstacle for multicenter clinical trials. Manufacturing of cell products for clinical application in the European Community must be conducted in compliance with Good Manufacturing Practice and requires a manufacturing license. In Germany, the Paul-Ehrlich-Institut as the Federal Authority for Vaccines and Biomedicines is critically involved in the approval process. This report summarizes a consensus meeting between researchers, clinicians and regulatory experts on standard quality requirements for MSC production. The strategy for quality control testing depends on the product's cell composition, the manufacturing process and the indication and target patient population. Important quality criteria in this sense are, among others, the immunophenotype of the cells, composition of the culture medium and the risk for malignant transformation, as well as aging and the immunosuppressive potential of the manufactured MSCs. This position paper intends to provide relevant information to interested parties regarding these criteria to foster the development of scientifically valid and harmonized quality standards and to support approval of MSC-based investigational medicinal products. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  1. Early osteoinductive human bone marrow mesenchymal stromal/stem cells support an enhanced hematopoietic cell expansion with altered chemotaxis- and adhesion-related gene expression profiles

    Energy Technology Data Exchange (ETDEWEB)

    Sugino, Noriko [Department of Hematology/Oncology, Graduate School of Medicine, Kyoto University, Kyoto 606-8507 (Japan); Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan); Miura, Yasuo, E-mail: ym58f5@kuhp.kyoto-u.ac.jp [Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan); Yao, Hisayuki [Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan); Iwasa, Masaki; Fujishiro, Aya [Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan); Division of Gastroenterology and Hematology, Shiga University of Medical Science, Shiga 520-2192 (Japan); Fujii, Sumie [Department of Hematology/Oncology, Graduate School of Medicine, Kyoto University, Kyoto 606-8507 (Japan); Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan); Hirai, Hideyo [Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan); Takaori-Kondo, Akifumi [Department of Hematology/Oncology, Graduate School of Medicine, Kyoto University, Kyoto 606-8507 (Japan); Ichinohe, Tatsuo [Department of Hematology and Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553 (Japan); Maekawa, Taira [Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto 606-8507 (Japan)

    2016-01-22

    Bone marrow (BM) microenvironment has a crucial role in supporting hematopoiesis. Here, by using a microarray analysis, we demonstrate that human BM mesenchymal stromal/stem cells (MSCs) in an early osteoinductive stage (e-MSCs) are characterized by unique hematopoiesis-associated gene expression with an enhanced hematopoiesis-supportive ability. In comparison to BM-MSCs without osteoinductive treatment, gene expression in e-MSCs was significantly altered in terms of their cell adhesion- and chemotaxis-related profiles, as identified with Gene Ontology and Gene Set Enrichment Analysis. Noteworthy, expression of the hematopoiesis-associated molecules CXCL12 and vascular cell adhesion molecule 1 was remarkably decreased in e-MSCs. e-MSCs supported an enhanced expansion of CD34{sup +} hematopoietic stem and progenitor cells, and generation of myeloid lineage cells in vitro. In addition, short-term osteoinductive treatment favored in vivo hematopoietic recovery in lethally irradiated mice that underwent BM transplantation. e-MSCs exhibited the absence of decreased stemness-associated gene expression, increased osteogenesis-associated gene expression, and apparent mineralization, thus maintaining the ability to differentiate into adipogenic cells. Our findings demonstrate the unique biological characteristics of e-MSCs as hematopoiesis-regulatory stromal cells at differentiation stage between MSCs and osteoprogenitor cells and have significant implications in developing new strategy for using pharmacological osteoinductive treatment to support hematopoiesis in hematopoietic stem and progenitor cell transplantation. - Highlights: • Human BM-MSCs in an early osteoinductive stage (e-MSCs) support hematopoiesis. • Adhesion- and chemotaxis-associated gene signatures are altered in e-MSCs. • Expression of CXCL12 and VCAM1 is remarkably decreased in e-MSCs. • e-MSCs are at differentiation stage between MSCs and osteoprogenitor cells. • Osteoinductive treatment

  2. Caspase-8 regulates the expression of pro- and anti-inflammatory cytokines in human bone marrow-derived mesenchymal stromal cells.

    Science.gov (United States)

    Moen, Siv H; Westhrin, Marita; Zahoor, Muhammad; Nørgaard, Nikolai N; Hella, Hanne; Størdal, Berit; Sundan, Anders; Nilsen, Nadra J; Sponaas, Anne-Marit; Standal, Therese

    2016-09-01

    Mesenchymal stem cells, also called mesenchymal stromal cells, MSCs, have great potential in stem cell therapy partly due to their immunosuppressive properties. How these cells respond to chronic inflammatory stimuli is therefore of importance. Toll-like receptors (TLR)s are innate immune receptors that mediate inflammatory signals in response to infection, stress, and damage. Caspase-8 is involved in activation of NF-kB downstream of TLRs in immune cells. Here we investigated the role of caspase-8 in regulating TLR-induced cytokine production from human bone marrow-derived mesenchymal stromal cells (hBMSCs). Cytokine expression in hBMCs in response to poly(I:C) and LPS was evaluated by PCR, multiplex cytokine assay, and ELISA. TLR3, TRIF, and caspase-8 were silenced using siRNA. Caspase-8 was also inhibited using a caspase-8 inhibitor, z-IEDT. We found that TLR3 agonist poly(I:C) and TLR4 agonist LPS induced secretion of several pro-inflammatory cytokines in a TLR-dependent manner which required the TLR signaling adaptor molecule TRIF. Further, poly(I:C) reduced the expression of anti-inflammatory cytokines HGF and TGFβ whereas LPS reduced HGF expression only. Notably, caspase-8 was involved in the induction of IL- IL-1β, IL-6, CXCL10, and in the inhibition of HGF and TGFβ. Caspase-8 appears to modulate hBMSCs into gaining a pro-inflammatory phenotype. Therefore, inhibiting caspase-8 in hBMSCs might promote an immunosuppressive phenotype which could be useful in clinical applications to treat inflammatory disorders.

  3. Comparative miRNA-Based Fingerprinting Reveals Biological Differences in Human Olfactory Mucosa- and Bone-Marrow-Derived Mesenchymal Stromal Cells

    Directory of Open Access Journals (Sweden)

    Susan Louise Lindsay

    2016-05-01

    Full Text Available Previously we reported that nestin-positive human mesenchymal stromal cells (MSCs derived from the olfactory mucosa (OM enhanced CNS myelination in vitro to a greater extent than bone-marrow-derived MSCs (BM-MSCs. miRNA-based fingerprinting revealed the two MSCs were 64% homologous, with 26 miRNAs differentially expressed. We focused on miR-146a-5p and miR-140-5p due to their reported role in the regulation of chemokine production and myelination. The lower expression of miR-140-5p in OM-MSCs correlated with higher secretion of CXCL12 compared with BM-MSCs. Addition of CXCL12 and its pharmacological inhibitors to neural co-cultures supported these data. Studies on related miR-146a-5p targets demonstrated that OM-MSCs had lower levels of Toll-like receptors and secreted less pro-inflammatory cytokines, IL-6, IL-8, and CCL2. OM-MSCs polarized microglia to an anti-inflammatory phenotype, illustrating potential differences in their inflammatory response. Nestin-positive OM-MSCs could therefore offer a cell transplantation alternative for CNS repair, should these biological behaviors be translated in vivo.

  4. Distribution and viability of fetal and adult human bone marrow stromal cells in a biaxial rotating vessel bioreactor after seeding on polymeric 3D additive manufactured scaffolds

    Directory of Open Access Journals (Sweden)

    Anne eLeferink

    2015-10-01

    Full Text Available One of the conventional approaches in tissue engineering is the use of scaffolds in combination with cells to obtain mechanically stable tissue constructs in vitro prior to implantation. Additive manufacturing by fused deposition modeling is a widely used technique to produce porous scaffolds with defined pore network, geometry, and therewith defined mechanical properties. Bone marrow derived mesenchymal stromal cells (MSCs are promising candidates for tissue engineering based cell therapies due to their multipotent character. One of the hurdles to overcome when combining additive manufactured scaffolds with MSCs is the resulting heterogeneous cell distribution and limited cell proliferation capacity. In this study, we show that the use of a biaxial rotating bioreactor, after static culture of human fetal MSCs (hfMSCs seeded on synthetic polymeric scaffolds, improved the homogeneity of cell and extracellular matrix (ECM distribution and increased the total cell number. Furthermore, we show that the relative mRNA expression levels of indicators for stemness and differentiation are not significantly changed upon this bioreactor culture, whereas static culture shows variations of several indicators for stemness and differentiation. The biaxial rotating bioreactor presented here offers a homogeneous distribution of hfMSCs, enabling studies on MSCs fate in additive manufactured scaffolds without inducing undesired differentiation.

  5. Feasibility and Efficiency of Human Bone Marrow Stromal Cell Culture with Allogeneic Platelet Lysate-Supplementation for Cell Therapy against Stroke

    Directory of Open Access Journals (Sweden)

    Chengbo Tan

    2016-01-01

    Full Text Available Currently, there is increasing interest in human bone marrow stromal cells (hBMSCs as regeneration therapy against cerebral stroke. The aim of the present study was to evaluate the feasibility and validity of hBMSC cultures with allogeneic platelet lysates (PLs. Platelet concentrates (PC were harvested from healthy volunteers and made into single donor-derived PL (sPL. The PL mixtures (mPL were made from three different sPL. Some growth factors and platelet cell surface antigens were detected by enzyme-linked immunosorbent assay (ELISA. The hBMSCs cultured with 10% PL were analyzed for their proliferative potential, surface markers, and karyotypes. The cells were incubated with superparamagnetic iron oxide (SPIO agents and injected into a pig brain. MRI and histological analysis were performed. Consequently, nine lots of sPL and three mPL were prepared. ELISA analysis showed that PL contained adequate growth factors and a particle of platelet surface antigens. Cell proliferation capacity of PLs was equivalent to or higher than that of fetal calf serum (FCS. No contradiction in cell surface markers and no chromosomal aberrations were found. The MRI detected the distribution of SPIO-labeled hBMSCs in the pig brain. In summary, the hBMSCs cultured with allogeneic PL are suitable for cell therapy against stroke.

  6. Bone marrow stromal cells spontaneously produce Flt3-ligand: influence of ionizing radiations and cytokine stimulation.

    Science.gov (United States)

    Bertho, Jean Marc; Demarquay, Christelle; Mouiseddine, Moubarak; Douenat, Noémie; Stefani, Johanna; Prat, Marie; Paquet, François

    2008-08-01

    To define the ability of human bone marrow (BM) stromal cells to produce fms-like tyrosine kinase 3 (Flt3)-ligand (FL), and the effect of irradiation, tumour necrosis factor-alpha (TNFalpha) or tumour growth factor beta (TGFbeta) on FL production. Primary BM stromal cell cultures were irradiated at 2-10 Gy or were stimulated with TNFalpha or TGFbeta1. The presence of FL was tested in culture supernatants and in cell lysate. The presence of a membrane-bound form of FL and the level of gene expression were also tested. Primary BM stromal cells spontaneously released FL. This production was increased by TNFalpha but not by TGFbeta1 or by irradiation. Chemical induction of osteoblastic differentiation from BM stromal cells also induced an increase in FL release. Our results suggest that the observed increase in FL concentration after in vivo irradiation is an indirect effect. The possible implication of BM stromal cells in these mechanisms is discussed.

  7. Multilineage differentiation of porcine bone marrow stromal cells associated with specific gene expression pattern

    DEFF Research Database (Denmark)

    Zou, Lijin; Zou, Xuenong; Chen, Li

    2007-01-01

    There are increasing reports regarding differentiation of bone marrow stromal cells (BMSC) from human and various species of animals including pigs. The phenotype and function of BMSC along a mesenchymal lineage differentiation are well characterized by specific transcription factors and marker g...

  8. Generation of human β-thalassemia induced pluripotent cell lines by reprogramming of bone marrow-derived mesenchymal stromal cells using modified mRNA.

    Science.gov (United States)

    Varela, Ioanna; Karagiannidou, Angeliki; Oikonomakis, Vasilis; Tzetis, Maria; Tzanoudaki, Marianna; Siapati, Elena-Konstantina; Vassilopoulos, George; Graphakos, Stelios; Kanavakis, Emmanuel; Goussetis, Evgenios

    2014-12-01

    Synthetic modified mRNA molecules encoding pluripotency transcription factors have been used successfully in reprogramming human fibroblasts to induced pluripotent stem cells (iPSCs). We have applied this method on bone marrow-derived mesenchymal stromal cells (BM-MSCs) obtained from a patient with β-thalassemia (β-thal) with the aim to generate trangene-free β-thal-iPSCs. Transfection of 10(4) BM-MSCs by lipofection with mRNA encoding the reprogramming factors Oct4, Klf4, Sox2, cMyc, and Lin28 resulted in formation of five iPSC colonies, from which three were picked up and expanded in β-thal-iPSC lines. After 10 serial passages in vitro, β-thal-iPSCs maintain genetic stability as shown by array comparative genomic hybridization (aCGH) and are capable of forming embryoid bodies in vitro and teratomas in vivo. Their gene expression profile compared to human embryonic stem cells (ESCs) and BM-MSCs seems to be similar to that of ESCs, whereas it differs from the profile of the parental BM-MSCs. Differentiation cultures toward a hematopoietic lineage showed the generation of CD34(+) progenitors up to 10%, but with a decreased hematopoietic colony-forming capability. In conclusion, we report herein the generation of transgene-free β-thal-iPSCs that could be widely used for disease modeling and gene therapy applications. Moreover, it was demonstrated that the mRNA-based reprogramming method, used mainly in fibroblasts, is also suitable for reprogramming of human BM-MSCs.

  9. Feline bone marrow-derived mesenchymal stromal cells (MSCs) show similar phenotype and functions with regards to neuronal differentiation as human MSCs.

    Science.gov (United States)

    Munoz, Jessian L; Greco, Steven J; Patel, Shyam A; Sherman, Lauren S; Bhatt, Suresh; Bhatt, Rekha S; Shrensel, Jeffrey A; Guan, Yan-Zhong; Xie, Guiqin; Ye, Jiang-Hong; Rameshwar, Pranela; Siegel, Allan

    2012-09-01

    Mesenchymal stromal cells (MSCs) show promise for treatment of a variety of neurological and other disorders. Cat has a high degree of linkage with the human genome and has been used as a model for analysis of neurological disorders such as stroke, Alzheimer's disease and motor disorders. The present study was designed to characterize bone marrow-derived MSCs from cats and to investigate the capacity to generate functional peptidergic neurons. MSCs were expanded with cells from the femurs of cats and then characterized by phenotype and function. Phenotypically, feline and human MSCs shared surface markers, and lacked hematopoietic markers, with similar morphology. As compared to a subset of human MSCs, feline MSCs showed no evidence of the major histocompatibility class II. Since the literature suggested Stro-1 as an indicator of pluripotency, we compared early and late passages feline MSCs and found its expression in >90% of the cells. However, the early passage cells showed two distinct populations of Stro-1-expressing cells. At passage 5, the MSCs were more homogeneous with regards to Stro-1 expression. The passage 5 MSCs differentiated to osteogenic and adipogenic cells, and generated neurons with electrophysiological properties. This correlated with the expression of mature neuronal markers with concomitant decrease in stem cell-associated genes. At day 12 induction, the cells were positive for MAP2, Neuronal Nuclei, tubulin βIII, Tau and synaptophysin. This correlated with electrophysiological maturity as presented by excitatory postsynaptic potentials (EPSPs). The findings indicate that the cat may constitute a promising biomedical model for evaluation of novel therapies such as stem cell therapy in such neurological disorders as Alzheimer's disease and stroke. Copyright © 2012 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

  10. Evaluation of GMP-compliant culture media for in vitro expansion of human bone marrow mesenchymal stromal cells.

    Science.gov (United States)

    Wuchter, Patrick; Vetter, Marcel; Saffrich, Rainer; Diehlmann, Anke; Bieback, Karen; Ho, Anthony D; Horn, Patrick

    2016-06-01

    Mesenchymal stromal cells (MSCs) from human bone marrow serve as a resource for cell-based therapies in regenerative medicine. Clinical applications require standardized protocols according to good manufacturing practice (GMP) guidelines. Donor variability as well as the intrinsic heterogeneity of MSC populations must be taken into consideration. The composition of the culture medium is a key factor in successful MSC expansion. The aim of this study was to comparatively assess the efficiency of xeno-free human platelet lysate (HPL)-based cell expansion with two commercially available media-StemPro MSC SFM CTS (for human ex vivo tissue and cell culture processing applications) and MSCGM (non-GMP-compliant, for research only)-in an academic setting as the first optimization step toward GMP-compliant manufacturing. We report the feasibility of MSC expansion up to the yielded cell number with all three media. MSCs exhibited the typical fibroblastoid morphology, with distinct differences in cell size depending on the medium. The differentiation capacity and characteristic immunophenotype were confirmed for all MSC populations. Proliferation was highest using StemPro MSC SFM CTS, whereas HPL medium was more cost-effective and its composition could be adjusted individually according to the respective needs. In summary, we present a comprehensive evaluation of GMP-compatible culture media for MSC expansion. Both StemPro and HPL medium proved to be suitable for clinical application and allowed sufficient cell proliferation. Specific differences were observed and should be considered according to the intended use. This study provides a detailed cost analysis and tools that may be helpful for the establishment of GMP-compliant MSC expansion. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

  11. TGF-β1, GDF-5, and BMP-2 stimulation induces chondrogenesis in expanded human articular chondrocytes and marrow-derived stromal cells.

    Science.gov (United States)

    Murphy, Meghan K; Huey, Daniel J; Hu, Jerry C; Athanasiou, Kyriacos A

    2015-03-01

    Replacement of degenerated cartilage with cell-based cartilage products may offer a long-term solution to halt arthritis' degenerative progression. Chondrocytes are frequently used in cell-based FDA-approved cartilage products; yet human marrow-derived stromal cells (hMSCs) show significant translational potential, reducing donor site morbidity and maintaining their undifferentiated phenotype with expansion. This study sought to investigate the effects of transforming growth factor β1 (TGF-β1), growth/differentiation factor 5 (GDF-5), and bone morphogenetic protein 2 (BMP-2) during postexpansion chondrogenesis in human articular chondrocytes (hACs) and to compare chondrogenesis in passaged hACs with that of passaged hMSCs. Through serial expansion, chondrocytes dedifferentiated, decreasing expression of chondrogenic genes while increasing expression of fibroblastic genes. However, following expansion, 10 ng/mL TGF-β1, 100 ng/mL GDF-5, or 100 ng/mL BMP-2 supplementation during three-dimensional aggregate culture each upregulated one or more markers of chondrogenic gene expression in both hACs and hMSCs. Additionally, in both cell types, the combination of TGF-β1, GDF-5, and BMP-2 induced the greatest upregulation of chondrogenic genes, that is, Col2A1, Col2A1/Col1A1 ratio, SOX9, and ACAN, and synthesis of cartilage-specific matrix, that is, glycosaminoglycans (GAGs) and ratio of collagen II/I. Finally, TGF-β1, GDF-5, and BMP-2 stimulation yielded mechanically robust cartilage rich in collagen II and GAGs in both cell types, following 4 weeks maturation. This study illustrates notable success in using the self-assembling method to generate robust, scaffold-free neocartilage constructs using expanded hACs and hMSCs. © 2014 AlphaMed Press.

  12. Chromosome copy number variation in telomerized human bone marrow stromal cells; insights for monitoring safe ex-vivo expansion of adult stem cells.

    Science.gov (United States)

    Burns, Jorge S; Harkness, Linda; Aldahmash, Abdullah; Gautier, Laurent; Kassem, Moustapha

    2017-12-01

    Adult human bone marrow stromal cells (hBMSC) cultured for cell therapy require evaluation of potency and stability for safe use. Chromosomal aberrations upsetting genomic integrity in such cells have been contrastingly described as "Limited" or "Significant". Previously reported stepwise acquisition of a spontaneous neoplastic phenotype during three-year continuous culture of telomerized cells (hBMSC-TERT20) didn't alter a diploid karyotype measured by spectral karyotype analysis (SKY). Such screening may not adequately monitor abnormal and potentially tumorigenic hBMSC in clinical scenarios. We here used array comparative genomic hybridization (aCGH) to more stringently compare non-tumorigenic parental hBMSC-TERT strains with their tumorigenic subcloned populations. Confirmation of a known chromosome 9p21 microdeletion at locus CDKN2A/B, showed it also impinged upon the adjacent MTAP gene. Compared to reference diploid human fibroblast genomic DNA, the non-tumorigenic hBMSC-TERT4 cells had a copy number variation (CNV) in at least 14 independent loci. The pre-tumorigenic hBMSC-TERT20 cell strain had further CNV including 1q44 gain enhancing SMYD3 expression and 11q13.1 loss downregulating MUS81 expression. Bioinformatic analysis of gene products reflecting 11p15.5 CNV gain in tumorigenic hBMSC-TERT20 cells highlighted networks implicated in tumorigenic progression involving cell cycle control and mis-match repair. We provide novel biomarkers for prospective risk assessment of expanded stem cell cultures. Copyright © 2017. Published by Elsevier B.V.

  13. Pulsed Electromagnetic Field Regulates MicroRNA 21 Expression to Activate TGF-β Signaling in Human Bone Marrow Stromal Cells to Enhance Osteoblast Differentiation

    Directory of Open Access Journals (Sweden)

    Nagarajan Selvamurugan

    2017-01-01

    Full Text Available Pulsed electromagnetic fields (PEMFs have been documented to promote bone fracture healing in nonunions and increase lumbar spinal fusion rates. However, the molecular mechanisms by which PEMF stimulates differentiation of human bone marrow stromal cells (hBMSCs into osteoblasts are not well understood. In this study the PEMF effects on hBMSCs were studied by microarray analysis. PEMF stimulation of hBMSCs’ cell numbers mainly affected genes of cell cycle regulation, cell structure, and growth receptors or kinase pathways. In the differentiation and mineralization stages, PEMF regulated preosteoblast gene expression and notably, the transforming growth factor-beta (TGF-β signaling pathway and microRNA 21 (miR21 were most highly regulated. PEMF stimulated activation of Smad2 and miR21-5p expression in differentiated osteoblasts, and TGF-β signaling was essential for PEMF stimulation of alkaline phosphatase mRNA expression. Smad7, an antagonist of the TGF-β signaling pathway, was found to be miR21-5p’s putative target gene and PEMF caused a decrease in Smad7 expression. Expression of Runx2 was increased by PEMF treatment and the miR21-5p inhibitor prevented the PEMF stimulation of Runx2 expression in differentiating cells. Thus, PEMF could mediate its effects on bone metabolism by activation of the TGF-β signaling pathway and stimulation of expression of miR21-5p in hBMSCs.

  14. Transforming growth factor-beta1 stimulates the production of insulin-like growth factor-I and insulin-like growth factor-binding protein-3 in human bone marrow stromal osteoblast progenitors

    DEFF Research Database (Denmark)

    Kveiborg, Marie; Flyvbjerg, Allan; Eriksen, E F

    2001-01-01

    While transforming growth factor-beta1 (TGF-beta1) regulates proliferation and differentiation of human osteoblast precursor cells, the mechanisms underlying these effects are not known. Several hormones and locally acting growth factors regulate osteoblast functions through changes in the insulin......-like growth factors (IGFs) and IGF-binding proteins (IGFBPs). Thus, we studied the effects of TGF-beta1 on IGFs and IGFBPs in human marrow stromal (hMS) osteoblast precursor cells. TGF-beta1 increased the steady-state mRNA level of IGF-I up to 8.5+/-0.6-fold (P...

  15. Different culture media affect growth characteristics, surface marker distribution and chondrogenic differentiation of human bone marrow-derived mesenchymal stromal cells.

    Science.gov (United States)

    Hagmann, Sebastien; Moradi, Babak; Frank, Sebastian; Dreher, Thomas; Kämmerer, Peer Wolfgang; Richter, Wiltrud; Gotterbarm, Tobias

    2013-07-30

    Bone marrow-derived mesenchymal stromal cells (BM-MSCs) play an important role in modern tissue engineering, while distinct variations of culture media compositions and supplements have been reported. Because MSCs are heterogeneous regarding their regenerative potential and their surface markers, these parameters were compared in four widely used culture media compositions. MSCs were isolated from bone marrow and expanded in four established cell culture media. MSC yield/1000 MNCs, passage time and growth index were observed. In P4, typical MSC surface markers were analysed by fluorescence cytometry. Additionally, chondrogenic, adipogenic and osteogenic differentiation potential were evaluated. Growth index and P0 cell yield varied importantly between the media. The different expansion media had a significant influence on the expression of CD10, CD90, CD105, CD140b CD146 and STRO-1. While no significant differences were observed regarding osteogenic and adipogenic differentiation, chondrogenic differentiation was superior in medium A as reflected by GAG/DNA content. The choice of expansion medium can have a significant influence on growth, differentiation potential and surface marker expression of mesenchymal stromal cells, which is of fundamental importance for tissue engineering procedures.

  16. Intrapancreatic injection of human bone marrow-derived mesenchymal stem/stromal cells alleviates hyperglycemia and modulates the macrophage state in streptozotocin-induced type 1 diabetic mice.

    Directory of Open Access Journals (Sweden)

    Norimitsu Murai

    Full Text Available Type 1 diabetes mellitus is a progressive disease caused by the destruction of pancreatic β-cells, resulting in insulin dependency and hyperglycemia. While transplanted bone marrow-derived mesenchymal stem/stromal cells (BMMSCs have been explored as an alternative therapeutic approach for diseases, the choice of delivery route may be a critical factor determining their sustainability. This study evaluated the effects of intrapancreatic and intravenous injection of human BMMSCs (hBMMSCs in streptozotocin (STZ-induced type 1 diabetic mouse model. C57/BL6 mice were intraperitoneally injected with 115 mg/kg STZ on day 0. hBMMSCs (1 × 106 cells or vehicle were injected into the pancreas or jugular vein on day 7. Intrapancreatic, but not intravenous, hBMMSC injection significantly reduced blood glucose levels on day 28 compared with vehicle injection by the same route. This glucose-lowering effect was not induced by intrapancreatic injection of human fibroblasts as the xenograft control. Intrapancreatically injected fluorescence-labeled hBMMSCs were observed in the intra- and extra-lobular spaces of the pancreas, and intravenously injected cells were in the lung region, although the number of cells mostly decreased within 2 weeks of injection. For hBMMSCs injected twice into the pancreatic region on days 7 and 28, the injected mice had further reduced blood glucose to borderline diabetic levels on day 56. Animals injected with hBMMSCs twice exhibited increases in the plasma insulin level, number and size of islets, insulin-positive proportion of the total pancreas area, and intensity of insulin staining compared with vehicle-injected animals. We found a decrease of Iba1-positive cells in islets and an increase of CD206-positive cells in both the endocrine and exocrine pancreas. The hBMMSC injection also reduced the number of CD40-positive cells merged with glucagon immunoreactions in the islets. These results suggest that intrapancreatic injection

  17. Plasmid-based genetic modification of human bone marrow-derived stromal cells: analysis of cell survival and transgene expression after transplantation in rat spinal cord.

    Science.gov (United States)

    Ronsyn, Mark W; Daans, Jasmijn; Spaepen, Gie; Chatterjee, Shyama; Vermeulen, Katrien; D'Haese, Patrick; Van Tendeloo, Viggo Fi; Van Marck, Eric; Ysebaert, Dirk; Berneman, Zwi N; Jorens, Philippe G; Ponsaerts, Peter

    2007-12-14

    Bone marrow-derived stromal cells (MSC) are attractive targets for ex vivo cell and gene therapy. In this context, we investigated the feasibility of a plasmid-based strategy for genetic modification of human (h)MSC with enhanced green fluorescent protein (EGFP) and neurotrophin (NT)3. Three genetically modified hMSC lines (EGFP, NT3, NT3-EGFP) were established and used to study cell survival and transgene expression following transplantation in rat spinal cord. First, we demonstrate long-term survival of transplanted hMSC-EGFP cells in rat spinal cord under, but not without, appropriate immune suppression. Next, we examined the stability of EGFP or NT3 transgene expression following transplantation of hMSC-EGFP, hMSC-NT3 and hMSC-NT3-EGFP in rat spinal cord. While in vivo EGFP mRNA and protein expression by transplanted hMSC-EGFP cells was readily detectable at different time points post-transplantation, in vivo NT3 mRNA expression by hMSC-NT3 cells and in vivo EGFP protein expression by hMSC-NT3-EGFP cells was, respectively, undetectable or declined rapidly between day 1 and 7 post-transplantation. Further investigation revealed that the observed in vivo decline of EGFP protein expression by hMSC-NT3-EGFP cells: (i) was associated with a decrease in transgenic NT3-EGFP mRNA expression as suggested following laser capture micro-dissection analysis of hMSC-NT3-EGFP cell transplants at day 1 and day 7 post-transplantation, (ii) did not occur when hMSC-NT3-EGFP cells were transplanted subcutaneously, and (iii) was reversed upon re-establishment of hMSC-NT3-EGFP cell cultures at 2 weeks post-transplantation. Finally, because we observed a slowly progressing tumour growth following transplantation of all our hMSC cell transplants, we here demonstrate that omitting immune suppressive therapy is sufficient to prevent further tumour growth and to eradicate malignant xenogeneic cell transplants. In this study, we demonstrate that genetically modified hMSC lines can survive

  18. Global MicroRNA Profiling in Human Bone Marrow Skeletal—Stromal or Mesenchymal–Stem Cells Identified Candidates for Bone Regeneration

    DEFF Research Database (Denmark)

    Chang, Chi Chih; Venø, Morten T.; Chen, Li

    2018-01-01

    Bone remodeling and regeneration are highly regulated multistep processes involving posttranscriptional regulation by microRNAs (miRNAs). Here, we performed a global profiling of differentially expressed miRNAs in bone-marrow-derived skeletal cells (BMSCs; also known as stromal or mesenchymal stem......RNAs for enhancing bone tissue regeneration. Scaffolds functionalized with miRNA nano-carriers enhanced osteoblastogenesis in 3D culture and retained this ability at least 2 weeks after storage. Additionally, anti-miR-222 enhanced in vivo ectopic bone formation through targeting the cell-cycle inhibitor CDKN1B...... cells) during in vitro osteoblast differentiation. We functionally validated the regulatory effects of several miRNAs on osteoblast differentiation and identified 15 miRNAs, most significantly miR-222 and miR-423, as regulators of osteoblastogenesis. In addition, we tested the possible targeting of mi...

  19. Bone Marrow Stromal Cells Generate Muscle Cells and Repair Muscle Degeneration

    Science.gov (United States)

    Dezawa, Mari; Ishikawa, Hiroto; Itokazu, Yutaka; Yoshihara, Tomoyuki; Hoshino, Mikio; Takeda, Shin-ichi; Ide, Chizuka; Nabeshima, Yo-ichi

    2005-07-01

    Bone marrow stromal cells (MSCs) have great potential as therapeutic agents. We report a method for inducing skeletal muscle lineage cells from human and rat general adherent MSCs with an efficiency of 89%. Induced cells differentiated into muscle fibers upon transplantation into degenerated muscles of rats and mdx-nude mice. The induced population contained Pax7-positive cells that contributed to subsequent regeneration of muscle upon repetitive damage without additional transplantation of cells. These MSCs represent a more ready supply of myogenic cells than do the rare myogenic stem cells normally found in muscle and bone marrow.

  20. Hematopoietic microenvironment. Origin, lineage, and transplantability of the stromal cells in long-term bone marrow cultures from chimeric mice

    International Nuclear Information System (INIS)

    Perkins, S.; Fleischman, R.A.

    1988-01-01

    Studies of bone marrow transplant patients have suggested that the stromal cells of the in vitro hematopoietic microenvironment are transplantable into conditioned recipients. Moreover, in patients with myeloproliferative disorders, all of the stromal cells, which include presumptive endothelial cells, appear to be derived from hematopoietic precursors. To confirm these findings, we have constructed two chimeric mouse models: (a) traditional radiation chimeras, and (b) fetal chimeras, produced by placental injection of bone marrow into genetically anemic Wx/Wv fetuses, a technique that essentially precludes engraftment of nonhematopoietic cells. Using two-color indirect immunofluorescence, the stromal cells in long-term bone marrow culture derived from these chimeras were analyzed for donor or host origin by strain-specific H-2 antigens, and for cell lineage by a variety of other specific markers. 75-95% of the stromal cells were shown to be hematopoietic cells of the monocyte-macrophage lineage, based upon donor origin, phagocytosis, and expression of specific hematopoietic surface antigens. The remaining 5-25% of the stromal cells were exclusively host in origin. Apart from occasional fat cells, these cells uniformly expressed collagen type IV, laminin, and a surface antigen associated with endothelial cells. Since these endothelial-like cells are not transplantable into radiation or fetal chimeras, they are not derived from hematopoietic stem cells. The contrast between our findings and human studies suggests either unexpected species differences in the origin of stromal lineages or limitations in the previous methodology used to detect nonhematopoietic stromal cells

  1. Radiosensitivity of marrow stromal cells and the effect of some radioprotective agents

    International Nuclear Information System (INIS)

    Liu Shuhua

    1992-01-01

    The results showed that marrow stromal cells include fibroblasts, reticular cells, macrophages and adipocytes. The capability of the adherent layer derived from marrow cells of 2 mouse femurs to support hematopoietic stem cells was stronger than those of layers derived from 0.5 or 1 mouse femurs. The radiosensitivity of bone marrow stromal cells was lower than that of hematopoietic stem cells. The radioprotective effect of AET and PLP (polysaccharide of Lobaria Pulmonaria Hoffm) on the bone marrow stromal cells and their capability to support hematopoietic stem cells was clearly demonstrated

  2. In vitro migration and proliferation ("wound healing") potential of mesenchymal stromal cells generated from human CD271(+) bone marrow mononuclear cells.

    Science.gov (United States)

    Latifi-Pupovci, Hatixhe; Kuçi, Zyrafete; Wehner, Sibylle; Bönig, Halvard; Lieberz, Ralf; Klingebiel, Thomas; Bader, Peter; Kuçi, Selim

    2015-09-25

    Emerging evidence indicates that mesenchymal stromal cells (MSCs) isolated from different tissue sources may be used in vivo as tissue restorative agents. To date, there is no evidence, however, on migration and proliferation ("wound healing") potential of different subsets of MSCs. The main goal of this study was therefore to compare the in vitro "wound healing" capacity of MSCs generated from positively selected CD271(+) bone marrow mononuclear cells (CD271-MSCs) and MSCs generated by plastic adherence (PA-MSCs). The in vitro model of wound healing (CytoSelect™ 24-Well Wound Healing Assay) was used in order to compare the migration and proliferation potential of CD271-MSCs and PA-MSCs of passage 2 and 4 cultured in presence or absence of growth factors or cytokines. CD271-MSCs of both passages when compared to PA-MSCs demonstrated a significantly higher potential to close the wound 12 and 24 h after initiation of the wound healing assay (P MSCs of second passage was significantly improved after stimulation with FGF-2 (P MSCs of P4 12 h after the treatment (P MSCs of both passages with growth factors or cytokines did not affect their migratory potential. Our in vitro data provide the first evidence that CD271-MSCs are significantly more potent in "wound healing" than their counterparts PA-MSCs.

  3. PS1/γ-Secretase-Mediated Cadherin Cleavage Induces β-Catenin Nuclear Translocation and Osteogenic Differentiation of Human Bone Marrow Stromal Cells

    Directory of Open Access Journals (Sweden)

    Danielle C. Bonfim

    2016-01-01

    Full Text Available Bone marrow stromal cells (BMSCs are considered a promising tool for bone bioengineering. However, the mechanisms controlling osteoblastic commitment are still unclear. Osteogenic differentiation of BMSCs requires the activation of β-catenin signaling, classically known to be regulated by the canonical Wnt pathway. However, BMSCs treatment with canonical Wnts in vitro does not always result in osteogenic differentiation and evidence indicates that a more complex signaling pathway, involving cadherins, would be required to induce β-catenin signaling in these cells. Here we showed that Wnt3a alone did not induce TCF activation in BMSCs, maintaining the cells at a proliferative state. On the other hand, we verified that, upon BMSCs osteoinduction with dexamethasone, cadherins were cleaved by the PS1/γ-secretase complex at the plasma membrane, and this event was associated with an enhanced β-catenin translocation to the nucleus and signaling. When PS1/γ-secretase activity was inhibited, the osteogenic process was impaired. Altogether, we provide evidence that PS1/γ-secretase-mediated cadherin cleavage has as an important role in controlling β-catenin signaling during the onset of BMSCs osteogenic differentiation, as part of a complex signaling pathway responsible for cell fate decision. A comprehensive map of these pathways might contribute to the development of strategies to improve bone repair.

  4. Breast carcinoma cells modulate the chemoattractive activity of human bone marrow-derived mesenchymal stromal cells by interfering with CXCL12.

    Science.gov (United States)

    Wobus, Manja; List, Catrin; Dittrich, Tobias; Dhawan, Abhishek; Duryagina, Regina; Arabanian, Laleh S; Kast, Karin; Wimberger, Pauline; Stiehler, Maik; Hofbauer, Lorenz C; Jakob, Franz; Ehninger, Gerhard; Anastassiadis, Konstantinos; Bornhäuser, Martin

    2015-01-01

    We investigated whether breast tumor cells can modulate the function of mesenchymal stromal cells (MSCs) with a special emphasis on their chemoattractive activity towards hematopoietic stem and progenitor cells (HSPCs). Primary MSCs as well as a MSC line (SCP-1) were cocultured with primary breast cancer cells, MCF-7, MDA-MB231 breast carcinoma or MCF-10A non-malignant breast epithelial cells or their conditioned medium. In addition, the frequency of circulating clonogenic hematopoietic progenitors was determined in 78 patients with breast cancer and compared with healthy controls. Gene expression analysis of SCP-1 cells cultured with MCF-7 medium revealed CXCL12 (SDF-1) as one of the most significantly downregulated genes. Supernatant from both MCF-7 and MDA-MB231 reduced the CXCL12 promoter activity in SCP-1 cells to 77% and 47%, respectively. Moreover, the CXCL12 mRNA and protein levels were significantly reduced. As functional consequence of lower CXCL12 levels, we detected a decreased trans-well migration of HSPCs towards MSC/tumor cell cocultures or conditioned medium. The specificity of this effect was confirmed by blocking studies with the CXCR4 antagonist AMD3100. Downregulation of SP1 and increased miR-23a levels in MSCs after contact with tumor cell medium as well as enhanced TGFβ1 expression were identified as potential molecular regulators of CXCL12 activity in MSCs. Moreover, we observed a significantly higher frequency of circulating colony-forming hematopoietic progenitors in patients with breast cancer compared with healthy controls. Our in vitro results propose a potential new mechanism by which disseminated tumor cells in the bone marrow may interfere with hematopoiesis by modulating CXCL12 in protected niches. © 2014 UICC.

  5. Isolation, culture and intraportal transplantation of rat marrow stromal cell

    International Nuclear Information System (INIS)

    Wang Ping; Wang Jianhua; Yan Zhiping; Li Wentao; Lin Genlai; Hu Meiyu; Wang Yanhong

    2004-01-01

    Objective: To observe the tracing and evolution of marrow stromal cell (MSC) after intraportal transplantation into the liver of homogenous rats, and to provide experimental data for MSC differentiation to hepatocyte in vivo. Methods: The MSC was isolated from the leg bone marrow of adult SD rats, and purified by culture-expanded in vitro. Before transplantation, MSC was labeled with DAPI. Then 10 5 MSC were intraportally transplanted into the homogenous rat liver. Rats were killed at 2 hours and 1, 2, 3 and 4 weeks after transplantation. The cryosection samples of liver and lung were observed under fluorescence microscopy. Results: MSC in vitro culture had high ability of proliferation. Except 4 rats were dead because of abdominal bleeding or infection, other recipients were healthy until sacrificed. The implantation cells were detected by identifying the DAPI labeled MSC in the host livers, but not in the host lungs. Conclusion: Intraportal transplanted MSC could immigrate and survive in the host livers at least for 4 weeks. They could immigrate from the small branches of portal veins to hepatic parenchyma

  6. Reducing macrophages to improve bone marrow stromal cell survival in the contused spinal cord.

    NARCIS (Netherlands)

    Ritfeld, G.J.; Nandoe Tewarie, R.D.S.; Rahiem, S.T.; Hurtado, A.; Roos, R.A.; Grotenhuis, A.; Oudega, M.

    2010-01-01

    We tested whether reducing macrophage infiltration would improve the survival of allogeneic bone marrow stromal cells (BMSC) transplanted in the contused adult rat thoracic spinal cord. Treatment with cyclosporine, minocycline, or methylprednisolone all resulted in a significant decrease in

  7. Bone marrow stromal elements in murine leukemia; Decreased CSF-producing fibroblasts and normal IL-1 expression by macrophages

    Energy Technology Data Exchange (ETDEWEB)

    Ben-Ishay, Z [Laboratory of Experimental Hematology, Department of Anatomy and Embryology, Hebrew University-Hadassah Medical School (Israel); Barak, V [Laboratory of Immunology, Department of Oncology, Hadassah University Hospital (Israel); Shoshan, S [Faculty of Dental Medicine, Connective Tissue Research Laboratory, Hebrew University, Jerusalem (Israel); Prindull, G [Department of Pediatrics, University of Gottingen, Gottingen (Germany, F.R.)

    1990-01-01

    A study of bone marrow stromal elements in murine acute myeloid leukemia (AML) was carried out. Our previous studies had indicated marrow stromal deficiency in murine AML. In the current investigation, separate stromal cells were cultured and the results obtained have shown that, while marrow stromal macrophages are normal in leukemia and express adequate amounts of IL-1, the fibroblasts are markedly reduced. However, if sufficient fibroblasts are pooled in vitro, they produce adequate amounts of CSF. Test of TNF{alpha} in leukemic cells CM, as possible cause of marrow stromal inhibition in leukemia, had not disclosed this cytokine. Further, it was observed that total body lethal irradiation of leukemic mice aggravates the stromal deficiency, confirming results of our previous investigations. It is concluded that bone marrow stromal deficiency in murine AML is due to decreased fibroblasts and, implicity, reduced CSF production. (author).

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

    OpenAIRE

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

    2014-01-01

    Background Iloprost, a stable prostacyclin I2 analogue, seems to have an osteoblast-protective potential, whereas indomethacin suppresses new bone formation. The aim of this study was to investigate human bone marrow stromal cell (BMSC) proliferation and differentiation towards the osteoblastic lineage by administration of indomethacin and/or iloprost. Material/Methods Human bone marrow cells were obtained from 3 different donors (A=26 yrs/m; B=25 yrs/f, C=35 yrs/m) via vacuum aspiration of t...

  9. CD34 defines an osteoprogenitor cell population in mouse bone marrow stromal cells

    DEFF Research Database (Denmark)

    Abdallah, Basem M; Al-Shammary, Asma; Skagen, Peter

    2015-01-01

    Bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) and their progenitors have been identified based on retrospective functional criteria. CD markers are employed to define cell populations with distinct functional characteristics. However, defining and pro...

  10. Stromal cell migration precedes hemopoietic repopulation of the bone marrow after irradiation

    International Nuclear Information System (INIS)

    Werts, E.D.; Gibson, D.P.; Knapp, S.A.; DeGowin, R.L.

    1980-01-01

    Circulation of hemopoietic stem cells into an irradiated site has been thoroughly documented, but migration of stromal cells to repair radiation damage has not. We determined the radiosensitivity of mouse bone marrow stroma and evaluated stromal and hemopoietic repopulation in x-irradiated marrow. The D 0 for growth of colonies of marrow stromal cells (MSC) was 215 to 230 rad. Total-body irradiation (TB) obliterated marrow stromal and hemopoietic cells within 3 days. In contrast, 1 day after 1000 rad leg irradiation (LI), MSC rose to 80% of normal, but fell to 34% by 3 days and recovered to 72% by 30 days. However, femoral nucleated cells diminished to 20% by 3 days and recovered to 74% of normal by 30 days. Likewise, differentiated marrow cells and hemopoietic stem cells were initially depleted. With 1000 rad LI followed 3 h later by 1000 rad to the body while shielding the leg, MSC and femoral nucleated cells recovered to values intermediate between 1000 rad TB and 1000 rad LI. We concluded that: (1) the D 0 for MSC was 215 to 230 rad, (2) stromal repopulation preceded hemopoietic recovery, and (3) immigration of stromal cells from an unirradiated sanctuary facilitated hemopoietic repopulation of a heavily irradiated site

  11. Insulin-producing Cells from Adult Human Bone Marrow Mesenchymal Stromal Cells Could Control Chemically Induced Diabetes in Dogs: A Preliminary Study.

    Science.gov (United States)

    Gabr, Mahmoud M; Zakaria, Mahmoud M; Refaie, Ayman F; Ismail, Amani M; Khater, Sherry M; Ashamallah, Sylvia A; Azzam, Maha M; Ghoneim, Mohamed A

    2018-01-01

    Ten mongrel dogs were used in this study. Diabetes was chemically induced in 7 dogs, and 3 dogs served as normal controls. For each diabetic dog, 5 million human bone marrow-derived mesenchymal stem cells/kg were differentiated to form insulin-producing cells using a trichostatin-based protocol. Cells were then loaded in 2 TheraCyte capsules which were transplanted under the rectus sheath. One dog died 4 d postoperatively from pneumonia. Six dogs were followed up with for 6 to 18 mo. Euglycemia was achieved in 4 dogs. Their glucose tolerance curves exhibited a normal pattern demonstrating that the encapsulated cells were glucose sensitive and insulin responsive. In the remaining 2 dogs, the fasting blood sugar levels were reduced but did not reach normal values. The sera of all transplanted dogs contained human insulin and C-peptide with a negligible amount of canine insulin. Removal of the transplanted capsules was followed by prompt return of diabetes. Intracytoplasmic insulin granules were seen by immunofluorescence in cells from the harvested capsules. Furthermore, all pancreatic endocrine genes were expressed. This study demonstrated that the TheraCyte capsule or a similar device can provide adequate immunoisolation, an important issue when stem cells are considered for the treatment of type 1 diabetes mellitus.

  12. Synthesis of embryonic tendon-like tissue by human marrow stromal/mesenchymal stem cells requires a three-dimensional environment and transforming growth factor β3.

    Science.gov (United States)

    Kapacee, Zoher; Yeung, Ching-Yan Chloé; Lu, Yinhui; Crabtree, David; Holmes, David F; Kadler, Karl E

    2010-10-01

    Tendon-like tissue generated from stem cells in vitro has the potential to replace tendons and ligaments lost through injury and disease. However, thus far, no information has been available on the mechanism of tendon formation in vitro and how to accelerate the process. We show here that human mesenchymal stem cells (MSCs) and bone marrow-derived mononuclear cells (BM-MNCs) can generate tendon-like tissue in 7days mediated by transforming growth factor (TGF) β3. MSCs cultured in fixed-length fibrin gels spontaneously synthesized narrow-diameter collagen fibrils and exhibited fibripositors (actin-rich, collagen fibril-containing plasma membrane protrusions) identical to those that occur in embryonic tendon. In contrast, BM-MNCs did not synthesize tendon-like tissue under these conditions. We performed real-time PCR analysis of MSCs and BM-MNCs. MSCs upregulated genes encoding type I collagen, TGFβ3, and Smad2 at the time of maximum contraction of the tendon-like tissue (7days). Western blot analysis showed phosphorylation of Smad2 at maximum contraction. The TGFβ inhibitor SB-431542, blocked the phosphorylation of Smad2 and stopped the formation of tendon-like tissue. Quantitative PCR showed that BM-MNCs expressed very low levels of TGFβ3 compared to MSCs. Therefore we added exogenous TGFβ3 protein to BM-MNCs in fibrin gels, which resulted in phosphorylation of Smad2, synthesis of collagen fibrils, the appearance of fibripositors at the plasma membrane, and the formation of tendon-like tissue. In conclusion, MSCs that self-generate TGFβ signaling or the addition of TGFβ3 protein to BM-MNCs in fixed-length fibrin gels spontaneously make embryonic tendon-like tissue in vitro within 7days. Copyright © 2010 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

  13. Bone Marrow Regeneration Promoted by Biophysically Sorted Osteoprogenitors From Mesenchymal Stromal Cells

    Science.gov (United States)

    Poon, Zhiyong; Lee, Wong Cheng; Guan, Guofeng; Nyan, Lin Myint; Lim, Chwee Teck; Han, Jongyoon

    2015-01-01

    Human tissue repair deficiencies can be supplemented through strategies to isolate, expand in vitro, and reimplant regenerative cells that supplant damaged cells or stimulate endogenous repair mechanisms. Bone marrow-derived mesenchymal stromal cells (MSCs), a subset of which is described as mesenchymal stem cells, are leading candidates for cell-mediated bone repair and wound healing, with hundreds of ongoing clinical trials worldwide. An outstanding key challenge for successful clinical translation of MSCs is the capacity to produce large quantities of cells in vitro with uniform and relevant therapeutic properties. By leveraging biophysical traits of MSC subpopulations and label-free microfluidic cell sorting, we hypothesized and experimentally verified that MSCs of large diameter within expanded MSC cultures were osteoprogenitors that exhibited significantly greater efficacy over other MSC subpopulations in bone marrow repair. Systemic administration of osteoprogenitor MSCs significantly improved survival rates (>80%) as compared with other MSC subpopulations (0%) for preclinical murine bone marrow injury models. Osteoprogenitor MSCs also exerted potent therapeutic effects as “cell factories” that secreted high levels of regenerative factors such as interleukin-6 (IL-6), interleukin-8 (IL-8), vascular endothelial growth factor A, bone morphogenetic protein 2, epidermal growth factor, fibroblast growth factor 1, and angiopoietin-1; this resulted in increased cell proliferation, vessel formation, and reduced apoptosis in bone marrow. This MSC subpopulation mediated rescue of damaged marrow tissue via restoration of the hematopoiesis-supporting stroma, as well as subsequent hematopoiesis. Together, the capabilities described herein for label-freeisolation of regenerative osteoprogenitor MSCs can markedly improve the efficacy of MSC-based therapies. PMID:25411477

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

    Czech Academy of Sciences Publication Activity Database

    Forostyak, Oksana; Butenko, Olena; Anděrová, Miroslava; Forostyak, Serhiy; Syková, Eva; Verkhratsky, A.; Dayanithi, Govindan

    2016-01-01

    Roč. 16, č. 3 (2016), s. 622-634 ISSN 1873-5061 R&D Projects: GA ČR(CZ) GA14-34077S; GA ČR(CZ) GAP304/11/2373; GA ČR(CZ) GBP304/12/G069 Institutional support: RVO:68378041 Keywords : adipose derived stromal cells * bone marrow stromal cell * Ca(2+) signaling * Ion channels Subject RIV: FH - Neurology Impact factor: 3.494, year: 2016

  15. Characterization of Cellular and Molecular Heterogeneity of Bone Marrow Stromal Cells

    DEFF Research Database (Denmark)

    Elsafadi, Mona; Manikandan, Muthurangan; Atteya, Muhammad

    2016-01-01

    and osteoblast differentiation genes which included several homeobox genes: TBX15, HOXA2 and HOXA10, and IGF1, FGFR3, BMP6, MCAM, ITGA10, IGFBP5, and ALP. siRNA-based downregulation of the ALP gene in CL1 impaired osteoblastic and adipocytic differentiation. Our studies demonstrate the existence of molecular......Human bone marrow-derived stromal stem cells (hBMSC) exhibit multiple functions, including differentiation into skeletal cells (progenitor function), hematopoiesis support, and immune regulation (nonprogenitor function). We have previously demonstrated the presence of morphological and functional...... and functional heterogeneity in cultured hBMSC. ALP can be employed to identify osteoblastic and adipocytic progenitor cells in the heterogeneous hBMSC cultures...

  16. Comparsion between Intravenous Delivered Human Fetal Bone Marrow Mesenchymal Stromal Cells and Mononuclear Cells in the Treatment of Rat Cerebral Infarct.

    Science.gov (United States)

    Huang, Ai-Hua; Zhang, Ping-Ping; Zhang, Bin; Ma, Bu-Qing; Guan, Yun-Qian; Zhou, Yi-Dan

    2016-10-10

    Objective To compare the effecacy of human mesenchymal stromal cell (hMSC) with human mononuclear cell (hMNC) in treating rat cerebral infarct.Methods The SD rat models of cerebral infarct were established by distal middle cerebral artery occlusion (dMCAO). Rats were divided into four groups: sham,ischemia vehicle,MSC,and MNC transplantation groups. For the transplantation group,1×10 6 hMSCs or hMNCs were intravascularly transplanted into the tail vein 1 hour after the ischemia onset. The ischemia vehicle group received dMCAO surgery and intravascular saline injection 1,3,5,and 7 days after the ischemia onset,and then behavioral tests were performed. At 48 h after the ischemia onset,the abundance of Iba- 1,the symbol of activated microglia,was evaluated in the peri-ischemia striatum area; meanwhile,the neurotrophic factors such as glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) in ipsilateral peri-ischemia striatum area were also measured. Results The relative infarct volume in ischemia vehicle group,hMSC group,and hMNC transplantation group were (37.85±4.40)%,(33.41±3.82)%,and (30.23±3.63)%,respectively. The infarct volumes of MSC group (t=2.100,P=0.034) and MNC group (t=2.109,P=0.0009) were significantly smaller than that of ischemia vehicle group,and that of MNC group was significantly smaller than that of MSC group (t=1.743,P=0.043). One day after transplantation,the score of ischemia vehicle group in limb placing test was (4.32±0.71)%,which was significantly lower than that in sham group (9.73±0.36)% (t=2.178,P=8.61×10 -11 ). The scores of MSC and MNC group,which were (5.09±0.62)% (t=2.1009,P=0.024) and (5.90±0.68)% (t=2.1008,P=0.0001),respectively,were significantly higher than that of ischemia vehicle group; also,the score of MNC group was significantly higher than that of MSC group(t=2.1009,P=0.0165). The contralateral forelimb scores of MSC and MNC groups in beam walking test were (5.56±0.86)% (t=2

  17. Bone marrow stromal and vascular smooth muscle cells have chemosensory capacity via bitter taste receptor expression.

    Directory of Open Access Journals (Sweden)

    Troy C Lund

    Full Text Available The ability of cells to detect changes in the microenvironment is important in cell signaling and responsiveness to environmental fluctuations. Our interest is in understanding how human bone marrow stromal-derived cells (MSC and their relatives, vascular smooth muscle cells (VSMC, interact with their environment through novel receptors. We found, through a proteomics screen, that MSC express the bitter taste receptor, TAS2R46, a protein more typically localized to the taste bud. Expression was also confirmed in VSMCs. A prototypical bitter compound that binds to the bitter taste receptor class, denatonium, increased intracellular calcium release and decreased cAMP levels as well as increased the extracellular release of ATP in human MSC. Denatonium also bound and activated rodent VSMC with a change in morphology upon compound exposure. Finally, rodents given denatonium in vivo had a significant drop in blood pressure indicating a vasodilator response. This is the first description of chemosensory detection by MSC and VSMCs via a taste receptor. These data open a new avenue of research into discovering novel compounds that operate through taste receptors expressed by cells in the marrow and vascular microenvironments.

  18. Low-frequency vibration treatment of bone marrow stromal cells induces bone repair in vivo.

    Science.gov (United States)

    He, Shengwei; Zhao, Wenzhi; Zhang, Lu; Mi, Lidong; Du, Guangyu; Sun, Chuanxiu; Sun, Xuegang

    2017-01-01

    To study the effect of low-frequency vibration on bone marrow stromal cell differentiation and potential bone repair in vivo . Forty New Zealand rabbits were randomly divided into five groups with eight rabbits in each group. For each group, bone defects were generated in the left humerus of four rabbits, and in the right humerus of the other four rabbits. To test differentiation, bones were isolated and demineralized, supplemented with bone marrow stromal cells, and implanted into humerus bone defects. Varying frequencies of vibration (0, 12.5, 25, 50, and 100 Hz) were applied to each group for 30 min each day for four weeks. When the bone defects integrated, they were then removed for histological examination. mRNA transcript levels of runt-related transcription factor 2, osteoprotegerin, receptor activator of nuclear factor κ-B ligan, and pre-collagen type 1 α were measured. Humeri implanted with bone marrow stromal cells displayed elevated callus levels and wider, more prevalent, and denser trabeculae following treatment at 25 and 50 Hz. The mRNA levels of runt-related transcription factor 2, osteoprotegerin, receptor activator of nuclear factor κ-B ligand, and pre-collagen type 1 α were also markedly higher following 25 and 50 Hz treatment. Low frequency (25-50 Hz) vibration in vivo can promote bone marrow stromal cell differentiation and repair bone injury.

  19. Low-frequency vibration treatment of bone marrow stromal cells induces bone repair in vivo

    Directory of Open Access Journals (Sweden)

    Shengwei He

    2017-01-01

    Full Text Available Objective(s:To study the effect of low-frequency vibration on bone marrow stromal cell differentiation and potential bone repair in vivo. Materials and Methods:Forty New Zealand rabbits were randomly divided into five groups with eight rabbits in each group. For each group, bone defects were generated in the left humerus of four rabbits, and in the right humerus of the other four rabbits. To test differentiation, bones were isolated and demineralized, supplemented with bone marrow stromal cells, and implanted into humerus bone defects. Varying frequencies of vibration (0, 12.5, 25, 50, and 100 Hz were applied to each group for 30 min each day for four weeks. When the bone defects integrated, they were then removed for histological examination. mRNA transcript levels of runt-related transcription factor 2, osteoprotegerin, receptor activator of nuclear factor k-B ligan, and pre-collagen type 1 a were measured. Results:Humeri implanted with bone marrow stromal cells displayed elevated callus levels and wider, more prevalent, and denser trabeculae following treatment at 25 and 50 Hz. The mRNA levels of runt-related transcription factor 2, osteoprotegerin, receptor activator of nuclear factor k-B ligand, and pre-collagen type 1 a were also markedly higher following 25 and 50 Hz treatment. Conclusion:Low frequency (25–50 Hz vibration in vivo can promote bone marrow stromal cell differentiation and repair bone injury.

  20. Aging of marrow stromal (skeletal) stem cells and their contribution to age-related bone loss

    DEFF Research Database (Denmark)

    Bellantuono, Ilaria; Aldahmash, Abdullah; Kassem, Moustapha

    2009-01-01

    Marrow stromal cells (MSC) are thought to be stem cells with osteogenic potential and therefore responsible for the repair and maintenance of the skeleton. Age related bone loss is one of the most prevalent diseases in the elder population. It is controversial whether MSC undergo a process of agi...

  1. Aging is associated with decreased maximal life span and accelerated senescence of bone marrow stromal cells

    DEFF Research Database (Denmark)

    Dokkedahl, Karin Stenderup; Justesen, Jeannette; Clausen, Christian

    2003-01-01

    Age-related decrease in bone formation is well described. However, the cellular causes are not known. Thus, we have established cultures of bone marrow stromal cells (MSC) from young (aged 18-29 years, n = 6) and old (aged 68-81 years, n = 5) donors. MSC were serially passaged until reaching maxi...

  2. Ceramic hydroxyapatite coating on titanium implants drives selective bone marrow stromal cell adhesion.

    NARCIS (Netherlands)

    Torensma, R.; Brugge, P.J. ter; Jansen, J.A.; Figdor, C.G.

    2003-01-01

    The aim of this study was to determine the cell characteristics that regulate implant osseointegration. The heterogeneity of bone marrow stromal cells obtained from 11 donors was assessed by measuring the expression of a large panel of adhesion molecules. Large differences in expression of adhesion

  3. [Bone marrow stromal damage mediated by immune response activity].

    Science.gov (United States)

    Vojinović, J; Kamenov, B; Najman, S; Branković, Lj; Dimitrijević, H

    1994-01-01

    The aim of this work was to estimate influence of activated immune response on hematopoiesis in vitro, using the experimental model of BCG immunized BALB/c mice and in patients with chronic immunoactivation: long-lasting infections, autoimmunity or malignancy. We correlated changes in long term bone marrow cultures (Dexter) and NBT reduction with appearance of anemia in patients and experimental model of immunization by BCG. Increased spontaneous NBT reduction pointed out role of macrophage activation in bone marrow stroma damage. Long-term bone marrow cultures showed reduced number of hematopoietic cells, with predomination of fibroblasts and loss of fat cells. This results correlated with anemia and leucocytosis with stimulated myelopoiesis in peripheral blood. Activation of immune response, or acting of any agent that directly changes extracellular matrix and cellularity of bone marrow, may result in microenviroment bone marrow damage that modify hematopoiesis.

  4. Establishing quiescence in human bone marrow stem cells leads to enhanced osteoblast marker expression

    DEFF Research Database (Denmark)

    Harkness, Linda; Rumman, Mohammad; Kassem, Moustapha

    Human bone marrow stromal (skeletal) stem cells (hBMSC) are cells that retain a multi-lineage differentiation potential and are thus increasingly being investigated for use in clinical applications. In vivo BMSC, which comprise approximately 0.1% of the bone marrow compartment, are thought to mai...

  5. Human multipotent mesenchymal stromal cells in the treatment of postoperative temporal bone defect: an animal model

    Czech Academy of Sciences Publication Activity Database

    Školoudík, L.; Chrobok, V.; Kalfert, D.; Kočí, Zuzana; Syková, Eva; Chumak, Tetyana; Popelář, Jiří; Syka, Josef; Laco, J.; Dědková, J.; Dayanithi, Govindan; Filip, S.

    2016-01-01

    Roč. 25, č. 7 (2016), s. 1405-1414 ISSN 0963-6897 R&D Projects: GA MŠk(CZ) LO1309 Institutional support: RVO:68378041 Keywords : Human bone marrow * Human mesenchymal stromal cells (hMSCs) * Middle ear surgery * Temporal bone Subject RIV: FP - Other Medical Disciplines Impact factor: 3.006, year: 2016

  6. Molecular characterisation of stromal populations derived from human embryonic stem cells

    DEFF Research Database (Denmark)

    Harkness, L.; Twine, N. A.; Abu Dawud, R.

    2015-01-01

    Human bone marrow-derived stromal (skeletal) stem cells (BM-hMSC) are being employed in an increasing number of clinical trials for tissue regeneration. A limiting factor for their clinical use is the inability to obtain sufficient cell numbers. Human embryonic stem cells (hESC) can provide an un...

  7. Cryopreservation and revival of human mesenchymal stromal cells

    DEFF Research Database (Denmark)

    Haack-Sørensen, Mandana; Ekblond, Annette; Kastrup, Jens

    2016-01-01

    Cell-based therapy is a promising and innovative new treatment for different degenerative and autoimmune diseases, and mesenchymal stromal cells (MSCs) from the bone marrow have demonstrated great therapeutic potential due to their immunosuppressive and regenerative capacities. The establishment ...

  8. Osteogenic potential of bone marrow stromal cells on smooth, roughened, and tricalcium phosphate-modified titanium alloy surfaces.

    LENUS (Irish Health Repository)

    Colombo, John S

    2012-09-01

    This study investigated the influence of smooth, roughened, and tricalcium phosphate (TCP)-coated roughened titanium-aluminum-vanadium (Ti-6Al-4V) surfaces on the osteogenic potential of rat bone marrow stromal cells (BMSCs).

  9. A proteome study of secreted prostatic factors affecting osteoblastic activity: galectin-1 is involved in differentiation of human bone marrow stromal cells

    DEFF Research Database (Denmark)

    Andersen, H; Jensen, Ole N; Moiseeva, Elena P

    2003-01-01

    Prostate cancer cells metastasize to bone causing a predominantly osteosclerotic response. It has been shown that cells from the human prostate cancer cell line PC3 secrete factors that influence the behavior of osteoblast-like cells. Some of these factors with mitogenic activity have been found...... to be proteins with molecular weights between 20 and 30 kDa, but the identity of the osteoblastic mitogenic factor or factors produced by prostate cancer cells is still unknown. Therefore, the aim of this study was to characterize the protein profile of conditioned medium (CM) from PC3 cells in the molecular......BMS) cells. Furthermore, we tested whether adhesion of PC3 cells to plastic, laminin, fibronectin, and collagen type I was influenced by lactose, which inhibits galectin-1. Galectin-1 (1000 ng/ml) inhibited the proliferation of hBMS cells up to 70 +/- 12% (treated/control) of control in contrast to PC3 CM...

  10. Silencing of RB1 and RB2/P130 during adipogenesis of bone marrow stromal cells results in dysregulated differentiation.

    Science.gov (United States)

    Capasso, Stefania; Alessio, Nicola; Di Bernardo, Giovanni; Cipollaro, Marilena; Melone, Mariarosa Ab; Peluso, Gianfranco; Giordano, Antonio; Galderisi, Umberto

    2014-01-01

    Bone marrow adipose tissue (BMAT) is different from fat found elsewhere in the body, and only recently have some of its functions been investigated. BMAT may regulate bone marrow stem cell niche and plays a role in energy storage and thermogenesis. BMAT may be involved also in obesity and osteoporosis onset. Given the paramount functions of BMAT, we decided to better clarify the human bone marrow adipogenesis by analyzing the role of the retinoblastoma gene family, which are key players in cell cycle regulation. Our data provide evidence that the inactivation of RB1 or RB2/P130 in uncommitted bone marrow stromal cells (BMSC) facilitates the first steps of adipogenesis. In cultures with silenced RB1 or RB2/P130, we observed an increase of clones with adipogenic potential and a higher percentage of cells accumulating lipid droplets. Nevertheless, the absence of RB1 or RB2/P130 impaired the terminal adipocyte differentiation and gave rise to dysregulated adipose cells, with alteration in lipid uptake and release. For the first time, we evidenced that RB2/P130 plays a role in bone marrow adipogenesis. Our data suggest that while the inactivation of retinoblastoma proteins may delay the onset of last cell division and allow more BMSC to be committed to adipocyte, it did not allow a permanent cell cycle exit, which is a prerequisite for adipocyte terminal maturation.

  11. On the role of subtype selective adenosine receptor agonists during proliferation and osteogenic differentiation of human primary bone marrow stromal cells.

    Science.gov (United States)

    Costa, M Adelina; Barbosa, A; Neto, E; Sá-e-Sousa, A; Freitas, R; Neves, J M; Magalhães-Cardoso, T; Ferreirinha, F; Correia-de-Sá, P

    2011-05-01

    Purines are important modulators of bone cell biology. ATP is metabolized into adenosine by human primary osteoblast cells (HPOC); due to very low activity of adenosine deaminase, the nucleoside is the end product of the ecto-nucleotidase cascade. We, therefore, investigated the expression and function of adenosine receptor subtypes (A(1) , A(2A) , A(2B) , and A(3) ) during proliferation and osteogenic differentiation of HPOC. Adenosine A(1) (CPA), A(2A) (CGS21680C), A(2B) (NECA), and A(3) (2-Cl-IB-MECA) receptor agonists concentration-dependently increased HPOC proliferation. Agonist-induced HPOC proliferation was prevented by their selective antagonists, DPCPX, SCH442416, PSB603, and MRS1191. CPA and NECA facilitated osteogenic differentiation measured by increases in alkaline phosphatase (ALP) activity. This contrasts with the effect of CGS21680C which delayed HPOC differentiation; 2-Cl-IB-MECA was devoid of effect. Blockade of the A(2B) receptor with PSB603 prevented osteogenic differentiation by NECA. In the presence of the A(1) antagonist, DPCPX, CPA reduced ALP activity at 21 and 28 days in culture. At the same time points, blockade of A(2A) receptors with SCH442416 transformed the inhibitory effect of CGS21680C into facilitation. Inhibition of adenosine uptake with dipyridamole caused a net increase in osteogenic differentiation. The presence of all subtypes of adenosine receptors on HPOC was confirmed by immunocytochemistry. Data show that adenosine is an important regulator of osteogenic cell differentiation through the activation of subtype-specific receptors. The most abundant A(2B) receptor seems to have a consistent role in cell differentiation, which may be balanced through the relative strengths of A(1) or A(2A) receptors determining whether osteoblasts are driven into proliferation or differentiation. Copyright © 2010 Wiley-Liss, Inc.

  12. Bone marrow stromal cell therapy for ischemic stroke: A meta-analysis of randomized control animal trials.

    Science.gov (United States)

    Wu, Qing; Wang, Yuexiang; Demaerschalk, Bart M; Ghimire, Saruna; Wellik, Kay E; Qu, Wenchun

    2017-04-01

    Background Results of animal studies assessing efficacy of bone marrow stromal cell therapy for ischemic stroke remain inconsistent. Aims The aims are to assess efficacy of bone marrow stromal cell therapy for ischemic stroke in animal studies. Methods Randomized controlled animal trials assessing efficacy of bone marrow stromal cell therapy were eligible. Stroke therapy academic industry round table was used to assess methodologic quality of included studies. Primary outcomes were total infarction volume and modified Neurological Severity Score. Multiple prespecified sensitivity analyses and subgroup analyses were conducted. Random effects models were used for meta-analysis. Results Thirty-three randomized animal trials were included with a total of 796 animals. The median quality score was 6 (interquartile range, 5-7). Bone marrow stromal cell therapy decreased total infarction volume (standardized mean difference, 0.897; 95% confidence interval, 0.553-1.241; P animals treated with bone marrow stromal cell and controls was 2.47 (95% confidence interval, 1.84-3.11; P animal studies. Conclusions Bone marrow stromal cell therapy significantly decreased total infarction volume and increased neural functional recovery in randomized controlled animal models of ischemic stroke.

  13. Analysis of bone marrow stromal cell transferred bacterial {beta}-galactosidase gene by PIXE

    Energy Technology Data Exchange (ETDEWEB)

    Kumakawa, Toshiro [Tokyo Metropolitan Geriatric Hospital, Tokyo (Japan). Dept. of Blood Transfusion and Hematology; Hibino, Hitoshi; Tani, Kenzaburo; Asano, Shigetaka; Futatugawa, Shouji; Sera, Kouichiro

    1997-12-31

    PIXE, Particle Induced X-ray Emission, is a powerful, multi-elemental analysis method which has many distinguishing features and has been used in varies research fields. Recently the method of applying baby cyclotrons for nuclear medicine to PIXE has been developed. This enables us to study biomedical phenomena from the physical point of view. Mouse bone marrow stromal cells were transferred bacterial {beta}-galactosidase gene (LacZ gene) by murine retroviral vectors. Analysis of the bone marrow stromal cells with the LacZ gene by PIXE revealed remarkable changes of intracellular trace elements compared with the normal control cells. These results indicate that gene transfer by retroviral vectors may bring about a dynamic change of intracellular circumstances of the target cell. (author)

  14. The globoseries glycosphingolipid SSEA-4 is a marker of bone marrow-derived clonal multipotent stromal cells in vitro and in vivo.

    Science.gov (United States)

    Rosu-Myles, Michael; McCully, Jennifer; Fair, Joel; Mehic, Jelica; Menendez, Pablo; Rodriguez, Rene; Westwood, Carole

    2013-05-01

    The therapeutic potential of multipotent stromal cells (MSC) may be enhanced by the identification of markers that allow their discrimination and enumeration both in vivo and in vitro. Here, we investigated the ability of embryonic stem cell-associated glycosphingolipids to isolate human MSC from both whole-bone-marrow (BM) and stromal cell cultures. Only SSEA-4 was consistently expressed on cells within the CD45loCD105hi marrow fraction and could be used to isolate cells with the capacity to give rise to stromal cultures containing MSC. Human stromal cultures, generated in either the presence or absence of serum, contained heterogeneous cell populations discriminated by the quantity of SSEA-4 epitopes detected on their surface. A low level of surface SSEA-4 (SSEA-4lo) correlated with undetectable levels of the α2,3-sialyltransferase-II enzyme required to synthesize SSEA-4; a reduced proliferative potential; and the loss of fat-, bone-, and cartilage-forming cells during long-term culture. In vitro, single cells with the capacity to generate multipotent stromal cultures were detected exclusively in the SSEA-4hi fraction. Our data demonstrate that a high level of surface epitopes for SSEA-4 provides a definitive marker of MSC from human BM.

  15. Ultrastructural and radiobiological characterization of stromal cells in continuous, long-term marrow culture

    International Nuclear Information System (INIS)

    Tavassoli, M.

    1982-01-01

    Hemopoietic stromal cells were studied in continuous, long-term marrow culture. A correlative study was carried out involving cytochemistry as well as scanning (SEM), and transmission electron microscopy (TEM) with sections cut either perpendicular or parallel to the substratum. Only two stromal cell types were identified: epithelioid cells and macrophages. The appearance of these cells, however, varied according to their topography in the culture and the method of observation; a finding that may explain the multiplicity of the cell types reported in these cultures. The two cell types displayed considerable interconnections and interactions which may be essential in their support function for the proliferation and maintenance of hemopoietic stem cells. They also demonstrated numerous coated pits and vesicles suggestive of extensive receptor-mediated endocytosis. Stromal cells, generally thought to be relatively radioresistant, demonstrated hitherto unrecognized radiosensitivity in culture. Doses of radiation as low as 500 rads interfered with their support function for the maintenance of the hemopoietic stem cell

  16. Superparamagnetic iron oxide nanoparticles labeling of bone marrow stromal (mesenchymal cells does not affect their "stemness".

    Directory of Open Access Journals (Sweden)

    Arun Balakumaran

    2010-07-01

    Full Text Available Superparamagnetic iron oxide nanoparticles (SPION are increasingly used to label human bone marrow stromal cells (BMSCs, also called "mesenchymal stem cells" to monitor their fate by in vivo MRI, and by histology after Prussian blue (PB staining. SPION-labeling appears to be safe as assessed by in vitro differentiation of BMSCs, however, we chose to resolve the question of the effect of labeling on maintaining the "stemness" of cells within the BMSC population in vivo. Assays performed include colony forming efficiency, CD146 expression, gene expression profiling, and the "gold standard" of evaluating bone and myelosupportive stroma formation in vivo in immuncompromised recipients. SPION-labeling did not alter these assays. Comparable abundant bone with adjoining host hematopoietic cells were seen in cohorts of mice that were implanted with SPION-labeled or unlabeled BMSCs. PB+ adipocytes were noted, demonstrating their donor origin, as well as PB+ pericytes, indicative of self-renewal of the stem cell in the BMSC population. This study confirms that SPION labeling does not alter the differentiation potential of the subset of stem cells within BMSCs.

  17. HOX and TALE signatures specify human stromal stem cell populations from different sources.

    Science.gov (United States)

    Picchi, Jacopo; Trombi, Luisa; Spugnesi, Laura; Barachini, Serena; Maroni, Giorgia; Brodano, Giovanni Barbanti; Boriani, Stefano; Valtieri, Mauro; Petrini, Mario; Magli, Maria Cristina

    2013-04-01

    Human stromal stem cell populations reside in different tissues and anatomical sites, however a critical question related to their efficient use in regenerative medicine is whether they exhibit equivalent biological properties. Here, we compared cellular and molecular characteristics of stromal stem cells derived from the bone marrow, at different body sites (iliac crest, sternum, and vertebrae) and other tissues (dental pulp and colon). In particular, we investigated whether homeobox genes of the HOX and TALE subfamilies might provide suitable markers to identify distinct stromal cell populations, as HOX proteins control cell positional identity and, together with their co-factors TALE, are involved in orchestrating differentiation of adult tissues. Our results show that stromal populations from different sources, although immunophenotypically similar, display distinct HOX and TALE signatures, as well as different growth and differentiation abilities. Stromal stem cells from different tissues are characterized by specific HOX profiles, differing in the number and type of active genes, as well as in their level of expression. Conversely, bone marrow-derived cell populations can be essentially distinguished for the expression levels of specific HOX members, strongly suggesting that quantitative differences in HOX activity may be crucial. Taken together, our data indicate that the HOX and TALE profiles provide positional, embryological and hierarchical identity of human stromal stem cells. Furthermore, our data suggest that cell populations derived from different body sites may not represent equivalent cell sources for cell-based therapeutical strategies for regeneration and repair of specific tissues. Copyright © 2012 Wiley Periodicals, Inc.

  18. Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice.

    Science.gov (United States)

    Maridas, David E; Rendina-Ruedy, Elizabeth; Le, Phuong T; Rosen, Clifford J

    2018-01-06

    Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

  19. Bone marrow stromal cell transplantation mitigates radiation-induced gastrointestinal syndrome in mice.

    Directory of Open Access Journals (Sweden)

    Subhrajit Saha

    Full Text Available Nuclear accidents and terrorism presents a serious threat for mass casualty. While bone-marrow transplantation might mitigate hematopoietic syndrome, currently there are no approved medical countermeasures to alleviate radiation-induced gastrointestinal syndrome (RIGS, resulting from direct cytocidal effects on intestinal stem cells (ISC and crypt stromal cells. We examined whether bone marrow-derived adherent stromal cell transplantation (BMSCT could restitute irradiated intestinal stem cells niche and mitigate radiation-induced gastrointestinal syndrome.Autologous bone marrow was cultured in mesenchymal basal medium and adherent cells were harvested for transplantation to C57Bl6 mice, 24 and 72 hours after lethal whole body irradiation (10.4 Gy or abdominal irradiation (16-20 Gy in a single fraction. Mesenchymal, endothelial and myeloid population were characterized by flow cytometry. Intestinal crypt regeneration and absorptive function was assessed by histopathology and xylose absorption assay, respectively. In contrast to 100% mortality in irradiated controls, BMSCT mitigated RIGS and rescued mice from radiation lethality after 18 Gy of abdominal irradiation or 10.4 Gy whole body irradiation with 100% survival (p<0.0007 and p<0.0009 respectively beyond 25 days. Transplantation of enriched myeloid and non-myeloid fractions failed to improve survival. BMASCT induced ISC regeneration, restitution of the ISC niche and xylose absorption. Serum levels of intestinal radioprotective factors, such as, R-Spondin1, KGF, PDGF and FGF2, and anti-inflammatory cytokines were elevated, while inflammatory cytokines were down regulated.Mitigation of lethal intestinal injury, following high doses of irradiation, can be achieved by intravenous transplantation of marrow-derived stromal cells, including mesenchymal, endothelial and macrophage cell population. BMASCT increases blood levels of intestinal growth factors and induces regeneration of the irradiated

  20. Transcriptional Profiling of Bone Marrow Stromal Cells in Response to Porphyromonas gingivalis Secreted Products

    Science.gov (United States)

    Reddi, Durga; Belibasakis, Georgios N.

    2012-01-01

    Periodontitis is an infectious inflammatory disease that destroys the tooth-supporting (periodontal) tissues. Porphyromonas gingivalis is an oral pathogen highly implicated in the pathogenesis of this disease. It can exert its effects to a number of cells, including osteogenic bone marrow stromal cells which are important for homeostastic capacity of the tissues. By employing gene microarray technology, this study aimed to describe the overall transcriptional events (>2-fold regulation) elicited by P. gingivalis secreted products in bone marrow stromal cells, and to dissect further the categories of genes involved in bone metabolism, inflammatory and immune responses. After 6 h of challenge with P. gingivalis, 271 genes were up-regulated whereas 209 genes were down-regulated, whereas after 24 h, these numbers were 259 and 109, respectively. The early (6 h) response was characterised by regulation of genes associated with inhibition of cell cycle, induction of apoptosis and loss of structural integrity, whereas the late (24 h) response was characterised by induction of chemokines, cytokines and their associated intracellular pathways (such as NF-κB), mediators of connective tissue and bone destruction, and suppression of regulators of osteogenic differentiation. The most strongly up-regulated genes were lipocalin 2 (LCN2) and serum amyloid A3 (SAA3), both encoding for proteins of the acute phase inflammatory response. Collectively, these transcriptional changes elicited by P. gingivalis denote that the fundamental cellular functions are hindered, and that the cells acquire a phenotype commensurate with propagated innate immune response and inflammatory-mediated tissue destruction. In conclusion, the global transcriptional profile of bone marrow stromal cells in response to P. gingivalis is marked by deregulated homeostatic functions, with implications in the pathogenesis of periodontitis. PMID:22937121

  1. The establishment of a bank of stored clinical bone marrow stromal cell products

    Directory of Open Access Journals (Sweden)

    Sabatino Marianna

    2012-02-01

    Full Text Available Abstract Background Bone marrow stromal cells (BMSCs are being used to treat a variety of conditions. For many applications a supply of cryopreserved products that can be used for acute therapy is needed. The establishment of a bank of BMSC products from healthy third party donors is described. Methods The recruitment of healthy subjects willing to donate marrow for BMSC production and the Good Manufacturing Practices (GMP used for assessing potential donors, collecting marrow, culturing BMSCs and BMSC cryopreservation are described. Results Seventeen subjects were enrolled in our marrow collection protocol for BMSC production. Six of the 17 subjects were found to be ineligible during the donor screening process and one became ill and their donation was cancelled. Approximately 12 ml of marrow was aspirated from one posterior iliac crest of 10 donors; one donor donated twice. The BMSCs were initially cultured in T-75 flasks and then expanded for three passages in multilayer cell factories. The final BMSC product was packaged into units of 100 × 106 viable cells, cryopreserved and stored in a vapor phase liquid nitrogen tank under continuous monitoring. BMSC products meeting all lot release criteria were obtained from 8 of the 11 marrow collections. The rate of growth of the primary cultures was similar for all products except those generated from the two oldest donors. One lot did not meet the criteria for final release; its CD34 antigen expression was greater than the cut off set at 5%. The mean number of BMSC units obtained from each donor was 17 and ranged from 3 to 40. Conclusions The production of large numbers of BMSCs from bone marrow aspirates of healthy donors is feasible, but is limited by the high number of donors that did not meet eligibility criteria and products that did not meet lot release criteria.

  2. A simple and efficient method for deriving neurospheres from bone marrow stromal cells

    International Nuclear Information System (INIS)

    Yang Qin; Mu Jun; Li Qi; Li Ao; Zeng Zhilei; Yang Jun; Zhang Xiaodong; Tang Jin; Xie Peng

    2008-01-01

    Bone marrow stromal cells (MSCs) can be differentiated into neuronal and glial-like cell types under appropriate experimental conditions. However, previously reported methods are complicated and involve the use of toxic reagents. Here, we present a simplified and nontoxic method for efficient conversion of rat MSCs into neurospheres that express the neuroectodermal marker nestin. These neurospheres can proliferate and differentiate into neuron, astrocyte, and oligodendrocyte phenotypes. We thus propose that MSCs are an emerging model cell for the treatment of a variety of neurological diseases

  3. Characteristics and function of bone marrow stromal adherent cells in normal and irradiated mice and guinea pigs

    Energy Technology Data Exchange (ETDEWEB)

    Changyu, Zheng; Ji, Liu; Xiaoying, Bi

    1986-04-01

    It has been shown from cytochemical and other characteristic studies of bone marrow stromal cells in CFU-F that there are seven types of stromal cells in the stromal adherent cell layer of normal and irradiated C/sub 57/ mice whereas there are only six types in guinea pigs. On the other hand, a radioresistant cell subtype appears in adherent layer after irradiation of both C/sub 57/ mice and guinea pig since the supernatant of cultured CFU-F of the normal and irradiated C/sub 57/ mice can stimulate production of CFU-Gm. It is justifiable that the bone marrow stromal adherent cells of the C/sub 57/ mice could produce CSF.

  4. Titanium biomaterials with complex surfaces induced aberrant peripheral circadian rhythms in bone marrow mesenchymal stromal cells.

    Science.gov (United States)

    Hassan, Nathaniel; McCarville, Kirstin; Morinaga, Kenzo; Mengatto, Cristiane M; Langfelder, Peter; Hokugo, Akishige; Tahara, Yu; Colwell, Christopher S; Nishimura, Ichiro

    2017-01-01

    Circadian rhythms maintain a high level of homeostasis through internal feed-forward and -backward regulation by core molecules. In this study, we report the highly unusual peripheral circadian rhythm of bone marrow mesenchymal stromal cells (BMSCs) induced by titanium-based biomaterials with complex surface modifications (Ti biomaterial) commonly used for dental and orthopedic implants. When cultured on Ti biomaterials, human BMSCs suppressed circadian PER1 expression patterns, while NPAS2 was uniquely upregulated. The Ti biomaterials, which reduced Per1 expression and upregulated Npas2, were further examined with BMSCs harvested from Per1::luc transgenic rats. Next, we addressed the regulatory relationship between Per1 and Npas2 using BMSCs from Npas2 knockout mice. The Npas2 knockout mutation did not rescue the Ti biomaterial-induced Per1 suppression and did not affect Per2, Per3, Bmal1 and Clock expression, suggesting that the Ti biomaterial-induced Npas2 overexpression was likely an independent phenomenon. Previously, vitamin D deficiency was reported to interfere with Ti biomaterial osseointegration. The present study demonstrated that vitamin D supplementation significantly increased Per1::luc expression in BMSCs, though the presence of Ti biomaterials only moderately affected the suppressed Per1::luc expression. Available in vivo microarray data from femurs exposed to Ti biomaterials in vitamin D-deficient rats were evaluated by weighted gene co-expression network analysis. A large co-expression network containing Npas2, Bmal1, and Vdr was observed to form with the Ti biomaterials, which was disintegrated by vitamin D deficiency. Thus, the aberrant BMSC peripheral circadian rhythm may be essential for the integration of Ti biomaterials into bone.

  5. Fate of bone marrow mesenchymal stromal cells following autologous transplantation in a rabbit model of osteonecrosis.

    Science.gov (United States)

    Sugaya, Hisashi; Mishima, Hajime; Gao, Ran; Kaul, Sunil C; Wadhwa, Renu; Aoto, Katsuya; Li, Meihua; Yoshioka, Tomokazu; Ogawa, Takeshi; Ochiai, Naoyuki; Yamazaki, Masashi

    2016-02-01

    Internalizing quantum dots (i-QDs) are a useful tool for tracking cells in vivo in models of tissue regeneration. We previously synthesized i-QDs by conjugating QDs with a unique internalizing antibody against a heat shock protein 70 family stress chaperone. In the present study, i-QDs were used to label rabbit mesenchymal stromal cells (MSCs) that were then transplanted into rabbits to assess differentiation potential in an osteonecrosis model. The i-QDs were taken up by bone marrow-derived MSCs collected from the iliac of 12-week-old Japanese white rabbits that were positive for cluster of differentiation (CD)81 and negative for CD34 and human leukocyte antigen DR. The average rate of i-QD internalization was 93.3%. At 4, 8, 12, and 24 weeks after transplantation, tissue repair was evaluated histologically and by epifluorescence and electron microscopy. The i-QDs were detected at the margins of the drill holes and in the necrotized bone trabecular. There was significant colocalization of the i-QD signal in transplanted cells and markers of osteoblast and mineralization at 4, 8, and 12 weeks post-transplantation, while i-QDs were detected in areas of mineralization at 12 and 24 weeks post-transplantation. Moreover, i-QDs were observed in osteoblasts in regenerated tissue by electron microscopy, demonstrating that the tissue was derived from transplanted cells. These results indicate that transplanted MSCs can differentiate into osteoblasts and induce tissue repair in an osteonecrosis model and can be tracked over the long term by i-QD labeling. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  6. Genetically engineered mesenchymal stromal cells produce IL-3 and TPO to further improve human scaffold-based xenograft models

    NARCIS (Netherlands)

    Carretta, M; Boer, de B.; Jaques, J.; Antonelli, A; Horton, S J; Yuan, H; de Bruijn, J D; Groen, R W J; Vellenga, E.; Schuringa, J J

    Recently, NOD-SLID IL2R gamma(-/-) (NSG) mice were implanted with human mesenchymal stromal cells (MSCs) in the presence of ceramic scaffolds or Matrigel to mimic the human bone marrow (BM) microenvironment. This approach allowed the engraftment of leukemic samples that failed to engraft in NSG mice

  7. Fetal Mesenchymal Stromal Cells Differentiating towards Chondrocytes Acquire a Gene Expression Profile Resembling Human Growth Plate Cartilage

    NARCIS (Netherlands)

    van Gool, S.A.; Emons, J.A.M.; Leijten, Jeroen Christianus Hermanus; Decker, E.; Sticht, C.; van Houwelingen, J.C.; Goeman, J.J.; Kleijburg, C.; Scherjon, S.; Gretz, N.; Wit, J.M.; Rappold, G.; Post, Janine Nicole; Karperien, Hermanus Bernardus Johannes

    2012-01-01

    Abstract We used human fetal bone marrow-derived mesenchymal stromal cells (hfMSCs) differentiating towards chondrocytes as an alternative model for the human growth plate (GP). Our aims were to study gene expression patterns associated with chondrogenic differentiation to assess whether

  8. Radiation effect on the generation and expression of bone marrow stromal cells

    International Nuclear Information System (INIS)

    Xu Xiaoya; Jin Weilao; Gao Jianjun; Zhou Yi; Sheng Hui

    2009-01-01

    In order to investigate focal flushing dose radiation effect on the generation, differentiation and gene expression of bone marrow stromal cells the femoral head of rats was irradiated at 30 Gy by 137 Cs γ-rays (dose rate: 0.83 Gy/min). Then the bone marrow stromal cells (BMSCs) was cultured. The ability of proliferation and colony formation was observed and the expression level of Cbf-α1, PPAR-γ, VEGF-a and KDR was detected by RT-PCR technology in two weeks later. It has been found that after partly irradiated by 137 Cs γ-rays the proliferation and the number of colony of BMSCs in irradiated group decreases obviously meanwhile the expression level of Cbf-α1, PPAR-γ and VEGF-a in irradiated BMSCs obviously decreases by 18.98 %, 9.46 %, 57.34 % and 5.56 % respectively compared to the normal BMSCs (p<0.05). It shows that the focal great radiation could damage the BMSCs and depress the generation, differentiation and the expression of the related genes obviously. (authors)

  9. Bone Marrow-Derived Mesenchymal Stromal Cells Enhanced by Platelet-Rich Plasma Maintain Adhesion to Scaffolds in Arthroscopic Simulation.

    Science.gov (United States)

    Hoberman, Alexander R; Cirino, Carl; McCarthy, Mary Beth; Cote, Mark P; Pauzenberger, Leo; Beitzel, Knut; Mazzocca, Augustus D; Dyrna, Felix

    2018-03-01

    To assess the response of bone marrow-derived mesenchymal stromal cells (bMSCs) enhanced by platelet-rich plasma (PRP) in the setting of a normal human tendon (NHT), a demineralized bone matrix (DBM), and a fibrin scaffold (FS) with simulated arthroscopic mechanical washout stress. Bone marrow was aspirated from the humeral head and concentrated. BMSCs were counted, plated, and grown to confluence. Cells were seeded onto 3 different scaffolds: (1) NHT, (2) DBM, and (3) FS. Each scaffold was treated with a combination of (+)/(-) PRP and (+)/(-) arthroscopic washout simulation. A period of 60 minutes was allotted before arthroscopic washout. Adhesion, proliferation, and differentiation assays were performed to assess cellular activity in each condition. Significant differences were seen in mesenchymal stromal cell adhesion, proliferation, and differentiation among the scaffolds. DBM and FS showed superior results to NHT for cell adhesion, proliferation, and differentiation. PRP significantly enhanced cellular adhesion, proliferation, and differentiation. Arthroscopic simulation did not significantly decrease bMSC adhesion. We found that the type of scaffold impacts bMSCs' behavior. Both scaffolds (DBM and FS) were superior to NHT. The use of an arthroscopic simulator did not significantly decrease the adhesion of bMSCs to the scaffolds nor did it decrease their biologic differentiation potential. In addition, PRP enhanced cellular adhesion, proliferation, and differentiation. Improved healing after tendon repair can lead to better clinical outcomes. BMSCs are attractive for enhancing healing given their accessibility and regenerative potential. Application of bMSCs using scaffolds as cell carriers relies on arthroscopic feasibility. Copyright © 2017 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.

  10. Early passage bone marrow stromal cells express genes involved in nervous system development supporting their relevance for neural repair

    NARCIS (Netherlands)

    Nandoe Tewarie, R.D.S.; Bossers, K.; Ritfeld, G.J.; Blits, B.; Grotenhuis, J.A.; Verhaagen, J.; Oudega, M.

    2011-01-01

    PURPOSE: The assessment of the capacity of bone marrow stromal cells (BMSC) to repair the nervous system using gene expression profiling. The evaluation of effects of long-term culturing on the gene expression profile of BMSC. METHODS: Fourty four k whole genome rat microarrays were used to study

  11. Structurally-diverse, PPARγ-activating environmental toxicants induce adipogenesis and suppress osteogenesis in bone marrow mesenchymal stromal cells

    International Nuclear Information System (INIS)

    Watt, James; Schlezinger, Jennifer J.

    2015-01-01

    Environmental obesogens are a newly recognized category of endocrine disrupting chemicals that have been implicated in contributing to the rising rates of obesity in the United States. While obesity is typically regarded as an increase in visceral fat, adipocyte accumulation in the bone has been linked to increased fracture risk, lower bone density, and osteoporosis. Exposure to environmental toxicants that activate peroxisome proliferator activated receptor γ (PPARγ), a critical regulator of the balance of differentiation between adipogenesis and osteogenesis, may contribute to the increasing prevalence of osteoporosis. However, induction of adipogenesis and suppression of osteogenesis are separable activities of PPARγ, and ligands may selectively alter these activities. It currently is unknown whether suppression of osteogenesis is a common toxic endpoint of environmental PPARγ ligands. Using a primary mouse bone marrow culture model, we tested the hypothesis that environmental toxicants acting as PPARγ agonists divert the differentiation pathway of bone marrow-derived multipotent mesenchymal stromal cells towards adipogenesis and away from osteogenesis. The toxicants tested included the organotins tributyltin and triphenyltin, a ubiquitous phthalate metabolite (mono-(2-ethylhexyl) phthalate, MEHP), and two brominated flame retardants (tetrabromobisphenol-a, TBBPA, and mono-(2-ethylhexyl) tetrabromophthalate, METBP). All of the compounds activated PPARγ1 and 2. All compounds increased adipogenesis (lipid accumulation, Fabp4 expression) and suppressed osteogenesis (alkaline phosphatase activity, Osx expression) in mouse primary bone marrow cultures, but with different potencies and efficacies. Despite structural dissimilarities, there was a strong negative correlation between efficacies to induce adipogenesis and suppress osteogenesis, with the organotins being distinct in their exceptional ability to suppress osteogenesis. As human exposure to a mixture of

  12. Nanofiber scaffolds influence organelle structure and function in bone marrow stromal cells.

    Science.gov (United States)

    Tutak, Wojtek; Jyotsnendu, Giri; Bajcsy, Peter; Simon, Carl G

    2017-07-01

    Recent work demonstrates that osteoprogenitor cell culture on nanofiber scaffolds can promote differentiation. This response may be driven by changes in cell morphology caused by the three-dimensional (3D) structure of nanofibers. We hypothesized that nanofiber effects on cell behavior may be mediated by changes in organelle structure and function. To test this hypothesis, human bone marrow stromal cells (hBMSCs) were cultured on poly(ε-caprolactone) (PCL) nanofibers scaffolds and on PCL flat spuncoat films. After 1 day-culture, hBMSCs were stained for actin, nucleus, mitochondria, and peroxisomes, and then imaged using 3D confocal microscopy. Imaging revealed that the hBMSC cell body (actin) and peroxisomal volume were reduced during culture on nanofibers. In addition, the nucleus and peroxisomes occupied a larger fraction of cell volume during culture on nanofibers than on films, suggesting enhancement of the nuclear and peroxisomal functional capacity. Organelles adopted morphologies with greater 3D-character on nanofibers, where the Z-Depth (a measure of cell thickness) was increased. Comparisons of organelle positions indicated that the nucleus, mitochondria, and peroxisomes were closer to the cell center (actin) for nanofibers, suggesting that nanofiber culture induced active organelle positioning. The smaller cell volume and more centralized organelle positioning would reduce the energy cost of inter-organelle vesicular transport during culture on nanofibers. Finally, hBMSC bioassay measurements (DNA, peroxidase, bioreductive potential, lactate, and adenosine triphosphate (ATP)) indicated that peroxidase activity may be enhanced during nanofiber culture. These results demonstrate that culture of hBMSCs on nanofibers caused changes in organelle structure and positioning, which may affect organelle functional capacity and transport. Published 2016. This article is a U.S. Government work and is in the public domain in the USA. J Biomed Mater Res Part B: Appl

  13. Rapid isolation of bone marrow mesenchymal stromal cells using integrated centrifuge-based technology.

    Science.gov (United States)

    Meppelink, Amanda M; Wang, Xing-Hua; Bradica, Gino; Barron, Kathryn; Hiltz, Kathleen; Liu, Xiang-Hong; Goldman, Scott M; Vacanti, Joseph P; Keating, Armand; Hoganson, David M

    2016-06-01

    The use of bone marrow-derived mesenchymal stromal cells (MSCs) in cell-based therapies is currently being developed for a number of diseases. Thus far, the clinical results have been inconclusive and variable, in part because of the variety of cell isolation procedures and culture conditions used in each study. A new isolation technique that streamlines the method of concentration and demands less time and attention could provide clinical and economic advantages compared with current methodologies. In this study, we evaluated the concentrating capability of an integrated centrifuge-based technology compared with standard Ficoll isolation. MSCs were concentrated from bone marrow aspirate using the new device and the Ficoll method. The isolation capabilities of the device and the growth characteristics, secretome production, and differentiation capacity of the derived cells were determined. The new MSC isolation device concentrated the bone marrow in 90 seconds and resulted in a mononuclear cell yield 10-fold higher and with a twofold increase in cell retention compared with Ficoll. The cells isolated using the device were shown to exhibit similar morphology and functional activity as assessed by growth curves and secretome production compared to the Ficoll-isolated cells. The surface marker and trilineage differentiation profile of the device-isolated cells was consistent with the known profile of MSCs. The faster time to isolation and greater cell yield of the integrated centrifuge-based technology may make this an improved approach for MSC isolation from bone marrow aspirates. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  14. Organotins Are Potent Activators of PPARγ and Adipocyte Differentiation in Bone Marrow Multipotent Mesenchymal Stromal Cells

    Science.gov (United States)

    Yanik, Susan C.; Baker, Amelia H.; Mann, Koren K.; Schlezinger, Jennifer J.

    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 rosiglitazone, a PPARγ agonist, bexarotene, an RXR agonist, or a series of organotins. Rosiglitazone and bexarotene potently activated adipocyte differentiation; however, bexarotene had a maximal efficacy of only 20% of that induced by rosiglitazone. Organotins (tributyltin [TBT], triphenyltin, and dibutyltin) also stimulated adipocyte differentiation (EC50 of 10–20nM) but with submaximal, structure-dependent efficacy. In coexposures, both bexarotene and TBT enhanced rosiglitazone-induced adipogenesis. To investigate the contribution of PPARγ to TBT-induced adipogenesis, we examined expression of PPARγ2, as well as its transcriptional target FABP4. TBT-induced PPARγ2 and FABP4 protein expression with an efficacy intermediate between rosiglitazone and bexarotene, similar to lipid accumulation. A PPARγ antagonist and PPARγ-specific small hairpin RNA suppressed TBT-induced differentiation, although to a lesser extent than rosiglitazone-induced differentiation, suggesting that TBT may engage alternate pathways. TBT and bexarotene, but not rosiglitazone, also induced the expression of TGM2 (an RXR target) and ABCA1 (a liver X receptor target). The results show that an environmental contaminant, acting with the same potency as a therapeutic drug, induces PPARγ-dependent adipocyte differentiation in bone marrow MSCs. Activation of multiple nuclear receptor pathways by organotins may have significant implications for bone physiology. PMID:21622945

  15. Aging of bone marrow mesenchymal stromal/stem cells: Implications on autologous regenerative medicine.

    Science.gov (United States)

    Charif, N; Li, Y Y; Targa, L; Zhang, L; Ye, J S; Li, Y P; Stoltz, J F; Han, H Z; de Isla, N

    2017-01-01

    With their proliferation, differentiation into specific cell types, and secretion properties, mesenchymal stromal/stem cells (MSC) are very interesting tools to be used in regenerative medicine. Bone marrow (BM) was the first MSC source characterized. In the frame of autologous MSC therapy, it is important to detect donor's parameters affecting MSC potency. Age of the donors appears as one parameter that could greatly affect MSC properties. Moreover, in vitro cell expansion is needed to obtain the number of cells necessary for clinical developments. It will lead to in vitro cell aging that could modify cell properties. This review recapitulates several studies evaluating the effect of in vitro and in vivo MSC aging on cell properties.

  16. Osterix enhances proliferation and osteogenic potential of bone marrow stromal cells

    International Nuclear Information System (INIS)

    Tu Qisheng; Valverde, Paloma; Chen, Jake

    2006-01-01

    Osterix (Osx) is a zinc-finger-containing transcription factor that is expressed in osteoblasts of all endochondral and membranous bones. In Osx null mice osteoblast differentiation is impaired and bone formation is absent. In this study, we hypothesized that overexpression of Osx in murine bone marrow stromal cells (BMSC) would be able to enhance their osteoblastic differentiation and mineralization in vitro. Retroviral transduction of Osx in BMSC cultured in non-differentiating medium did not affect expression of Runx2/Cbfa1, another key transcription factor of osteoblast differentiation, but induced an increase in the expression of other markers associated with the osteoblastic lineage including alkaline phosphatase, bone sialoprotein, osteocalcin, and osteopontin. Retroviral transduction of Osx in BMSC also increased their proliferation, alkaline phosphatase activity, and ability to form bone nodules. These events occurred without significant changes in the expression of α1(II) procollagen or lipoprotein lipase, which are markers of chondrogenic and adipogenic differentiation, respectively

  17. Bone marrow-derived mesenchymal stromal cell treatment in patients with severe ischaemic heart failure

    DEFF Research Database (Denmark)

    Mathiasen, Anders Bruun; Qayyum, Abbas Ali; Jørgensen, Erik

    2015-01-01

    AIMS: Regenerative treatment with mesenchymal stromal cells (MSCs) has been promising in patients with ischaemic heart failure but needs confirmation in larger randomized trials. We aimed to study effects of intra-myocardial autologous bone marrow-derived MSC treatment in patients with severe isc...... identified. CONCLUSION: Intra-myocardial injections of autologous culture expanded MSCs were safe and improved myocardial function in patients with severe ischaemic heart failure. STUDY REGISTRATION NUMBER: NCT00644410 (ClinicalTrials.gov)....... ischaemic heart failure. METHODS AND RESULTS: The MSC-HF trial is a randomized, double-blind, placebo-controlled trial. Patients were randomized 2 : 1 to intra-myocardial injections of MSC or placebo, respectively. The primary endpoint was change in left ventricular end-systolic volume (LVESV), measured...

  18. Induction of quiescence (G0) in bone marrow stromal stem cells enhances their stem cell characteristics

    DEFF Research Database (Denmark)

    Rumman, Mohammad; Majumder, Abhijit; Harkness, Linda

    2018-01-01

    Several studies have suggested that bone marrow stromal steam cells (BMSC) exist in a quiescent state (G0) within the in vivo niche; however, an explicit analysis of the biology of G0 state-BMSC has not been reported. We hypothesized that induction of G0 in BMSC might enhance their stem cell...... properties. Thus, we induced quiescence in BMSC in vitro by (a) suspension culture in a viscous medium or (b) culture on soft polyacrylamide substrate; and examined their molecular and functional phenotype. Induction of G0 was confirmed by bromo-deoxyuridine (BrdU) labelling and analysis of cell cycle gene...... expression. Upon reactivation and re-entry into cell cycle, G0 state-BMSC exhibited enhanced clonogenic self-renewal, preferential differentiation into osteoblastic rather than adipocytic cells and increased ectopic bone formation when implanted subcutaneously in vivo in immune-deficient mice, compared...

  19. In vitro inhibitory effects of imatinib mesylate on stromal cells and hematopoietic progenitors from bone marrow

    Directory of Open Access Journals (Sweden)

    P.B. Soares

    2013-01-01

    Full Text Available Imatinib mesylate (IM is used to treat chronic myeloid leukemia (CML because it selectively inhibits tyrosine kinase, which is a hallmark of CML oncogenesis. Recent studies have shown that IM inhibits the growth of several non-malignant hematopoietic and fibroblast cells from bone marrow (BM. The aim of the present study was to evaluate the effects of IM on stromal and hematopoietic progenitor cells, specifically in the colony-forming units of granulocyte/macrophage (CFU-GM, using BM cultures from 108 1.5- to 2-month-old healthy Swiss mice. The results showed that low concentrations of IM (1.25 µM reduced the growth of CFU-GM in clonogenic assays. In culture assays with stromal cells, fibroblast proliferation and α-SMA expression by immunocytochemistry analysis were also reduced in a concentration-dependent manner, with a survival rate of approximately 50% with a dose of 2.5 µM. Cell viability and morphology were analyzed using MTT and staining with acrydine orange/ethidium bromide. Most cells were found to be viable after treatment with 5 µM IM, although there was gradual growth inhibition of fibroblastic cells while the number of round cells (macrophage-like cells increased. At higher concentrations (15 µM, the majority of cells were apoptotic and cell growth ceased completely. Oil red staining revealed the presence of adipocytes only in untreated cells (control. Cell cycle analysis of stromal cells by flow cytometry showed a blockade at the G0/G1 phases in groups treated with 5-15 µM. These results suggest that IM differentially inhibits the survival of different types of BM cells since toxic effects were achieved.

  20. Transcriptome analysis of bone marrow mesenchymal stromal cells from patients with primary myelofibrosis

    Directory of Open Access Journals (Sweden)

    Christophe Martinaud

    2015-09-01

    Full Text Available Primary myelofibrosis (PMF is a clonal myeloproliferative neoplasm whose severity and treatment complexity are attributed to the presence of bone marrow (BM fibrosis and alterations of stroma impairing the production of normal blood cells. Despite the recently discovered mutations including the JAK2V617F mutation in about half of patients, the primitive event responsible for the clonal proliferation is still unknown. In the highly inflammatory context of PMF, the presence of fibrosis associated with a neoangiogenesis and an osteosclerosis concomitant to the myeloproliferation and to the increase number of circulating hematopoietic progenitors suggests that the crosstalk between hematopoietic and stromal cells is deregulated in the PMF BM microenvironmental niches. Within these niches, mesenchymal stromal cells (BM-MSC play a hematopoietic supportive role in the production of growth factors and extracellular matrix which regulate the proliferation, differentiation, adhesion and migration of hematopoietic stem/progenitor cells. A transcriptome analysis of BM-MSC in PMF patients will help to characterize their molecular alterations and to understand their involvement in the hematopoietic stem/progenitor cell deregulation that features PMF.

  1. Bone marrow-derived multipotent mesenchymal stromal cells from horses after euthanasia.

    Science.gov (United States)

    Schröck, Carmen; Eydt, Carina; Geburek, Florian; Kaiser, Lena; Päbst, Felicitas; Burk, Janina; Pfarrer, Christiane; Staszyk, Carsten

    2017-11-01

    Allogeneic equine multipotent mesenchymal stromal cells (eMSCs) have been proposed for use in regenerative therapies in veterinary medicine. A source of allogeneic eMSCs might be the bone marrow from euthanized horses. The purpose of this study was to compare in vitro characteristics of equine bone marrow derived eMSC (eBM-MSCs) from euthanized horses (eut-MSCs) and from narcotized horses (nar-MSCs). Eut-MSCs and nar-MSCs showed typical eMSC marker profiles (positive: CD44, CD90; negative: CD11a/CD18 and MHCII) and possessed tri-lineage differentiation characteristics. Although CD105 and MHCI expression varied, no differences were detected between eut-MSCs and nar-MSCs. Proliferation characteristics did not differ between eut-MSCs and nar-MSCs, but age dependent decrease in proliferation and increase in MHCI expression was detected. These results suggest the possible use of eut-MSCs for therapeutic applications and production of commercial available eBM-MSC products.

  2. Destiny of autologous bone marrow-derived stromal cells implanted in the vocal fold.

    Science.gov (United States)

    Kanemaru, Shin-ichi; Nakamura, Tatsuo; Yamashita, Masaru; Magrufov, Akhmar; Kita, Tomoko; Tamaki, Hisanobu; Tamura, Yoshihiro; Iguchi, Fuku-ichiro; Kim, Tae Soo; Kishimoto, Masanao; Omori, Koichi; Ito, Juichi

    2005-12-01

    The aim of this study was to investigate the destiny of implanted autologous bone marrow-derived stromal cells (BSCs) containing mesenchymal stem cells. We previously reported the successful regeneration of an injured vocal fold through implantation of BSCs in a canine model. However, the fate of the implanted BSCs was not examined. In this study, implanted BSCs were traced in order to determine the type of tissues resulting at the injected site of the vocal fold. After harvest of bone marrow from the femurs of green fluorescent transgenic mice, adherent cells were cultured and selectively amplified. By means of a fluorescence-activated cell sorter, it was confirmed that some cells were strongly positive for mesenchymal stem cell markers, including CD29, CD44, CD49e, and Sca-1. These cells were then injected into the injured vocal fold of a nude rat. Immunohistologic examination of the resected vocal folds was performed 8 weeks after treatment. The implanted cells were alive in the host tissues and showed positive expression for keratin and desmin, markers for epithelial tissue and muscle, respectively. The implanted BSCs differentiated into more than one tissue type in vivo. Cell-based tissue engineering using BSCs may improve the quality of the healing process in vocal fold injuries.

  3. Effects of Na/K-ATPase and its ligands on bone marrow stromal cell differentiation

    Directory of Open Access Journals (Sweden)

    Moustafa Sayed

    2014-07-01

    Full Text Available Endogenous ligands of Na/K-ATPase have been demonstrated to increase in kidney dysfunction and heart failure. It is also reported that Na/K-ATPase signaling function effects stem cell differentiation. This study evaluated whether Na/K-ATPase activation through its ligands and associated signaling functions affect bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells differentiation capacity. BMSCs were isolated from male Sprague–Dawley rats and cultured in minimal essential medium alpha (MEM-α supplemented with 15% Fetal Bovine serum (FBS. The results showed that marinobufagenin (MBG, a specific Na/K-ATPase ligand, potentiated rosiglitazone-induced adipogenesis in these BMSCs. Meanwhile, it attenuated BMSC osteogenesis. Mechanistically, MBG increased CCAAT/enhancer binding protein alpha (C/EBPα protein expression through activation of an extracellular regulated kinase (ERK signaling pathway, which leads to enhanced rosiglitazone-induced adipogenesis. Inhibition of ERK activation by U0126 blocks the effect of MBG on C/EBPα expression and on rosiglitazone-induced adipogenesis. Reciprocally, MBG reduced runt-related transcription factor 2 (RunX2 expression, which resulted in the inhibition of osteogenesis induced by β-glycerophosphate/ascorbic acid. MBG also potentiated rosiglitazone-induced adipogenesis in 3T3-L1 cells and in mouse BMSCs. These results suggest that Na/K-ATPase and its signaling functions are involved in the regulation of BMSCs differentiation.

  4. Radiosensitivity of stromal cells responsible for in vitro maintenance of hemopoietic stem cells in continuous, long-term marrow culture. [/sup 137/Cs; Mice

    Energy Technology Data Exchange (ETDEWEB)

    Tavassoli, M

    1982-05-01

    Marrow stromal cells are generally thought to be radioresistant. However, when the marrow was irradiated in vivo or in vitro before its use for the continuous longterm marrow culture, doses of radiation as low as 500 rad interfered with the establishment of the adherent stromal layer. Moreover, when the stromal layer was permitted to establish, similar doses of radiation interfered with its potential to support the proliferation and maintenance of the hemopoietic stem cell. Thus, marrow stromal cells appear to be more radiosensitive than hitherto thought. The type of damage may vary, however, according to the dose of radiation. Small doses may interfere with such functions as adhesion or cell division while larger doses may completely destroy the cell.

  5. Transforming growth factor-β inhibits CCAAT/enhancer-binding protein expression and PPARγ activity in unloaded bone marrow stromal cells

    International Nuclear Information System (INIS)

    Ahdjoudj, S.; Kaabeche, K.; Holy, X.; Fromigue, O.; Modrowski, D.; Zerath, E.; Marie, P.J.

    2005-01-01

    The molecular mechanisms regulating the adipogenic differentiation of bone marrow stromal cells in vivo remain largely unknown. In this study, we investigated the regulatory effects of transforming growth factor beta-2 (TGF-β2) on transcription factors involved in adipogenic differentiation induced by hind limb suspension in rat bone marrow stromal cells in vivo. Time course real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis of gene expression showed that skeletal unloading progressively increases the expression of CCAAT/enhancer-binding protein (C/EBP)α and C/EBPβ α at 5 days in bone marrow stromal cells resulting in increased peroxisome proliferator-activated receptor γ (PPARγ2) transcripts at 7 days. TGF-β2 administration in unloaded rats corrected the rise in C/EBPα and C/EBPβ transcripts induced by unloading in bone marrow stromal cells. This resulted in inhibition of PPARγ2 expression that was associated with increased Runx2 expression. Additionally, the inhibition of C/EBPα and C/EBPβ expression by TGF-β2 was associated with increased PPARγ serine phosphorylation in bone marrow stromal cells, a mechanism that inhibits PPARγ transactivating activity. The sequential inhibitory effect of TGF-β2 on C/EBPα, C/EBPβ, and PPARγ2 resulted in reduced LPL expression and abolition of bone marrow stromal cell adipogenic differentiation, which contributed to prevent bone loss induced by skeletal unloading. We conclude that TGF-β2 inhibits the excessive adipogenic differentiation of bone marrow stromal cells induced by skeletal unloading by inhibiting C/EBPα, C/EBPβ, and PPARγ expression and activity, which provides a sequential mechanism by which TGF-β2 regulates adipogenic differentiation of bone marrow stromal cells in vivo

  6. Aryl hydrocarbon receptor (AhR agonists suppress interleukin-6 expression by bone marrow stromal cells: an immunotoxicology study

    Directory of Open Access Journals (Sweden)

    Schlezinger Jennifer J

    2003-12-01

    Full Text Available Abstract Background Bone marrow stromal cells produce cytokines required for the normal growth and development of all eight hematopoietic cell lineages. Aberrant cytokine production by stromal cells contributes to blood cell dyscrasias. Consequently, factors that alter stromal cell cytokine production may significantly compromise the development of normal blood cells. We have shown that environmental chemicals, such as aromatic hydrocarbon receptor (AhR agonists, suppress B lymphopoiesis by modulating bone marrow stromal cell function. Here, we extend these studies to evaluate the potential for two prototypic AhR agonists, 7,12-dimethylbenz [a]anthracene (DMBA and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, to alter stromal cell cytokine responses. Methods Bone marrow stromal cells were treated with AhR agonists and bacterial lipopolysaccharide (LPS to mimic innate inflammatory cytokine responses and to study the effects of AhR ligands on those responses. Steady state cytokine RNA levels were screened by RNAse protection assays (RPA and quantified by real-time PCR. Cytokine (IL-6 protein production was measured by ELISA. NF-κB EMSAs were used to study IL-6 transcriptional regulation. Results RPAs indicated that AhR+ bone marrow stromal cells consistently up-regulated genes encoding IL-6 and LIF in response to LPS, presumably through activation of Toll-like receptor 4. Pre-treatment with low doses of DMBA or TCDD selectively abrogated IL-6 gene induction but had no effect on LIF mRNA. Real-time-PCR indicated a significant inhibition of IL-6 mRNA by AhR ligands within 1 hour of LPS challenge which was reflected in a profound down-regulation of IL-6 protein induction, with DMBA and TCDD suppressing IL-6 levels as much as 65% and 88%, respectively. This potent inhibitory effect persisted for at least 72 hours. EMSAs measuring NF-κB binding to IL-6 promoter sequences, an event known to induce IL-6 transcription, indicated a significant decrease in

  7. Mouse Embryonic Fibroblasts (MEF) Exhibit a Similar but not Identical Phenotype to Bone Marrow Stromal Stem Cells (BMSC)

    DEFF Research Database (Denmark)

    Saeed, Hamid; Taipaleenmäki, Hanna; Aldahmash, Abdullah M

    2012-01-01

    Mouse embryonic fibroblasts have been utilized as a surrogate stem cell model for the postnatal bone marrow-derived stromal stem cells (BMSC) to study mesoderm-type cell differentiation e.g. osteoblasts, adipocytes and chondrocytes. However, no formal characterization of MEF phenotype has been...... by real-time PCR analysis. Compared to BMSC, MEF exhibited a more enhanced differentiation into adipocyte and chondrocyte lineages. Interestingly, both MEF and BMSC formed the same amount of heterotopic bone and bone marrow elements upon in vivo subcutaneous implantation with hydroxyapatite...... and differentiation to osteoblasts, adipocytes and chondrocytes....

  8. Primary observation on adherent function of bone marrow stromal cells in mice post combined radiation-burn injury

    International Nuclear Information System (INIS)

    Chen Xinghua; Luo Chengji; Guo Chaohua; Wang Ping; Deng Xuecai

    1999-01-01

    Objective: To investigate the adherent function of bone marrow stromal cells in hematopoietic inductive microenvironment post combined radiation-burn injury. Methods: The expression of cell adhesion molecules including vascular cell adhesion molecule-1 (VCAM-1), fibro-connection (Fn), laminin (Ln) and collagen type IV (Col IV) on bone marrow stromal cells cultured in vitro was detected by flow cytometry and the binding capacity of bone marrow mononuclear cells to stromal cell adherence layer was tested by cell binding assay and cell binding blocking assay respectively from mice treated with 5.0 Gy γ-ray 15% of total body surface area (TBSA), third-degree burn injury and combined irradiation-burn injury, respectively. Results: 1. The expression levels of molecules mentioned above in burn-injured mice were the highest. The molecules levels in control mice were greater than those in radiation-injured mice, which were lower than those in mice with combined radiation-burn injury. 2. The binding capacity of stromal cell adherence layer in burn-injured mice was greater than that in control mice, and significantly increased from 3 to 7 days post injury as compared with that in controls, radiation-injured mice and combined radiation-burn-injured mice, respectively (P < 0.05-0.01). Contrarily, the capacity of binding in the radiation-injured and combined radiation-burn-injured mice was the lowest from 3 to 7 days post injury. 3. The binding rate of bone marrow mononuclear cells to stromal cell adherence layer descended in different degrees after pre-treatment with monoclonal antibodies directed to VCAM-1, Fn, Ln, or Col IV respectively or VCAM-1 combined with anti-Fn, anti-Ln or anti-Col IV, respectively, in stromal cell adherence layer. Conclusion: The damage of cell adherent function for bone marrow hematopoietic inductive microenvironment post combined radiation-burn injury might be one of the important factors in hematopoietic disorder in combined radiation-burn injury

  9. The effect of two novel amino acid-coated magnetic nanoparticles on survival in vascular endothelial cells, bone marrow stromal cells, and macrophages

    Science.gov (United States)

    Wu, Qinghua; Meng, Ning; Zhang, Yanru; Han, Lei; Su, Le; Zhao, Jing; Zhang, Shangli; Zhang, Yun; Zhao, Baoxiang; Miao, Junying

    2014-09-01

    Magnetic nanoparticles (MNPs) have been popularly used in many fields. Recently, many kinds of MNPs are modified as new absorbents, which have attracted considerable attention and are promising to be applied in waste water. In our previous study, we synthesized two novel MNPs surface-coated with glycine or lysine, which could efficiently remove many anionic and cationic dyes under severe conditions. It should be considered that MNP residues in water may exert some side effects on human health. In the present study, we evaluated the potential nanotoxicity of MNPs in human endothelial cells, macrophages, and rat bone marrow stromal cells. The results showed that the two kinds of nanoparticles were consistently absorbed into the cell cytoplasm. The concentration of MNPs@Gly that could distinctly decrease survival was 15 μg/ml in human umbilical vascular endothelial cells (HUVECs) or bone marrow stromal cells (BMSCs) and 10 μg/ml in macrophages. While the concentration of MNPs@Lys that obviously reduced viability was 15 μg/ml in HUVECs or macrophages and 50 μg/ml in BMSCs. Furthermore, cell nucleus staining and cell integrity assay indicated that the nanoparticles induced cell apoptosis, but not necrosis even at a high concentration. Altogether, these data suggest that the amino acid-coated magnetic nanoparticles exert relatively high cytotoxicity. By contrast, lysine-coated magnetic nanoparticles are more secure than glycine-coated magnetic nanoparticles.

  10. Marrow stromal cells administrated intracisternally to rats after traumatic brain injury migrate into the brain and improve neurological function

    Institute of Scientific and Technical Information of China (English)

    胡德志; 周良辅; 朱剑虹

    2004-01-01

    @@ Marrow stromal cells(MSCs) have been reported to transplant into injured brain via intravenous or intraarterial or direct intracerebral administration.1-3 In the present study, we observed that MSCs migrated into the brain, survived and diffeneriated into neural cells after they were injected into the cisterna magna of rats, and that the behavior of the rats after traumatic brain injury (TBI) was improved.

  11. Stable subcutaneous cartilage regeneration of bone marrow stromal cells directed by chondrocyte sheet.

    Science.gov (United States)

    Li, Dan; Zhu, Lian; Liu, Yu; Yin, Zongqi; Liu, Yi; Liu, Fangjun; He, Aijuan; Feng, Shaoqing; Zhang, Yixin; Zhang, Zhiyong; Zhang, Wenjie; Liu, Wei; Cao, Yilin; Zhou, Guangdong

    2017-05-01

    In vivo niche plays an important role in regulating differentiation fate of stem cells. Due to lack of proper chondrogenic niche, stable cartilage regeneration of bone marrow stromal cells (BMSCs) in subcutaneous environments is always a great challenge. This study explored the feasibility that chondrocyte sheet created chondrogenic niche retained chondrogenic phenotype of BMSC engineered cartilage (BEC) in subcutaneous environments. Porcine BMSCs were seeded into biodegradable scaffolds followed by 4weeks of chondrogenic induction in vitro to form BEC, which were wrapped with chondrocyte sheets (Sheet group), acellular small intestinal submucosa (SIS, SIS group), or nothing (Blank group) respectively and then implanted subcutaneously into nude mice to trace the maintenance of chondrogenic phenotype. The results showed that all the constructs in Sheet group displayed typical cartilaginous features with abundant lacunae and cartilage specific matrices deposition. These samples became more mature with prolonged in vivo implantation, and few signs of ossification were observed at all time points except for one sample that had not been wrapped completely. Cell labeling results in Sheet group further revealed that the implanted BEC directly participated in cartilage formation. Samples in both SIS and Blank groups mainly showed ossified tissue at all time points with partial fibrogenesis in a few samples. These results suggested that chondrocyte sheet could create a chondrogenic niche for retaining chondrogenic phenotype of BEC in subcutaneous environment and thus provide a novel research model for stable ectopic cartilage regeneration based on stem cells. In vivo niche plays an important role in directing differentiation fate of stem cells. Due to lack of proper chondrogenic niche, stable cartilage regeneration of bone marrow stromal cells (BMSCs) in subcutaneous environments is always a great challenge. The current study demonstrated that chondrocyte sheet generated by

  12. Role of marrow architecture and stromal cells in the recovery process of aplastic marrow of lethally irradiated rats parabiosed with healthy litter mates

    International Nuclear Information System (INIS)

    Hayashi, K.; Kagawa, K.; Awai, M.; Irino, S.

    1986-01-01

    Bone marrow aplasia was induced in rats by whole body lethal irradiation (1,000 rads by x-ray), and rats died of irradiation injury within 7 days. Correlative studies at light (LM), transmission (TEM) and scanning electron microscopy (SEM) demonstrated swelling of endothelial and reticular cells and hemorrhage due to detachment of sinus endothelial cells on days 1 and 2. With time, structural recovery occurred without hemopoietic recovery. Reticular cells developed small intracytoplasmic lipid droplets on days 3 and 4. This resulted in fatty aplastic marrow within 7 days. On the other hand, in the marrow of irradiated rats parabiosed with healthy mates by aortic anastomosis, hemopoiesis was initiated by adhesion of nucleated blood cells to fine cytoplasmic pseudopods of fat-stored cells on days 1 and 2 after parabiosis. On days 3 to 5, reticular cells with large lipid droplets and fine pseudopods increased, then hemopoietic foci became clear and extensive. On day 8 after parabiosis, the aplastic bone marrow recovered completely both its structure and hemopoietic activity. Thus, hemopoietic recovery in lethally irradiated marrow begins with recovery of vascular endothelial cells, re-establishment of sinusoidal structure, and morphological and functional recoveries of reticular cells from fat-storage cells by releasing intracytoplasmic lipid droplets. Marrow stromal cells, namely reticular, fat-storage and fibroblastoid cells, share a common cellular origin, and regain their structure and function when fat-storage cells and fibroid cells are placed in contact with hemopoietic precursor cells

  13. Characterization and Immunomodulatory Effects of Canine Adipose Tissue- and Bone Marrow-Derived Mesenchymal Stromal Cells.

    Directory of Open Access Journals (Sweden)

    Keith A Russell

    Full Text Available Mesenchymal stromal cells (MSC hold promise for both cell replacement and immune modulation strategies owing to their progenitor and non-progenitor functions, respectively. Characterization of MSC from different sources is an important and necessary step before clinical use of these cells is widely adopted. Little is known about the biology and function of canine MSC compared to their mouse or human counterparts. This knowledge-gap impedes development of canine evidence-based MSC technologies.We hypothesized that canine adipose tissue (AT and bone marrow (BM MSC (derived from the same dogs will have similar differentiation and immune modulatory profiles. Our objectives were to evaluate progenitor and non-progenitor functions as well as other characteristics of AT- and BM-MSC including 1 proliferation rate, 2 cell surface marker expression, 3 DNA methylation levels, 4 potential for trilineage differentiation towards osteogenic, adipogenic, and chondrogenic cell fates, and 5 immunomodulatory potency in vitro.1 AT-MSC proliferated at more than double the rate of BM-MSC (population doubling times in days for passage (P 2, AT: 1.69, BM: 3.81; P3, AT: 1.80, BM: 4.06; P4, AT: 2.37, BM: 5.34; P5, AT: 3.20, BM: 7.21. 2 Canine MSC, regardless of source, strongly expressed cell surface markers MHC I, CD29, CD44, and CD90, and were negative for MHC II and CD45. They also showed moderate expression of CD8 and CD73 and mild expression of CD14. Minor differences were found in expression of CD4 and CD34. 3 Global DNA methylation levels were significantly lower in BM-MSC compared to AT-MSC. 4 Little difference was found between AT- and BM-MSC in their potential for adipogenesis and osteogenesis. Chondrogenesis was poor to absent for both sources in spite of adding varying levels of bone-morphogenic protein to our standard transforming growth factor (TGF-β3-based induction medium. 5 Immunomodulatory capacity was equal regardless of cell source when tested in

  14. Sequential growth factor application in bone marrow stromal cell ligament engineering.

    Science.gov (United States)

    Moreau, Jodie E; Chen, Jingsong; Horan, Rebecca L; Kaplan, David L; Altman, Gregory H

    2005-01-01

    In vitro bone marrow stromal cell (BMSC) growth may be enhanced through culture medium supplementation, mimicking the biochemical environment in which cells optimally proliferate and differentiate. We hypothesize that the sequential administration of growth factors to first proliferate and then differentiate BMSCs cultured on silk fiber matrices will support the enhanced development of ligament tissue in vitro. Confluent second passage (P2) BMSCs obtained from purified bone marrow aspirates were seeded on RGD-modified silk matrices. Seeded matrices were divided into three groups for 5 days of static culture, with medium supplement of basic fibroblast growth factor (B) (1 ng/mL), epidermal growth factor (E; 1 ng/mL), or growth factor-free control (C). After day 5, medium supplementation was changed to transforming growth factor-beta1 (T; 5 ng/mL) or C for an additional 9 days of culture. Real-time RT-PCR, SEM, MTT, histology, and ELISA for collagen type I of all sample groups were performed. Results indicated that BT supported the greatest cell ingrowth after 14 days of culture in addition to the greatest cumulative collagen type I expression measured by ELISA. Sequential growth factor application promoted significant increases in collagen type I transcript expression from day 5 of culture to day 14, for five of six groups tested. All T-supplemented samples surpassed their respective control samples in both cell ingrowth and collagen deposition. All samples supported spindle-shaped, fibroblast cell morphology, aligning with the direction of silk fibers. These findings indicate significant in vitro ligament development after only 14 days of culture when using a sequential growth factor approach.

  15. Bone marrow stromal cells with a combined expression of BMP-2 and VEGF-165 enhanced bone regeneration

    International Nuclear Information System (INIS)

    Xiao Caiwen; Zhou Huifang; Fu Yao; Gu Ping; Fan Xianqun; Liu Guangpeng; Zhang Peng; Hou Hongliang; Tang Tingting

    2011-01-01

    Bone graft substitutes with osteogenic factors alone often exhibit poor bone regeneration due to inadequate vascularization. Combined delivery of osteogenic and angiogenic factors from biodegradable scaffolds may enhance bone regeneration. We evaluated the effects of bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor (VEGF), combined with natural coral scaffolds, on the repair of critical-sized bone defects in rabbit orbits. In vitro expanded rabbit bone marrow stromal cells (BMSCs) were transfected with human BMP2 and VEGF165 genes. Target protein expression and osteogenic differentiation were confirmed after gene transduction. Rabbit orbital defects were treated with a coral scaffold loaded with BMP2-transduced and VEGF-transduced BMSCs, BMP2-expressing BMSCs, VEGF-expressing BMSCs, or BMSCs without gene transduction. Volume and density of regenerated bone were determined by micro-computed tomography at 4, 8, and 16 weeks after implantation. Neovascularity, new bone deposition rate, and new bone formation were measured by immunostaining, tetracycline and calcein labelling, and histomorphometric analysis at different time points. The results showed that VEGF increased blood vessel formation relative to groups without VEGF. Combined delivery of BMP2 and VEGF increased new bone deposition and formation, compared with any single factor. These findings indicate that mimicking the natural bone development process by combined BMP2 and VEGF delivery improves healing of critical-sized orbital defects in rabbits.

  16. Proliferation and differentiation of bone marrow stromal cells under hypoxic conditions

    Energy Technology Data Exchange (ETDEWEB)

    Hongying, Ren; Huiguo, Cai; Zhongchao, Han; Renchi, Yang; Zhao, Qinjun [State Key Lab of Experimental Hematology, Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union of Medical College, Tianjin (China); Ying, Cao; Jing, Li [Institute of Basic Medical Sciences and School of Basic Medicine, Center of Excellence in Tissue Engineering, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing (China); Cixiang, Zhou [Health Science Center, Shanghai Institutes of Biological Sciences, Chinese Academy of Science-SSMU, Shanghai (China); Lianming, Liao; Mingyue, Jia [Institute of Basic Medical Sciences and School of Basic Medicine, Center of Excellence in Tissue Engineering, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing (China); Qian, Zhao [Health Science Center, Shanghai Institutes of Biological Sciences, Chinese Academy of Science-SSMU, Shanghai (China); Guoqiang, Chen [Health Science Center, Shanghai Institutes of Biological Sciences, Chinese Academy of Science-SSMU, Shanghai (China); Zhao, R C [State Key Lab of Experimental Hematology, Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union of Medical College, Tianjin (China); [Institute of Basic Medical Sciences and School of Basic Medicine, Center of Excellence in Tissue Engineering, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing (China)]. E-mail: chunhuaz@public.tpt.tj.cn

    2006-08-18

    Low oxygen tension is a potent differentiation inducer of numerous cell types and an effective stimulus of many gene expressions. Here, we described that under 8% O{sub 2}, bone marrow stromal cells (MSCs) exhibited proliferative and morphologic changes. The level of differentiated antigen H-2Dd and the number of G{sub 2}/S/M phase cells increased evidently under 8% O{sub 2} condition. Also, the proportion of wide, flattened, and epithelial-like cells (which were alkaline phosphatase staining positive) in MSCs increased significantly. When cultured in adipogenic medium, there was a 5- to 6-fold increase in the number of lipid droplets under hypoxic conditions compared with that in normoxic culture. We also demonstrated the existence of MSC differentiation under hypoxic conditions by electron microscopy. Expression of Oct4 was inhibited under 8% O{sub 2} condition, but after adipocyte differentiation in normoxic culture and hypoxia-mimicking agents cobalt chloride (CoCl{sub 2}) and deferoxamine mesylate (DFX) treatments, Oct4 was still expressed in MSCs. These results indicate hypoxia accelerates MSC differentiation and hypoxia and hypoxia-mimicking agents exert different effects on MSC differentiation.

  17. Fate of bone marrow stromal cells in a syngenic model of bone formation.

    Science.gov (United States)

    Boukhechba, Florian; Balaguer, Thierry; Bouvet-Gerbettaz, Sébastien; Michiels, Jean-François; Bouler, Jean-Michel; Carle, Georges F; Scimeca, Jean-Claude; Rochet, Nathalie

    2011-09-01

    Bone marrow stromal cells (BMSCs) have been demonstrated to induce bone formation when associated to osteoconductive biomaterials and implanted in vivo. Nevertheless, their role in bone reconstruction is not fully understood and rare studies have been conducted to follow their destiny after implantation in syngenic models. The aim of the present work was to use sensitive and quantitative methods to track donor and recipient cells after implantation of BMSCs in a syngenic model of ectopic bone formation. Using polymerase chain reaction (PCR) amplification of the Sex determining Region Y (Sry) gene and in situ hybridization of the Y chromosome in parallel to histological analysis, we have quantified within the implants the survival of the donor cells and the colonization by the recipient cells. The putative migration of the BMSCs in peripheral organs was also analyzed. We show here that grafted cells do not survive more than 3 weeks after implantation and might migrate in peripheral lymphoid organs. These cells are responsible for the attraction of host cells within the implants, leading to the centripetal colonization of the biomaterial by new bone.

  18. Inhibition of phosphatidylcholine-specific phospholipase C prevents bone marrow stromal cell senescence in vitro.

    Science.gov (United States)

    Sun, Chunhui; Wang, Nan; Huang, Jie; Xin, Jie; Peng, Fen; Ren, Yinshi; Zhang, Shangli; Miao, Junying

    2009-10-01

    Bone marrow stromal cells (BMSCs) can proliferate in vitro and can be transplanted for treating many kinds of diseases. However, BMSCs become senescent with long-term culture, which inhibits their application. To understand the mechanism underlying the senescence, we investigated the activity of phosphatidylcholine-specific phospholipase C (PC-PLC) and levels of integrin beta4, caveolin-1 and ROS with BMSC senescence. The activity of PC-PLC and levels of integrin beta4, caveolin-1 and ROS increased greatly during cell senescence. Selective inhibition of increased PC-PLC activity with D609 significantly decreased the number of senescence-associated beta galactosidase positive cells in BMSCs. Furthermore, D609 restored proliferation of BMSCs and their differentiation into adipocytes. Moreover, D609 suppressed the elevated levels of integrin beta4, caveolin-1 and ROS. The data suggest that PC-PLC is involved in senescence of BMSCs, and its function is associated with integrin beta4, caveolin-1 and ROS. (c) 2009 Wiley-Liss, Inc.

  19. [Effects of nicotine on bone marrow stromal cells proliferation and differentiation of chondrocyte in vitro].

    Science.gov (United States)

    Ying, Xiao-zhou; Peng, Lei; Cheng, Shao-wen; Chen, Qing-yu; Zhang, Wei; Kou, Dong-quan; Shen, Yue

    2011-11-01

    To examine the effects of various concentration of nicotine on bone marrow stromal cells (BMSCs) proliferation and differentiation of cartilaginous in vitro. BMSCs was obtained from femoral bone and tibia of New-Zealand albino rabbit. The cells of the 3rd generation were used in study. Different concentration of nicotine (0, 1 x 10(-7), 1 x 10(-6), 1 x 10(-5) M) were added into BMSCs. BMSCs proliferation was analyzed by MTT assay at the 1, 4, 7, 14 days. The expression of collagen type II and aggrecan as the marker genes of cartilaginous differentiation from BMSCs were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Microscope showed that BMSCs transformed from round to fusiform shape. The concentration of nicotine in 1 x 10(-7), 1 x 10(-6) M had a significant positive effect on cell proliferation and the expression of type II collagen in a time-dependent manner when supplemented in commonly used induction media (P<0.05). Concentrations of nicotine in 1 x 10(-7) can promote the expression of aggrecan at the 7th day after induction,and in 1 x 10(-5) M may inhibit the expression of type II collagen and aggrecan. It was implied that local application of nicotine at an appropriate concentration may be a promising approach for enhancing cartilaginous differentiation capacity of BMSCs in cartilage tissue engineering.

  20. Effects of extracellular calcium on viability and osteogenic differentiation of bone marrow stromal cells in vitro.

    Science.gov (United States)

    Cheng, Shaowen; Wang, Wei; Lin, Zhongqin; Zhou, Ping; Zhang, Xiaolei; Zhang, Wei; Chen, Qingyu; Kou, Dongquan; Ying, Xiaozhou; Shen, Yue; Cheng, Xiaojie; Yu, Ziming; Peng, Lei; Lu, Chuanzhu

    2013-09-01

    Bone marrow stromal cells (BMSCs) have been extensively used for tissue engineering. However, the effect of Ca(2+) on the viability and osteogenic differentiation of BMSCs has yet to be evaluated. To determine the dose-dependent effect of Ca(2+) on viability and osteogenesis of BMSCs in vitro, BMSCs were cultured in calcium-free DMEM medium supplemented with various concentrations of Ca(2+) (0, 1, 2, 3, 4, and 5 mM) from calcium citrate. Cell viability was analyzed by MTT assay and osteogenic differentiation was evaluated by alkaline phosphatase (ALP) assay, Von Kossa staining, and real-time PCR. Ca(2+) stimulated BMSCs viability in a dose-dependent manner. At slightly higher concentrations (4 and 5 mM) in the culture, Ca(2+) significantly inhibited the activity of ALP on days 7 and 14 (P < 0.01 or P < 0.05), significantly suppressed collagen synthesis (P < 0.01 or P < 0.05), and significantly elevated calcium deposition (P < 0.01) and mRNA levels of osteocalcin (P < 0.01 or P < 0.05) and osteopontin (P < 0.01 or P < 0.05). Therefore, elevated concentrations of extracellular calcium may promote cell viability and late-stage osteogenic differentiation, but may suppress early-stage osteogenic differentiation in BMSCs.

  1. Secreted Factors from Bone Marrow Stromal Cells Upregulate IL-10 and Reverse Acute Kidney Injury

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    Jack M. Milwid

    2012-01-01

    Full Text Available Acute kidney injury is a devastating syndrome that afflicts over 2,000,000 people in the US per year, with an associated mortality of greater than 70% in severe cases. Unfortunately, standard-of-care treatments are not sufficient for modifying the course of disease. Many groups have explored the use of bone marrow stromal cells (BMSCs for the treatment of AKI because BMSCs have been shown to possess unique anti-inflammatory, cytoprotective, and regenerative properties in vitro and in vivo. It is yet unresolved whether the primary mechanisms controlling BMSC therapy in AKI depend on direct cell infusion, or whether BMSC-secreted factors alone are sufficient for mitigating the injury. Here we show that BMSC-secreted factors are capable of providing a survival benefit to rats subjected to cisplatin-induced AKI. We observed that when BMSC-conditioned medium (BMSC-CM is administered intravenously, it prevents tubular apoptosis and necrosis and ameliorates AKI. In addition, we observed that BMSC-CM causes IL-10 upregulation in treated animals, which is important to animal survival and protection of the kidney. In all, these results demonstrate that BMSC-secreted factors are capable of providing support without cell transplantation, and the IL-10 increase seen in BMSC-CM-treated animals correlates with attenuation of severe AKI.

  2. Adult rat bone marrow stromal cells express genes associated with dopamine neurons

    International Nuclear Information System (INIS)

    Kramer, Brian C.; Woodbury, Dale; Black, Ira B.

    2006-01-01

    An intensive search is underway to identify candidates to replace the cells that degenerate in Parkinson's disease (PD). To date, no suitable substitute has been found. We have recently found that adult rat bone marrow stromal cells (MSCs) can be induced to assume a neuronal phenotype in vitro. These findings may have particular relevance to the treatment of PD. We now report that adult MSCs express multiple dopaminergic genes, suggesting that they are potential candidates for cell therapy. Using RT-PCR, we have examined families of genes that are associated with the development and/or survival of dopaminergic neurons. MSCs transcribe a variety of dopaminergic genes including patched and smoothened (components of the Shh receptor), Gli-1 (downstream mediator of Shh), and Otx-1, a gene associated with formation of the mesencephalon during development. Furthermore, Shh treatment elicits a 1.5-fold increase in DNA synthesis in cultured MSCs, suggesting the presence of a functional Shh receptor complex. We have also found that MSCs transcribe and translate Nurr-1, a nuclear receptor essential for the development of dopamine neurons. In addition, MSCs express a variety of growth factor receptors including the glycosyl-phosphatidylinositol-anchored ligand-binding subunit of the GDNF receptor, GFRα1, as well as fibroblast growth factor receptors one and four. The expression of genes that are associated with the development and survival of dopamine neurons suggests a potential role for these cells in the treatment of Parkinson's disease

  3. Tensile loading modulates bone marrow stromal cell differentiation and the development of engineered fibrocartilage constructs.

    Science.gov (United States)

    Connelly, John T; Vanderploeg, Eric J; Mouw, Janna K; Wilson, Christopher G; Levenston, Marc E

    2010-06-01

    Mesenchymal progenitors such as bone marrow stromal cells (BMSCs) are an attractive cell source for fibrocartilage tissue engineering, but the types or combinations of signals required to promote fibrochondrocyte-specific differentiation remain unclear. The present study investigated the influences of cyclic tensile loading on the chondrogenesis of BMSCs and the development of engineered fibrocartilage. Cyclic tensile displacements (10%, 1 Hz) were applied to BMSC-seeded fibrin constructs for short (24 h) or extended (1-2 weeks) periods using a custom loading system. At early stages of chondrogenesis, 24 h of cyclic tension stimulated both protein and proteoglycan synthesis, but at later stages, tension increased protein synthesis only. One week of intermittent cyclic tension significantly increased the total sulfated glycosaminoglycan and collagen contents in the constructs, but these differences were lost after 2 weeks of loading. Constraining the gels during the extended culture periods prevented contraction of the fibrin matrix, induced collagen fiber alignment, and increased sulfated glycosaminoglycan release to the media. Cyclic tension specifically stimulated collagen I mRNA expression and protein synthesis, but had no effect on collagen II, aggrecan, or osteocalcin mRNA levels. Overall, these studies suggest that the combination of chondrogenic stimuli and tensile loading promotes fibrochondrocyte-like differentiation of BMSCs and has the potential to direct fibrocartilage development in vitro.

  4. Activation of GLP-1 Receptor Promotes Bone Marrow Stromal Cell Osteogenic Differentiation through β-Catenin

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    Jingru Meng

    2016-04-01

    Full Text Available Glucagon-like peptide 1 (GLP-1 plays an important role in regulating bone remodeling, and GLP-1 receptor agonist shows a positive relationship with osteoblast activity. However, GLP-1 receptor is not found in osteoblast, and the mechanism of GLP-1 receptor agonist on regulating bone remodeling is unclear. Here, we show that the GLP-1 receptor agonist exendin-4 (Ex-4 promoted bone formation and increased bone mass and quality in a rat unloading-induced bone loss model. These functions were accompanied by an increase in osteoblast number and serum bone formation markers, while the adipocyte number was decreased. Furthermore, GLP-1 receptor was detected in bone marrow stromal cells (BMSCs, but not in osteoblast. Activation of GLP-1 receptor by Ex-4 promoted the osteogenic differentiation and inhibited BMSC adipogenic differentiation through regulating PKA/β-catenin and PKA/PI3K/AKT/GSK3β signaling. These findings reveal that GLP-1 receptor regulates BMSC osteogenic differentiation and provide a molecular basis for therapeutic potential of GLP-1 against osteoporosis.

  5. Efficacy of Surgery Combined with Autologous Bone Marrow Stromal Cell Transplantation for Treatment of Intracerebral Hemorrhage

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    Jianxin Zhu

    2015-01-01

    Full Text Available Bone marrow stromal cells (BMSCs may differentiate into nerve cells under a certain condition; however, the clinical application for treating nervous system disease remains unclear. The aim is to assess the safety profile, feasibility, and effectiveness of surgery combined with autologous BMSCs transplantation for treating ICH. 206 ICH patients who had received surgical procedure were divided into transplantation (n=110 or control group (n=96. For transplantation group, BMSCs were injected into the perihemorrhage area in the base ganglia through an intracranial drainage tube 5.5 (3.01–6.89 days after surgery, followed by a second injection into the subarachnoid space through lumbar puncture 4 weeks later. Neurologic impairment and daily activities were assessed with National Institute Stroke Scale (NIHSS, Barthel index, and Rankin scale before transplantation and 6 months and 12 months after transplantation. Our results revealed that, compared with control group, NIHSS score and Rankin scale were both significantly decreased but Barthel index was increased in transplantation group after 6 months. Interestingly, no significant difference was observed between 12 months and 6 months. No transplantation-related adverse effects were investigated during follow-up assessments. Our findings suggest that surgery combined with autologous BMSCs transplantation is safe for treatment of ICH, providing short-term therapeutic benefits.

  6. Proliferation and differentiation of bone marrow stromal cells under hypoxic conditions

    International Nuclear Information System (INIS)

    Ren Hongying; Cao Ying; Zhao, Qinjun; Li Jing; Zhou Cixiang; Liao Lianming; Jia Mingyue; Zhao Qian; Cai Huiguo; Han Zhongchao; Yang Renchi; Chen Guoqiang; Zhao, R.C.

    2006-01-01

    Low oxygen tension is a potent differentiation inducer of numerous cell types and an effective stimulus of many gene expressions. Here, we described that under 8% O 2 , bone marrow stromal cells (MSCs) exhibited proliferative and morphologic changes. The level of differentiated antigen H-2Dd and the number of G 2 /S/M phase cells increased evidently under 8% O 2 condition. Also, the proportion of wide, flattened, and epithelial-like cells (which were alkaline phosphatase staining positive) in MSCs increased significantly. When cultured in adipogenic medium, there was a 5- to 6-fold increase in the number of lipid droplets under hypoxic conditions compared with that in normoxic culture. We also demonstrated the existence of MSC differentiation under hypoxic conditions by electron microscopy. Expression of Oct4 was inhibited under 8% O 2 condition, but after adipocyte differentiation in normoxic culture and hypoxia-mimicking agents cobalt chloride (CoCl 2 ) and deferoxamine mesylate (DFX) treatments, Oct4 was still expressed in MSCs. These results indicate hypoxia accelerates MSC differentiation and hypoxia and hypoxia-mimicking agents exert different effects on MSC differentiation

  7. Effects of continuous and pulsatile PTH treatments on rat bone marrow stromal cells

    International Nuclear Information System (INIS)

    Yang Chiming; Frei, Hanspeter; Burt, Helen M.; Rossi, Fabio

    2009-01-01

    Bone marrow stromal cells (MSCs) differentiation and proliferation are controlled by numerous growth factors and hormones. Continuous parathyroid hormone (PTH) treatment has been shown to decrease osteoblast differentiation, whereas pulsatile PTH increases osteoblast differentiation. However, the effects of PTH treatments on MSCs have not been investigated. This study showed continuous PTH treatment in the presence of dexamethasone (DEX) promoted osteogenic differentiation of rat MSCs in vitro, as demonstrated by increased alkaline phosphatase (ALP) activity, number of ALP expressing cells, and up-regulation of PTH receptor-1, ALP, and osteocalcin mRNA expressions. In contrast, pulsatile PTH treatment was found to suppress osteogenesis of rat MSCs, possibly by promoting the maintenance of undifferentiated cells. Additionally, the observed effects of PTH were strongly dependent on the presence of DEX. MSC proliferation however was not influenced by PTH independent of treatment regimen and presence or absence of DEX. Furthermore, our work raised the possibility that PTH treatment may modulate stem/progenitor cell activity within MSC cultures.

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Science.gov (United States)

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

    2009-01-01

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

  10. Changes in compartments of hemospoietic and stromal marrow progenitor cells after continuous low dose gamma-irradiation

    Science.gov (United States)

    Domaratskaya, E.; Starostin, V.

    The low dose continuous gamma-irradiation chosen corresponded with that affected the organisms onboard a spacecraft (Mitrikas, Tsetlin, 2000). F1 (CBAxC57Bl/6) male and female mice were used at 3 4 months of age. Experimental mice were- irradiated during 10 days to a total dose of 15 mGy (Co60 gamma-sources, mean dose rate of 1.5-2.0 mGy/day). Another group of intact mice served as control. Younger and advanced hemopoietic progenitors measured at day 11 (i.e. CFU -S-11) and day 7 (i.e. CFU-S-7), respectively, after transplantation of test donor cells were assayed by the method of Till and McCulloch (1961). Stromal changes were evaluated by estimation of in vitro fibroblastic colony-forming units (CFU -F ) content and by the ability of ectopically grafted (under renal capsule) stroma to regenerate the new bone marrow organ. CFU-S-11 number increased of 40% as compared with control and almost 2-fold higher than that of CFU-S-7. The CFU-F content increased almost of 3-fold. Size of ectopic marrow transplants was estimated at day 70 following grafting by counting myelokariocyte and CFU -S number that repopulated the newly formed bone marrow organ. It was found more than 2-fold increase of myelokariocytes in transplants produced by marrow stroma of irradiated donors. CFU -S contents in transplants increased strikingly in comparison to control level. CFU-S-7 and CFU-S-11 increased of 7.5- and of 3.7-fold, respectively, i.e. the rate of advanced CFU - S predominated. It should be noted a good correlation between number of stromal progenitor cells (CFU-F) and ectopic transplant sizes evaluated as myelokaryocyte counts when irradiated donors used. In the same time, if sizes of transplants was measured as CFU-S-7 and CFU - S-11 numbers, their increases were more pronounced. Therefore, continuous low dose gamma- irradiation augments significantly both hemopoietic and stromal progenitor cell number in bone marrow. Additionally, the ratio of distinct CFU -S subpopulations

  11. Bone Marrow Mesenchymal Stromal Cells Stimulate Skeletal Myoblast Proliferation through the Paracrine Release of VEGF

    Science.gov (United States)

    Chellini, Flaminia; Mazzanti, Benedetta; Nistri, Silvia; Nosi, Daniele; Saccardi, Riccardo; Quercioli, Franco; Zecchi-Orlandini, Sandra; Formigli, Lucia

    2012-01-01

    Mesenchymal stromal cells (MSCs) are the leading cell candidates in the field of regenerative medicine. These cells have also been successfully used to improve skeletal muscle repair/regeneration; however, the mechanisms responsible for their beneficial effects remain to be clarified. On this basis, in the present study, we evaluated in a co-culture system, the ability of bone-marrow MSCs to influence C2C12 myoblast behavior and analyzed the cross-talk between the two cell types at the cellular and molecular level. We found that myoblast proliferation was greatly enhanced in the co-culture as judged by time lapse videomicroscopy, cyclin A expression and EdU incorporation. Moreover, myoblasts immunomagnetically separated from MSCs after co-culture expressed higher mRNA and protein levels of Notch-1, a key determinant of myoblast activation and proliferation, as compared with the single culture. Notch-1 intracellular domain and nuclear localization of Hes-1, a Notch-1 target gene, were also increased in the co-culture. Interestingly, the myoblastic response was mainly dependent on the paracrine release of vascular endothelial growth factor (VEGF) by MSCs. Indeed, the addition of MSC-derived conditioned medium (CM) to C2C12 cells yielded similar results as those observed in the co-culture and increased the phosphorylation and expression levels of VEGFR. The treatment with the selective pharmacological VEGFR inhibitor, KRN633, resulted in a marked attenuation of the receptor activation and concomitantly inhibited the effects of MSC-CM on C2C12 cell growth and Notch-1 signaling. In conclusion, this study provides novel evidence for a role of MSCs in stimulating myoblast cell proliferation and suggests that the functional interaction between the two cell types may be exploited for the development of new and more efficient cell-based skeletal muscle repair strategies. PMID:22815682

  12. Engineering of hyaline cartilage with a calcified zone using bone marrow stromal cells.

    Science.gov (United States)

    Lee, W D; Hurtig, M B; Pilliar, R M; Stanford, W L; Kandel, R A

    2015-08-01

    In healthy joints, a zone of calcified cartilage (ZCC) provides the mechanical integration between articular cartilage and subchondral bone. Recapitulation of this architectural feature should serve to resist the constant shear force from the movement of the joint and prevent the delamination of tissue-engineered cartilage. Previous approaches to create the ZCC at the cartilage-substrate interface have relied on strategic use of exogenous scaffolds and adhesives, which are susceptible to failure by degradation and wear. In contrast, we report a successful scaffold-free engineering of ZCC to integrate tissue-engineered cartilage and a porous biodegradable bone substitute, using sheep bone marrow stromal cells (BMSCs) as the cell source for both cartilaginous zones. BMSCs were predifferentiated to chondrocytes, harvested and then grown on a porous calcium polyphosphate substrate in the presence of triiodothyronine (T3). T3 was withdrawn, and additional predifferentiated chondrocytes were placed on top of the construct and grown for 21 days. This protocol yielded two distinct zones: hyaline cartilage that accumulated proteoglycans and collagen type II, and calcified cartilage adjacent to the substrate that additionally accumulated mineral and collagen type X. Constructs with the calcified interface had comparable compressive strength to native sheep osteochondral tissue and higher interfacial shear strength compared to control without a calcified zone. This protocol improves on the existing scaffold-free approaches to cartilage tissue engineering by incorporating a calcified zone. Since this protocol employs no xenogeneic material, it will be appropriate for use in preclinical large-animal studies. Copyright © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

  13. Application of cell sheet technology to bone marrow stromal cell transplantation for rat brain infarct.

    Science.gov (United States)

    Ito, Masaki; Shichinohe, Hideo; Houkin, Kiyohiro; Kuroda, Satoshi

    2017-02-01

    Bone marrow stromal cells (BMSC) transplantation enhances functional recovery after cerebral infarct, but the optimal delivery route is undetermined. This study was aimed to assess whether a novel cell-sheet technology non-invasively serves therapeutic benefits to ischemic stroke. First, the monolayered cell sheet was engineered by culturing rat BMSCs on a temperature-responsive dish. The cell sheet was analysed histologically and then transplanted onto the ipsilateral neocortex of rats subjected to permanent middle cerebral artery occlusion at 7 days after the insult. Their behaviours and histology were compared with those in the animals treated with direct injection of BMSCs or vehicle over 4 weeks post-transplantation. The cell sheet was 27.9 ± 8.0 μm thick and was composed of 9.8 ± 2.4 × 10 5 cells. Cell sheet transplantation significantly improved motor function when compared with the vehicle-injected animals. Histological analysis revealed that the BMSCs were densely distributed to the neocortex adjacent to the cerebral infarct and expressed neuronal phenotype in the cell sheet-transplanted animals. These findings were almost equal to those for the animals treated with direct BMSC injection. The attachment of the BMSC sheet to the brain surface did not induce reactive astrocytes in the adjacent neocortex, although direct injection of BMSCs profoundly induced reactive astrocytes around the injection site. These findings suggest that the BMSCs in cell sheets preserve their biological capacity of migration and neural differentiation. Cell-sheet technology may enhance functional recovery after ischaemic stroke, using a less invasive method. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

  14. Transcription factor and bone marrow stromal cells in osseointegration of dental implants

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    SG Yan

    2018-05-01

    Full Text Available Titanium implants are widely used in dental clinics and orthopaedic surgery. However, bone formation surrounding the implant is relatively slow after inserting the implant. The current study assessed the effects of bone marrow stromal cells (BMSCs with forced expression of special AT-rich sequence-binding protein 2 (SATB2 on the osseointegration of titanium implants. To determine whether SATB2 overexpression in BMSCs can enhance the osseointegration of implants, BMSCs were infected with the retrovirus encoding Satb2 (pBABE-Satb2 and were locally applied to bone defects before implanting the titanium implants in the mouse femur. Seven and twenty-one days after implantation, the femora were isolated for immunohistochemical (IHC staining, haematoxylin eosin (H&E staining, real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR, and micro-computed tomography (μCT analysis. IHC staining analysis revealed that SATB2-overexpressing BMSCs were intensely distributed in the bone tissue surrounding the implant. Histological analysis showed that SATB2-overexpressing BMSCs significantly enhanced new bone formation and bone-to-implant contact 3 weeks after implantation. Real-time qRT-PCR results showed that the local delivery of SATB2-overexpressing BMSCs enhanced expression levels of potent osteogenic transcription factors and bone matrix proteins in the implantation sites. μCT analysis demonstrated that SATB2-overexpressing BMSCs significantly increased the density of the newly formed bone surrounding the implant 3 weeks post-operatively. These results conclude that local delivery of SATB2-overexpressing BMSCs significantly accelerates osseointegration of titanium implants. These results provide support for future pharmacological and clinical applications of SATB2, which accelerates bone regeneration around titanium implants.

  15. 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.

  16. Silencing of RB1 and RB2/P130 during adipogenesis of bone marrow stromal cells results in dysregulated differentiation

    OpenAIRE

    Capasso, Stefania; Alessio, Nicola; Di Bernardo, Giovanni; Cipollaro, Marilena; Melone, Mariarosa A. B.; Peluso, Gianfranco; Giordano, Antonio; Galderisi, Umberto

    2014-01-01

    Bone marrow adipose tissue (BMAT) is different from fat found elsewhere in the body, and only recently have some of its functions been investigated. BMAT may regulate bone marrow stem cell niche and plays a role in energy storage and thermogenesis. BMAT may be involved also in obesity and osteoporosis onset. Given the paramount functions of BMAT, we decided to better clarify the human bone marrow adipogenesis by analyzing the role of the retinoblastoma gene family, which are key players in ce...

  17. Conditioned media from differentiating craniofacial bone marrow stromal cells influence mineralization and proliferation in periodontal ligament stem cells.

    Science.gov (United States)

    Jin, Zhenyu; Feng, Yuan; Liu, Hongwei

    2016-10-01

    Previous reports have mainly focused on the behavioral responses of human periodontal ligament stem cells (hPDLSCs) in interaction with tibia bone marrow stromal cells (BMSCs). However, there is little study on the biologic features of hPDLSCs under the induction of maxilla BMSCs (M-BMSCs) at different phases of osteogenic differentiation. We hypothesized that M-BMSCs undergoing osteogenic differentiation acted on the proliferation, differentiation, and bone-forming capacity of hPDLSCs. In this paper, primary hPDLSCs and human M-BMSCs (hM-BMSCs) were expanded in vitro. After screening of surface markers for characterization, hPDLSCs were cocultured with different phases of differentiating hM-BMSCs. Cell proliferation and alkaline phosphatase activity were examined, and mineralization-associated markers such as osteocalcin and runt-related transcription factor 2 of hPDLSCs in coculture with uninduced/osteoinduced hM-BMSCs were evaluated. hPDLSCs in hM-BMSCs-conditioned medium (hM-BMSCs-CM) group showed a reduction in proliferation compared with untreated hPDLSCs, while osteoinduced hM-BMSCs for 10 day-conditioned medium (hM-BMSCs-CM-10ds) and osteoinduced hM-BMSCs for 15 day-conditioned medium (hM-BMSCs-CM-15ds) enhance the proliferation of hPDLSCs. hM-BMSCs of separate differentiation stages temporarily inhibited osteogenesis of hPDLSCs in the early days. Upon extending time periods, uninduced/osteoinduced hM-BMSCs markedly enhanced osteogenesis of hPDLSCs to different degrees. The transplantation results showed hM-BMSCs-CM-15ds treatment promoted tissue regeneration to generate cementum/periodontal ligament-like structure characterized by hard-tissue formation. This research supported the notion that hM-BMSCs triggered osteogenesis of hPDLSCs suggesting important implications for periodontal engineering.

  18. Hypoxia impedes hypertrophic chondrogenesis of human multipotent stromal cells.

    Science.gov (United States)

    Gawlitta, Debby; van Rijen, Mattie H P; Schrijver, Edmée J M; Alblas, Jacqueline; Dhert, Wouter J A

    2012-10-01

    Within the field of bone tissue engineering, the endochondral approach to forming bone substitutes represents a novel concept, where cartilage will undergo hypertrophic differentiation before its conversion into bone. For this purpose, clinically relevant multipotent stromal cells (MSCs), MSCs, can be differentiated into the chondrogenic lineage before stimulating hypertrophy. Controversy exists in literature on the oxygen tensions naturally present during this transition in, for example, the growth plate. Therefore, the present study focused on the effects of different oxygen tensions on the progression of the hypertrophic differentiation of MSCs. Bone marrow-derived MSCs of four human donors were expanded, and differentiation was induced in aggregate cultures. Normoxic (20% oxygen) and hypoxic (5%) conditions were imposed on the cultures in chondrogenic or hypertrophic differentiation media. After 4 weeks, the cultures were histologically examined and by real-time polymerase chain reaction. Morphological assessment showed the chondrogenic differentiation of cultures from all donors under normoxic chondrogenic conditions. In addition, hypertrophic differentiation was observed in cultures derived from all but one donor. The deposition of collagen type X was evidenced in both chondrogenically and hypertrophically stimulated cultures. However, mineralization was exclusively observed in hypertrophically stimulated, normoxic cultures. Overall, the progression of hypertrophy was delayed in hypoxic compared with normoxic groups. The observed delay was supported by the gene expression patterns, especially showing the up-regulation of the late hypertrophic markers osteopontin and osteocalcin under normoxic hypertrophic conditions. Concluding, normoxic conditions are more beneficial for hypertrophic differentiation of MSCs than are hypoxic conditions, as long as the MSCs possess hypertrophic potential. This finding has implications for cartilage tissue engineering as well

  19. Effect of cotransplantation of hematopoietic stem cells and embryonic AGM stromal cells on hematopoietic reconstitution in mice after bone marrow transplantation

    International Nuclear Information System (INIS)

    Tao Si; Sun Hanying; Liu Wenli

    2007-01-01

    Objective: To explore the effects of cotransplantation of hematopoietic stem cells and stromal cells derived from aorta-gonad-mesonephros (AGM) region on hematopoietic reconstitution in mice after bone marrow transplantation (BMT). Methods: The typical mice model of syngeneic BMT was established and the mice were randomly divided into 4 groups: the control group, the BMT group, the group of cotransplantation of HSC with AGM stromal cells (the cotransplantation group) and the ligustrazine group (the LT group). On days 3, 7, 10, 14, 21 and 28 after BMT, the peripheral blood cells and bone marrow mononuclear cells (BMMNC) were counted, and histology changes of bone marrow were detected. Results: The levels of peripheral WBC, RBC, platelet, and BMMNC in the contransplantation group were significantly higher than those in the single BMT group and the LT group (P<0.05). Conclusions: Cotransplantation with AGM stromal cells could significantly promote hematopoietic reconstruction in mice after BMT. (authors)

  20. Molecular signature and in vivo behavior of bone marrow endosteal and subendosteal stromal cell populations and their relevance to hematopoiesis

    Energy Technology Data Exchange (ETDEWEB)

    Balduino, Alex, E-mail: balduino@uva.edu.br [School of Dentistry, Veiga de Almeida University, Rio de Janeiro, RJ (Brazil); Mello-Coelho, Valeria [Biomedical Science Institute, Federal University of Rio de Janeiro, Rio de Janeiro, RJ (Brazil); National Institute on Aging, National Institute of Health, Baltimore, MD (United States); Wang, Zhou; Taichman, Russell S.; Krebsbach, Paul H. [Department of Periodontics, Prevention and Geriatrics, University of Michigan School of Dentistry, Ann Arbor, MI (United States); Weeraratna, Ashani T.; Becker, Kevin G. [National Institute on Aging, National Institute of Health, Baltimore, MD (United States); Mello, Wallace de [Instituto Oswaldo Cruz, Rio de Janeiro, RJ (Brazil); Taub, Dennis D. [National Institute on Aging, National Institute of Health, Baltimore, MD (United States); Borojevic, Radovan [Biomedical Science Institute, Federal University of Rio de Janeiro, Rio de Janeiro, RJ (Brazil)

    2012-11-15

    In the bone marrow cavity, hematopoietic stem cells (HSC) have been shown to reside in the endosteal and subendosteal perivascular niches, which play specific roles on HSC maintenance. Although cells with long-term ability to reconstitute full hematopoietic system can be isolated from both niches, several data support a heterogenous distribution regarding the cycling behavior of HSC. Whether this distinct behavior depends upon the role played by the stromal populations which distinctly create these two niches is a question that remains open. In the present report, we used our previously described in vivo assay to demonstrate that endosteal and subendosteal stromal populations are very distinct regarding skeletal lineage differentiation potential. This was further supported by a microarray-based analysis, which also demonstrated that these two stromal populations play distinct, albeit complementary, roles in HSC niche. Both stromal populations were preferentially isolated from the trabecular region and behave distinctly in vitro, as previously reported. Even though these two niches are organized in a very close range, in vivo assays and molecular analyses allowed us to identify endosteal stroma (F-OST) cells as fully committed osteoblasts and subendosteal stroma (F-RET) cells as uncommitted mesenchymal cells mainly represented by perivascular reticular cells expressing high levels of chemokine ligand, CXCL12. Interestingly, a number of cytokines and growth factors including interleukin-6 (IL-6), IL-7, IL-15, Hepatocyte growth factor (HGF) and stem cell factor (SCF) matrix metalloproteases (MMPs) were also found to be differentially expressed by F-OST and F-RET cells. Further microarray analyses indicated important mechanisms used by the two stromal compartments in order to create and coordinate the 'quiescent' and 'proliferative' niches in which hematopoietic stem cells and progenitors reside.

  1. Molecular signature and in vivo behavior of bone marrow endosteal and subendosteal stromal cell populations and their relevance to hematopoiesis

    International Nuclear Information System (INIS)

    Balduino, Alex; Mello-Coelho, Valeria; Wang, Zhou; Taichman, Russell S.; Krebsbach, Paul H.; Weeraratna, Ashani T.; Becker, Kevin G.; Mello, Wallace de; Taub, Dennis D.; Borojevic, Radovan

    2012-01-01

    In the bone marrow cavity, hematopoietic stem cells (HSC) have been shown to reside in the endosteal and subendosteal perivascular niches, which play specific roles on HSC maintenance. Although cells with long-term ability to reconstitute full hematopoietic system can be isolated from both niches, several data support a heterogenous distribution regarding the cycling behavior of HSC. Whether this distinct behavior depends upon the role played by the stromal populations which distinctly create these two niches is a question that remains open. In the present report, we used our previously described in vivo assay to demonstrate that endosteal and subendosteal stromal populations are very distinct regarding skeletal lineage differentiation potential. This was further supported by a microarray-based analysis, which also demonstrated that these two stromal populations play distinct, albeit complementary, roles in HSC niche. Both stromal populations were preferentially isolated from the trabecular region and behave distinctly in vitro, as previously reported. Even though these two niches are organized in a very close range, in vivo assays and molecular analyses allowed us to identify endosteal stroma (F-OST) cells as fully committed osteoblasts and subendosteal stroma (F-RET) cells as uncommitted mesenchymal cells mainly represented by perivascular reticular cells expressing high levels of chemokine ligand, CXCL12. Interestingly, a number of cytokines and growth factors including interleukin-6 (IL-6), IL-7, IL-15, Hepatocyte growth factor (HGF) and stem cell factor (SCF) matrix metalloproteases (MMPs) were also found to be differentially expressed by F-OST and F-RET cells. Further microarray analyses indicated important mechanisms used by the two stromal compartments in order to create and coordinate the “quiescent” and “proliferative” niches in which hematopoietic stem cells and progenitors reside.

  2. The effects of canine bone marrow stromal cells on neuritogenesis from dorsal root ganglion neurons in vitro.

    Science.gov (United States)

    Kamishina, Hiroaki; Cheeseman, Jennifer A; Clemmons, Roger M

    2009-10-01

    The present in vitro study was designed to evaluate whether canine bone marrow stromal cells (BMSCs) promote neurite outgrowth from dorsal root ganglion (DRG) neurons. Bone marrow aspirates were collected from iliac crests of three young adult dogs. DRG neurons were cultured on BMSCs, fibroblasts, or laminin substrates. DRG neurons were also cultured in BMSC- or fibroblast-conditioned media. DRG neurons grown on BMSCs extended longer neurites and developed a much more elaborate conformation of branching neurites compared to those on fibroblasts or laminin. Quantitative analysis revealed that these effects were associated with the emergence of increased numbers of primary and branching neurites. The effect appears to be dependent upon cell-cell interactions rather than by elaboration of diffusible molecules. With more extensive investigations into the basic biology of canine BMSCs, their ability for promoting neurite outgrowth may be translated into a novel therapeutic strategy for dogs with a variety of neurological disorders.

  3. Effect of transplantation of olfactory ensheathing cell conditioned medium induced bone marrow stromal cells on rats with spinal cord injury

    Science.gov (United States)

    Feng, Linjie; Gan, Hongquan; Zhao, Wenguo; Liu, Yingjie

    2017-01-01

    Spinal cord injury is a serious threat to human health and various techniques have been deployed to ameliorate or cure its effects. Stem cells transplantation is one of the promising methods. The primary aim of the present study was to investigate the effect of the transplantation of olfactory ensheathing cell (OEC) conditioned medium-induced bone marrow stromal cells (BMSCs) on spinal cord injury. Rat spinal cord compression injury animal models were generated, and the rats divided into the following three groups: Group A, (control) Dulbecco's modified Eagle's medium-treated group; group B, normal BMSC-treated group; group C, OEC conditioned medium-induced BMSC-treated group. The animals were sacrificed at 2, 4 and 8 weeks following transplantation for hematoxylin and eosin staining, and fluorescence staining of neurofilament protein, growth associated protein-43 and neuron-specific nuclear protein. The cavity area of the spinal cord injury was significantly reduced at 2 and 4 weeks following transplantation in group C, and a significant difference between the Basso, Beattie and Bresnahan score in group C and groups A and B was observed. Regenerated nerve fibers were observed in groups B and C; however, a greater number of regenerated nerve fibers were observed in group C. BMSCs induced by OEC conditioned medium survived in vivo, significantly reduced the cavity area of spinal cord injury, promoted nerve fiber regeneration following spinal cord injury and facilitated recovery of motor function. The present study demonstrated a novel method to repair spinal cord injury by using induced BMSCs, with satisfactory results. PMID:28656221

  4. 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. © 2015 International Federation for Cell Biology.

  5. Potential Effect of CD271 on Human Mesenchymal Stromal Cell Proliferation and Differentiation

    Directory of Open Access Journals (Sweden)

    Giovanna Calabrese

    2015-07-01

    Full Text Available The Low-Affinity Nerve Growth Factor Receptor (LNGFR, also known as CD271, is a member of the tumor necrosis factor receptor superfamily. The CD271 cell surface marker defines a subset of multipotential mesenchymal stromal cells and may be used to isolate and enrich cells derived from bone marrow aspirate. In this study, we compare the proliferative and differentiation potentials of CD271+ and CD271− mesenchymal stromal cells. Mesenchymal stromal cells were isolated from bone marrow aspirate and adipose tissue by plastic adherence and positive selection. The proliferation and differentiation potentials of CD271+ and CD271− mesenchymal stromal cells were assessed by inducing osteogenic, adipogenic and chondrogenic in vitro differentiation. Compared to CD271+, CD271− mesenchymal stromal cells showed a lower proliferation rate and a decreased ability to give rise to osteocytes, adipocytes and chondrocytes. Furthermore, we observed that CD271+ mesenchymal stromal cells isolated from adipose tissue displayed a higher efficiency of proliferation and trilineage differentiation compared to CD271+ mesenchymal stromal cells isolated from bone marrow samples, although the CD271 expression levels were comparable. In conclusion, these data show that both the presence of CD271 antigen and the source of mesenchymal stromal cells represent important factors in determining the ability of the cells to proliferate and differentiate.

  6. Potential Effect of CD271 on Human Mesenchymal Stromal Cell Proliferation and Differentiation.

    Science.gov (United States)

    Calabrese, Giovanna; Giuffrida, Raffaella; Lo Furno, Debora; Parrinello, Nunziatina Laura; Forte, Stefano; Gulino, Rosario; Colarossi, Cristina; Schinocca, Luciana Rita; Giuffrida, Rosario; Cardile, Venera; Memeo, Lorenzo

    2015-07-09

    The Low-Affinity Nerve Growth Factor Receptor (LNGFR), also known as CD271, is a member of the tumor necrosis factor receptor superfamily. The CD271 cell surface marker defines a subset of multipotential mesenchymal stromal cells and may be used to isolate and enrich cells derived from bone marrow aspirate. In this study, we compare the proliferative and differentiation potentials of CD271+ and CD271- mesenchymal stromal cells. Mesenchymal stromal cells were isolated from bone marrow aspirate and adipose tissue by plastic adherence and positive selection. The proliferation and differentiation potentials of CD271+ and CD271- mesenchymal stromal cells were assessed by inducing osteogenic, adipogenic and chondrogenic in vitro differentiation. Compared to CD271+, CD271- mesenchymal stromal cells showed a lower proliferation rate and a decreased ability to give rise to osteocytes, adipocytes and chondrocytes. Furthermore, we observed that CD271+ mesenchymal stromal cells isolated from adipose tissue displayed a higher efficiency of proliferation and trilineage differentiation compared to CD271+ mesenchymal stromal cells isolated from bone marrow samples, although the CD271 expression levels were comparable. In conclusion, these data show that both the presence of CD271 antigen and the source of mesenchymal stromal cells represent important factors in determining the ability of the cells to proliferate and differentiate.

  7. Normal human bone marrow and its variations in MRI

    International Nuclear Information System (INIS)

    Vahlensieck, M.; Schmidt, H.M.

    2000-01-01

    Physiology and age dependant changes of human bone marrow are described. The resulting normal distribution patterns of active and inactive bone marrow including the various contrasts on different MR-sequences are discussed. (orig.) [de

  8. Immunocytochemical characterization of explant cultures of human prostatic stromal cells

    NARCIS (Netherlands)

    A. Kooistra (Anko); A.M.J. Elissen (Arianne ); J.J. Konig (Josee); M. Vermey; Th.H. van der Kwast (Theo); J.C. Romijn (Johannes); F.H. Schröder (Fritz)

    1995-01-01

    textabstractThe study of stromal-epithelial interactions greatly depends on the ability to culture both cell types separately, in order to permit analysis of their interactions under defined conditions in reconstitution experiments. Here we report the establishment of explant cultures of human

  9. Endogenous collagen influences differentiation of human multipotent mesenchymal stromal cells

    NARCIS (Netherlands)

    Fernandes, H.; Mentink, A.; Bank, R.; Stoop, R.; Blitterswijk, C. van; Boer, J. de

    2010-01-01

    Human multipotent mesenchymal stromal cells (hMSCs) are multipotent cells that, in the presence of appropriate stimuli, can differentiate into different lineages such as the osteogenic, chondrogenic, and adipogenic lineages. In the presence of ascorbic acid, MSCs secrete an extracellular matrix

  10. Endogenous Collagen Influences Differentiation of Human Multipotent Mesenchymal Stromal Cells

    NARCIS (Netherlands)

    Fernandes, Hugo; Mentink, Anouk; Bank, Ruud; Stoop, Reinout; van Blitterswijk, Clemens; de Boer, Jan

    Human multipotent mesenchymal stromal cells (hMSCs) are multipotent cells that, in the presence of appropriate stimuli, can differentiate into different lineages such as the osteogenic, chondrogenic, and adipogenic lineages. In the presence of ascorbic acid, MSCs secrete an extracellular matrix

  11. Endogenous Collagen Influences Differentiation of Human Multipotent Mesenchymal Stromal Cells

    NARCIS (Netherlands)

    Fernandes, H.A.M.; Mentink-Leusink, Anouk; Bank, Ruud; Stoop, Reinout; van Blitterswijk, Clemens; de Boer, Jan

    2010-01-01

    Human multipotent mesenchymal stromal cells (hMSCs) are multipotent cells that, in the presence of appropriate stimuli, can differentiate into different lineages such as the osteogenic, chondrogenic, and adipogenic lineages. In the presence of ascorbic acid, MSCs secrete an extracellular matrix

  12. Human mesenchymal stromal cells : biological characterization and clinical application

    NARCIS (Netherlands)

    Bernardo, Maria Ester

    2010-01-01

    This thesis focuses on the characterization of the biological and functional properties of human mesenchymal stromal cells (MSCs), isolated from different tissue sources. The differentiation capacity of MSCs from fetal and adult tissues has been tested and compared. Umbilical cord blood (UCB) has

  13. Bone marrow stromal cells elicit tissue sparing after acute but not delayed transplantation into the contused adult rat thoracic spinal cord.

    NARCIS (Netherlands)

    Tewarie, R.D.; Hurtado, A.; Ritfeld, G.J.; Rahiem, S.T.; Wendell, D.F.; Barroso, M.M.; Grotenhuis, J.A.; Oudega, M.

    2009-01-01

    Bone marrow stromal cells (BMSC) transplanted into the contused spinal cord may support repair by improving tissue sparing. We injected allogeneic BMSC into the moderately contused adult rat thoracic spinal cord at 15 min (acute) and at 3, 7, and 21 days (delayed) post-injury and quantified tissue

  14. New aspects of galectin functionality in nuclei of cultured bone marrow stromal and epidermal cells: biotinylated galectins as tool to detect specific bindong sites

    Czech Academy of Sciences Publication Activity Database

    Purkrábková, T.; Smetana Jr., K.; Dvořánková, B.; Holíková, Z.; Böck, C.; Lensch, M.; André, S.; Pytlík, R.; Liu, F.; Klíma, Jiří; Smetana, K.; Motlík, Jan; Gabius, H. J.

    2003-01-01

    Roč. 95, - (2003), s. 535-545 ISSN 0248-4900 R&D Projects: GA MŠk LN00A065 Institutional research plan: CEZ:AV0Z5045916 Keywords : biotinylation * bone marrow stromal cells * galectin Subject RIV: ED - Physiology Impact factor: 2.159, year: 2003

  15. Migration of acute lymphoblastic leukemia cells into human bone marrow stroma.

    Science.gov (United States)

    Makrynikola, V; Bianchi, A; Bradstock, K; Gottlieb, D; Hewson, J

    1994-10-01

    Most cases of acute lymphoblastic leukemia (ALL) arise from malignant transformation of B-cell precursors in the bone marrow. Recent studies have shown that normal and leukemic B-cell precursors bind to bone marrow stromal cells through the beta-1 integrins VLA-4 and VLA-5, thereby exposing early lymphoid cells to regulatory cytokines. It has been recently reported that the pre-B cell line NALM-6 is capable of migrating under layers of murine stromal cells in vitro (Miyake et al. J Cell Biol 1992;119:653-662). We have further analyzed leukemic cell motility using human bone marrow fibroblasts (BMF) as a stromal layer. The precursor-B ALL cell line NALM-6 rapidly adhered to BMF, and underwent migration or tunneling into BMF layers within 5 h, as demonstrated by light and electron microscopy, and confirmed by a chromium-labeling assay. Migration was also observed with the precursor-B ALL lines Reh and KM-3, with a T leukemia line RPMI-8402, the monocytic line U937, and the mature B line Daudi. In contrast, mature B (Raji), myeloid (K562, HL-60), and T lines (CCRF-CEM, MOLT-4) did not migrate. When cases of leukemia were analyzed, BMF migration was largely confined to precursor-B ALL, occurring in eight of 13 cases tested. Of other types of leukemia, migration was observed in one of four cases of T-ALL, but no evidence was seen in six acute myeloid leukemias and two patients with chronic lymphocytic leukemia. Only minimal migration into BMF was observed with purified sorted CD10+ CD19+ early B cells from normal adult marrow, while normal mature B lymphocytes from peripheral blood did not migrate. ALL migration was inhibited by monoclonal antibodies to the beta sub-unit of the VLA integrin family, and by a combination of antibodies to VLA-4 and VLA-5. Partial inhibition was also observed when leukemic cells were incubated with antibodies to VLA-4, VLA-5, or VLA-6 alone. In contrast, treatment of stromal cells with antibodies to vascular cell adhesion molecule or

  16. Secretomes from bone marrow-derived mesenchymal stromal cells enhance periodontal tissue regeneration.

    Science.gov (United States)

    Kawai, Takamasa; Katagiri, Wataru; Osugi, Masashi; Sugimura, Yukiko; Hibi, Hideharu; Ueda, Minoru

    2015-04-01

    Periodontal tissue regeneration with the use of mesenchymal stromal cells (MSCs) has been regarded as a future cell-based therapy. However, low survival rates and the potential tumorigenicity of implanted MSCs could undermine the efficacy of cell-based therapy. The use of conditioned media from MSCs (MSC-CM) may be a feasible approach to overcome these limitations. The aim of this study was to confirm the effect of MSC-CM on periodontal regeneration. MSC-CM were collected during their cultivation. The concentrations of the growth factors in MSC-CM were measured with the use of enzyme-linked immunoassay. Rat MSCs (rMSCs) and human umbilical vein endothelial cells cultured in MSC-CM were assessed on wound-healing and angiogenesis. The expressions of osteogenetic- and angiogenic-related genes of rMSCs cultured in MSC-CM were quantified by means of real-time reverse transcriptase-polymerase chain reaction analysis. In vivo, periodontal defects were prepared in the rat models and the collagen sponges with MSC-CM were implanted. MSC-CM includes insulin-like growth factor-1, vascular endothelial growth factor, transforming growth factor-β1 and hepatocyte growth factor. In vitro, wound-healing and angiogenesis increased significantly in MSC-CM. The levels of expression of osteogenetic- and angiogenic-related genes were significantly upregulated in rMSCs cultured with MSC-CM. In vivo, in the MSC-CM group, 2 weeks after implantation, immunohistochemical analysis showed several CD31-, CD105-or FLK-1-positive cells occurring frequently. At 4 weeks after implantation, regenerated periodontal tissue was observed in MSC-CM groups. The use of MSC-CM may be an alternative therapy for periodontal tissue regeneration because several cytokines included in MSC-CM will contribute to many processes of complicated periodontal tissue regeneration. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  17. Acquisition and Expansion of Adult Rat Bone Marrow Multipotent Mesenchymal Stromal Cells

    Directory of Open Access Journals (Sweden)

    Šulla I.

    2017-03-01

    Full Text Available This study was initiated in order to test a mini-invasive method of mesenchymal stem/progenitor cells (MS/PCs isolation from a rat bone marrow (BM, and subsequently their expansion, differentiation, and evaluation of their immunophenotypic characteristics; and later their preservation as donor cells in an optimal condition for potential autotransplantation. The study group comprised of 6 adult male Sprague-Dawley (S-D rats, weighing 480—690 g. The rats were anaesthetised by isoflurane with room air in a Plexiglas box and maintained by inhalation of a mixture of isoflurane and O2. Their femurs were surgically exposed and their diaphyses double-trephined. Then BM cells were flushed out by saline with heparin and aspirated into a syringe with a solution of DMEM (Dulbecco’s modified eagle’s medium and heparin. The mononuclear cells from the BM were isolated by centrifugation and expanded in a standard culture medium supplemented with ES-FBS (es-cell-qualified foetal bovine serum, L-glutamine and rh LIF (recombinant human leukemia inhibitory factor. Following 14 days of passaging cultures, the cells were split into 2 equal parts. The first culture continued with the original medium. The second culture received additional supplementation with a human FGFβ (fibroblast growth factor beta and EGF (epidermal growth factor. The populations of these cells were analysed by light-microscopy, then the mean fluorescence intensities (MFIs of CD90 and Nestin were evaluated by a tricolour flow cytometry using monoclonal antibodies. The type of general anaesthesia used proved to be appropriate for the surgical phase of the experiments. All rats survived the harvesting of the BM without complications. The total number of mononuclear cells was 1.5—4.0 × 106 per sample and the proportion of CD90/Nestin expressing cells was < 1 %. Following 14 days of expansion, the cells became larger, adherent, with fibrillary morphology; the proportion of cells expressing

  18. The Effects of Bone Marrow Stromal Cells Therapy on the Improvement of Epilepsy Model in Rat Induced by pilocarpine

    Directory of Open Access Journals (Sweden)

    Ali Reza Abdani-Pour

    2008-01-01

    Full Text Available Objective: Temporal lobe epilepsy is the most common type of epilepsy in adult humans, which is characterized clinically by a progressive development of spontaneous recurrent seizures of temporal lobe origin. Because of the side effects of current treatment protocols, the resistance to pharmacological therapy in this investigation, bone marrow stromal cells were used to evaluate the recovery of epileptic rats induced by pilocarpine. Materials & Methods: In this randomized experimental research, the rats were divided into five groups, controls (untreated, three treated groups (12, 24 and 36 hours and a group treated with the vehicle only. The animals in chronic phase were monitored via video monitoring system for three weeks (day & night. For assessment of the response for the treatment of epileptic’s rat using BMSCs therapy, Racine scale was used as a behavioral test. BMSCs (2 – 3 million cells were labeled with BrdU and were injected via tail vein rat 12, 24, 36 hours after first seizure. After 6 week all rats sacrificed and processed for paraffin, sectioned and for Cresyl violet staining. Data were analyzed by use of Wilcoxon signed Ranks test and Tukey’s test. Results: The result of the study showed that the behavioral test in three week as follows: in control group (untreated, number of seizure is 6.25±1.3 in vehicle group, number of seizure was 6.2±0.8 the group which received BMSCs 12 h after seizure all rats died the group which received BMSCs (24 h after seizure, the number was 2±0.4 and the group which received BMSCs (36 h after seizure, the number of seizure was 2.25±0.47. There was significant difference between treated (2 and 36 hours groups and other groups in number of seizure (P<0/01. Also, cells number was different significantly between same groups (P<0/01. Conclusion: Intravenous injection of BMSCs can improve number of attacks in epileptic’s rats. Also, BMSCs injection can prevent from decrease of cells numerical

  19. FGF7 supports hematopoietic stem and progenitor cells and niche-dependent myeloblastoma cells via autocrine action on bone marrow stromal cells in vitro

    International Nuclear Information System (INIS)

    Ishino, Ruri; Minami, Kaori; Tanaka, Satowa; Nagai, Mami; Matsui, Keiji; Hasegawa, Natsumi; Roeder, Robert G.; Asano, Shigetaka; Ito, Mitsuhiro

    2013-01-01

    Highlights: •FGF7 is downregulated in MED1-deficient mesenchymal cells. •FGF7 produced by mesenchymal stromal cells is a novel hematopoietic niche molecule. •FGF7 supports hematopoietic progenitor cells and niche-dependent leukemia cells. •FGF7 activates FGFR2IIIb of bone marrow stromal cells in an autocrine manner. •FGF7 indirectly acts on hematopoietic cells lacking FGFR2IIIb via stromal cells. -- Abstract: FGF1 and FGF2 support hematopoietic stem and progenitor cells (HSPCs) under stress conditions. In this study, we show that fibroblast growth factor (FGF7) may be a novel niche factor for HSPC support and leukemic growth. FGF7 expression was attenuated in mouse embryonic fibroblasts (MEFs) deficient for the MED1 subunit of the Mediator transcriptional coregulator complex. When normal mouse bone marrow (BM) cells were cocultured with Med1 +/+ MEFs or BM stromal cells in the presence of anti-FGF7 antibody, the growth of BM cells and the number of long-time culture-initiating cells (LTC-ICs) decreased significantly. Anti-FGF7 antibody also attenuated the proliferation and cobblestone formation of MB1 stromal cell-dependent myeloblastoma cells. The addition of recombinant FGF7 to the coculture of BM cells and Med1 −/− MEFs increased BM cells and LTC-ICs. FGF7 and its cognate receptor, FGFR2IIIb, were undetectable in BM cells, but MEFs and BM stromal cells expressed both. FGF7 activated downstream targets of FGFR2IIIb in Med1 +/+ and Med1 −/− MEFs and BM stromal cells. Taken together, we propose that FGF7 supports HSPCs and leukemia-initiating cells indirectly via FGFR2IIIb expressed on stromal cells

  20. FGF7 supports hematopoietic stem and progenitor cells and niche-dependent myeloblastoma cells via autocrine action on bone marrow stromal cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Ishino, Ruri; Minami, Kaori; Tanaka, Satowa [Laboratory of Hematology, Division of Medical Biophysics, Kobe University Graduate School of Health Sciences, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142 (Japan); Nagai, Mami [Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 159-8555 (Japan); Matsui, Keiji; Hasegawa, Natsumi [Laboratory of Hematology, Division of Medical Biophysics, Kobe University Graduate School of Health Sciences, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142 (Japan); Roeder, Robert G. [Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Asano, Shigetaka [Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 159-8555 (Japan); Ito, Mitsuhiro, E-mail: itomi@med.kobe-u.ac.jp [Laboratory of Hematology, Division of Medical Biophysics, Kobe University Graduate School of Health Sciences, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142 (Japan); Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 159-8555 (Japan); Department of Family and Community Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 654-0142 (Japan)

    2013-10-11

    Highlights: •FGF7 is downregulated in MED1-deficient mesenchymal cells. •FGF7 produced by mesenchymal stromal cells is a novel hematopoietic niche molecule. •FGF7 supports hematopoietic progenitor cells and niche-dependent leukemia cells. •FGF7 activates FGFR2IIIb of bone marrow stromal cells in an autocrine manner. •FGF7 indirectly acts on hematopoietic cells lacking FGFR2IIIb via stromal cells. -- Abstract: FGF1 and FGF2 support hematopoietic stem and progenitor cells (HSPCs) under stress conditions. In this study, we show that fibroblast growth factor (FGF7) may be a novel niche factor for HSPC support and leukemic growth. FGF7 expression was attenuated in mouse embryonic fibroblasts (MEFs) deficient for the MED1 subunit of the Mediator transcriptional coregulator complex. When normal mouse bone marrow (BM) cells were cocultured with Med1{sup +/+} MEFs or BM stromal cells in the presence of anti-FGF7 antibody, the growth of BM cells and the number of long-time culture-initiating cells (LTC-ICs) decreased significantly. Anti-FGF7 antibody also attenuated the proliferation and cobblestone formation of MB1 stromal cell-dependent myeloblastoma cells. The addition of recombinant FGF7 to the coculture of BM cells and Med1{sup −/−} MEFs increased BM cells and LTC-ICs. FGF7 and its cognate receptor, FGFR2IIIb, were undetectable in BM cells, but MEFs and BM stromal cells expressed both. FGF7 activated downstream targets of FGFR2IIIb in Med1{sup +/+} and Med1{sup −/−} MEFs and BM stromal cells. Taken together, we propose that FGF7 supports HSPCs and leukemia-initiating cells indirectly via FGFR2IIIb expressed on stromal cells.

  1. Tissue engineering of ligaments: a comparison of bone marrow stromal cells, anterior cruciate ligament, and skin fibroblasts as cell source.

    Science.gov (United States)

    Van Eijk, F; Saris, D B F; Riesle, J; Willems, W J; Van Blitterswijk, C A; Verbout, A J; Dhert, W J A

    2004-01-01

    Anterior cruciate ligament (ACL) reconstruction surgery still has important problems to overcome, such as "donor site morbidity" and the limited choice of grafts in revision surgery. Tissue engineering of ligaments may provide a solution for these problems. Little is known about the optimal cell source for tissue engineering of ligaments. The aim of this study is to determine the optimal cell source for tissue engineering of the anterior cruciate ligament. Bone marrow stromal cells (BMSCs), ACL, and skin fibroblasts were seeded onto a resorbable suture material [poly(L-lactide/glycolide) multifilaments] at five different seeding densities, and cultured for up to 12 days. All cell types tested attached to the suture material, proliferated, and synthesized extracellular matrix rich in collagen type I. On day 12 the scaffolds seeded with BMSCs showed the highest DNA content (p engineered ligament.

  2. Neurospheres induced from bone marrow stromal cells are multipotent for differentiation into neuron, astrocyte, and oligodendrocyte phenotypes

    International Nuclear Information System (INIS)

    Suzuki, Hidenori; Taguchi, Toshihiko; Tanaka, Hiroshi; Kataoka, Hideo; Li Zhenglin; Muramatsu, Keiichi; Gondo, Toshikazu; Kawai, Shinya

    2004-01-01

    Bone marrow stromal cells (MSCs) can be expanded rapidly in vitro and have the potential to be differentiated into neuronal, glial and endodermal cell types. However, induction for differentiation does not always have stable result. We present a new method for efficient induction and acquisition of neural progenitors, neuronal- and glial-like cells from MSCs. We demonstrate that rat MSCs can be induced to neurospheres and most cells are positive for nestin, which is an early marker of neuronal progenitors. In addition, we had success in proliferation of these neurospheres with undifferentiated characteristics and finally we could obtain large numbers of neuronal and glial phenotypes. Many of the cells expressed β-tubulin III when they were cultivated with our method. MSCs can become a valuable cell source as an autograft for clinical application involving regeneration of the central nervous system

  3. Increased survival of CBA pluripotent haemopoietic stem cells in vitro induced by a marrow stromal factor in Sl/Sl/sup d/ mice

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    Blackburn, M J; Patt, H M

    1979-07-01

    Media conditioned by marrow adherent cells from anaemic Sl/Sl/sup d/ and W/W/sup v/ mice increased the 24-h survival of CBA CFU/sub s/ in vitro compared to fresh medium to about the same extent as marrow-conditioned medium from normal Sl/sup +//Sl/sup +/, W/sup +//W/sup +/, and CBA mice. Sl/Sl/sup d/ marrow-conditioned medium also increased the percentage of CFU/sub s/ in DNA synthesis to the same extent as CBA marrow-conditioned medium. These results demonstrate that Sl/Sl/sup d/ mice produce a marrow stromal factor that increases both survival of CFU/sub s/ and the percentage of CFU/sub s/ in DNA synthesis in vitro. Therefore, the defective haemopoietic microenvironment of Sl/Sl/sup d/ mice is not due to a deficiency in the production of this factor.

  4. 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...

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

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    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.

  6. Thioredoxin-1 Protects Bone Marrow-Derived Mesenchymal Stromal Cells from Hyperoxia-Induced Injury In Vitro

    Science.gov (United States)

    Zhang, Lei; Wang, Jin; Zeng, Lingkong; Li, Qiong; Liu, Yalan

    2018-01-01

    Background The poor survival rate of mesenchymal stromal cells (MSC) transplanted into recipient lungs greatly limits their therapeutic efficacy for diseases like bronchopulmonary dysplasia (BPD). The aim of this study is to evaluate the effect of thioredoxin-1 (Trx-1) overexpression on improving the potential for bone marrow-derived mesenchymal stromal cells (BMSCs) to confer resistance against hyperoxia-induced cell injury. Methods 80% O2 was used to imitate the microenvironment surrounding-transplanted cells in the hyperoxia-induced lung injury in vitro. BMSC proliferation and apoptotic rates and the levels of reactive oxygen species (ROS) were measured. The effects of Trx-1 overexpression on the level of antioxidants and growth factors were investigated. We also investigated the activation of apoptosis-regulating kinase-1 (ASK1) and p38 mitogen-activated protein kinases (MAPK). Result Trx-1 overexpression significantly reduced hyperoxia-induced BMSC apoptosis and increased cell proliferation. We demonstrated that Trx-1 overexpression upregulated the levels of superoxide dismutase and glutathione peroxidase as well as downregulated the production of ROS. Furthermore, we illustrated that Trx-1 protected BMSCs against hyperoxic injury via decreasing the ASK1/P38 MAPK activation rate. Conclusion These results demonstrate that Trx-1 overexpression improved the ability of BMSCs to counteract hyperoxia-induced injury, thus increasing their potential to treat hyperoxia-induced lung diseases such as BPD. PMID:29599892

  7. Long-Term Engraftment of Primary Bone Marrow Stromal Cells Repairs Niche Damage and Improves Hematopoietic Stem Cell Transplantation.

    Science.gov (United States)

    Abbuehl, Jean-Paul; Tatarova, Zuzana; Held, Werner; Huelsken, Joerg

    2017-08-03

    Hematopoietic stem cell (HSC) transplantation represents a curative treatment for various hematological disorders. However, delayed reconstitution of innate and adaptive immunity often causes fatal complications. HSC maintenance and lineage differentiation are supported by stromal niches, and we now find that bone marrow stroma cells (BMSCs) are severely and permanently damaged by the pre-conditioning irradiation required for efficient HSC transplantation. Using mouse models, we show that stromal insufficiency limits the number of donor-derived HSCs and B lymphopoiesis. Intra-bone transplantation of primary, but not cultured, BMSCs quantitatively reconstitutes stroma function in vivo, which is mediated by a multipotent NT5E + (CD73) + ENG - (CD105) - LY6A + (SCA1) + BMSC subpopulation. BMSC co-transplantation doubles the number of functional, donor-derived HSCs and significantly reduces clinically relevant side effects associated with HSC transplantation including neutropenia and humoral immunodeficiency. These data demonstrate the potential of stroma recovery to improve HSC transplantation. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Isolation of Mesenchymal Stromal Cells (MSCs from Human Adenoid Tissue

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    Yoon Se Lee

    2013-04-01

    Full Text Available Background: Mesenchymal stromal cells (MSCs are multipotent progenitor cells that originally derived from bone marrow. Clinical use of bone marrow-derived MSC is difficult due to morbidity and low MSC abundance and isolation efficiency. Recently, MSCs have been isolated from various adult tissues. Here we report the isolation of adenoid tissue-derived MSCs (A-MSCs and their characteristics. Methods: We compared the surface markers, morphologies, and differentiation and proliferation capacities of previously established tonsil-derived MSCs (T-MSCs and bone marrow-derived MSCs (BM-MSCs with cells isolated from adenoid tissue. The immunophenotype of A-MSCs was investigated upon interferon (IFN-γ stimulation. Results: A-MSCs, T-MSCs, and BM-MSCs showed negative CD45, CD31 HLA-DR, CD34, CD14, CD19 and positive CD 90, CD44, CD73, CD105 expression. A-MSCs were fibroblast-like, spindle-shaped non-adherent cells, similar to T-MSCs and BM-MSCs. Adipogenesis was observed in A-MSCs by the formation of lipid droplets after Oil Red O staining. Osteogenesis was observed by the formation of the matrix mineralization in Alizarin Red staining. Chondrogenesis was observed by the accumulation of sulfated glycosaminoglycan-rich matrix in collagen type II staining. These data were similar to those of T-MSCs and BM-MSCs. Expression of marker genes (i.e., adipogenesis; lipoprotein lipase, proliferator-activator receptor-gamma, osteogenesis; osteocalcin, alkaline phasphatase, chondrogenesis; aggrecan, collagen type II α1 in A-MSCs were not different from those in T-MSCs and BM-MSCs. Conclusions: A-MSCs possess the characteristics of MSCs in terms of morphology, multipotent differentiation capacity, cell surface markers, and immunogeneity. Therefore, A-MSCs fulfill the definition of MSCs and represent an alternate source of MSCs.

  9. Equine allogeneic bone marrow-derived mesenchymal stromal cells elicit antibody responses in vivo.

    Science.gov (United States)

    Pezzanite, Lynn M; Fortier, Lisa A; Antczak, Douglas F; Cassano, Jennifer M; Brosnahan, Margaret M; Miller, Donald; Schnabel, Lauren V

    2015-04-12

    This study tested the hypothesis that Major Histocompatibility Complex (MHC) incompatible equine mesenchymal stromal cells (MSCs) would induce cytotoxic antibodies to donor MHC antigens in recipient horses after intradermal injection. No studies to date have explored recipient antibody responses to allogeneic donor MSC transplantation in the horse. This information is critical because the horse is a valuable species for assessing the safety and efficacy of MSC treatment prior to human clinical application. Six MHC heterozygote horses were identified as non-ELA-A2 haplotype by microsatellite typing and used as allogeneic MHC-mismatched MSC recipients. MHC homozygote horses of known ELA-A2 haplotype were used as MSC and peripheral blood leukocyte (PBL) donors. One MHC homozygote horse of the ELA-A2 haplotype was the recipient of ELA-A2 donor MSCs as an MHC-matched control. Donor MSCs, which were previously isolated and immunophenotyped, were thawed and culture expanded to achieve between 30x10(6) and 50x10(6) cells for intradermal injection into the recipient's neck. Recipient serum was collected and tested for the presence of anti-donor antibodies prior to MSC injection and every 7 days after MSC injection for the duration of the 8-week study using the standard two-stage lymphocyte microcytotoxicity dye-exclusion test. In addition to anti-ELA-A2 antibodies, recipient serum was examined for the presence of cross-reactive antibodies including anti-ELA-A3 and anti-RBC antibodies. All MHC-mismatched recipient horses produced anti-ELA-A2 antibodies following injection of ELA-A2 MSCs and developed a wheal at the injection site that persisted for the duration of the experiment. Anti-ELA-A2 antibody responses were varied both in terms of strength and timing. Four recipient horses had high-titered anti-ELA-A2 antibody responses resulting in greater than 80% donor PBL death in the microcytotoxicity assays and one of these horses also developed antibodies that cross

  10. Comparison of different culture conditions for human mesenchymal stromal cells for clinical stem cell therapy

    DEFF Research Database (Denmark)

    Haack-Sorensen, M.; Friis, T.; Bindslev, L.

    2008-01-01

    OBJECTIVE: Mesenchymal stromal cells (MSCs) from adult bone marrow (BM) are considered potential candidates for therapeutic neovascularization in cardiovascular disease. When implementing results from animal trials in clinical treatment, it is essential to isolate and expand the MSCs under...... conditions following good manufacturing practice (GMP). The aims of the study were first to establish culture conditions following GMP quality demands for human MSC expansion and differentiation for use in clinical trials, and second to compare these MSCs with MSCs derived from culture in four media commonly...... analysis showed that the plastic-adherent MSCs cultured in EMEA medium or in the other four media were identically negative for the haematopoietic surface markers CD45 and CD34 and positive for CD105, CD73, CD90, CD166 and CD13, which in combined expression is characteristic of MSCs. MSC stimulation...

  11. Evaluation of median survival patients with acute lymphoblastic leukemia exposed ionizing radiation by the Chornobyl accident, depending on the aminoacid composition of stromal fibroblasts bone marrow and indicators of myelogram

    International Nuclear Information System (INIS)

    Bebeshko, V.G.; Bruslova, K.M.; Tsvjetkova, N.M.; Pushkar'ova, T.Yi.; Panchenko, L.M.; Yatsemirs'kij, S.N.; Volodyina, T.T.; Lyashenko, L.O.

    2015-01-01

    The aminoacid composition in the stromal fibroblasts of bone marrow and indexes of hemogram in children with acute lymphoblastic leukemias, which exposed to radiation from the Chornobyl accident, for evaluation of the median survival was investigated. In the study 54 patients with acute lymphoblastic leukemia, residents of Kyiv, Zhytomyr and Chernihiv regions of Ukraine were involved. Effectiveness of stromal fibroblasts colony forming units of bone marrow and aminoacid composition of fibroblasts were studied. Bone marrow examination was conducted on day 33 and after stop of chemotherapy, the median survival of patients and exposure dose were evaluated. In children with acute leukemias effectiveness of stromal fibroblasts of bone marrow was 2-2.3 times lower than normative. Correlation (r_s = +0.83) between the number of erythroid progenitor cells in the bone marrow and proline content in stromal fibroblasts established . The correlation between patients median survival and number of granulocytes in myelogram (r_s = + 0.82) was proven. Mean exposure doses in patients was (5.86 ±1.11) mSv and it did not correlate with myelogram results, effectiveness of stromal fibroblasts of bone marrow and median survival. The median survival of patients with acute leukemias depends on the content of amino acids in stromal fibroblasts of bone marrow and recover of hematopoiesis after chemotherapy

  12. Colonic complications following human bone marrow transplantation

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    Paulino Martínez Hernández-Magro

    2015-01-01

    Full Text Available Background: Human bone marrow transplantation (BMT becomes an accepted treatment of leukemia, aplastic anemia, immunodeficiency syndromes, and hematologic malignancies. Colorectal surgeons must know how to determine and manage the main colonic complications. Objective: To review the clinical features, clinical and pathological staging of graft vs host disease (GVHD, and treatment of patients suffering with colonic complications of human bone marrow transplantation. Patients and methods: We have reviewed the records of all patients that received an allogeneic bone marrow transplant and were evaluated at our Colon and Rectal Surgery department due to gastrointestinal symptoms, between January 2007 and January 2012. The study was carried out in patients who developed colonic complications, all of them with clinical, histopathological or laboratory diagnosis. Results: The study group was constituted by 77 patients, 43 male and 34 female patients. We identified colonic complications in 30 patients (38.9%; five patients developed intestinal toxicity due to pretransplant chemotherapy (6.4%; graft vs. host disease was present in 16 patients (20%; 13 patients (16.8% developed acute colonic GVHD, and 3 (3.8% chronic GVHD. Infection was identified in 9 patients (11.6%. Conclusions: The three principal colonic complications are the chemotherapy toxicity, GVHD, and superinfection; the onset of symptoms could help to suspect the type of complication (0–20 day chemotherapy toxicity, 20 and more GVHD, and infection could appear in any time of transplantation. Resumo: Experiência: O transplante de medula óssea humana (MOH passou a ser um tratamento adotado para leucemia, anemia aplástica, síndromes de imunodeficiência e neoplasias hematológicas. Cirurgiões colorretais devem saber como determinar e tratar as principais complicações do cólon. Objetivo: Revisar as características clínicas, estadiamentos clínico e patológico da doença do enxerto

  13. * Human Amniotic Mesenchymal Stromal Cells as Favorable Source for Cartilage Repair.

    Science.gov (United States)

    Muiños-López, Emma; Hermida-Gómez, Tamara; Fuentes-Boquete, Isaac; de Toro-Santos, Javier; Blanco, Francisco Javier; Díaz-Prado, Silvia María

    2017-09-01

    Localized trauma-derived breakdown of the hyaline articular cartilage may progress toward osteoarthritis, a degenerative condition characterized by total loss of articular cartilage and joint function. Tissue engineering technologies encompass several promising approaches with high therapeutic potential for the treatment of these focal defects. However, most of the research in tissue engineering is focused on potential materials and structural cues, while little attention is directed to the most appropriate source of cells endowing these materials. In this study, using human amniotic membrane (HAM) as scaffold, we defined a novel static in vitro model for cartilage repair. In combination with HAM, four different cell types, human chondrocytes, human bone marrow-derived mesenchymal stromal cells (hBMSCs), human amniotic epithelial cells, and human amniotic mesenchymal stromal cells (hAMSCs) were assessed determining their therapeutic potential. A chondral lesion was drilled in human cartilage biopsies simulating a focal defect. A pellet of different cell types was implanted inside the lesion and covered with HAM. The biopsies were maintained for 8 weeks in culture. Chondrogenic differentiation in the defect was analyzed by histology and immunohistochemistry. HAM scaffold showed good integration and adhesion to the native cartilage in all groups. Although all cell types showed the capacity of filling the focal defect, hBMSCs and hAMSCs demonstrated higher levels of new matrix synthesis. However, only the hAMSCs-containing group presented a significant cytoplasmic content of type II collagen when compared with chondrocytes. More collagen type I was identified in the new synthesized tissue of hBMSCs. In accordance, hBMSCs and hAMSCs showed better International Cartilage Research Society scoring although without statistical significance. HAM is a useful material for articular cartilage repair in vitro when used as scaffold. In combination with hAMSCs, HAM showed better

  14. Human adipose-derived stromal cells in a clinically applicable injectable alginate hydrogel

    DEFF Research Database (Denmark)

    Larsen, Bjarke Follin; Juhl, Morten; Cohen, Smadar

    2015-01-01

    BACKGROUND AIMS: Clinical trials have documented beneficial effects of mesenchymal stromal cells from bone marrow and adipose tissue (ASCs) as treatment in patients with ischemic heart disease. However, retention of transplanted cells is poor. One potential way to increase cell retention is to in...

  15. Using Hydroxyapatite-Gelatin Scaffold Seeded with Bone Marrow Stromal Cells as a Bone Graft in Animal Model

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    Mahsoumeh Behruzi

    2016-11-01

    Full Text Available Background: Nowadays, composite scaffolds with some desired characteristics have a numerous applications in hard tissue engineering. In present study, the role of composite hydroxyapatite - gelatin was examined in both alone and coated by Bone Marrow Stromal Stem Cells (BMSCs conditions in the process of healing bone defects, reduction of time repair and the immune response of body by laboratory studies (in vitro and in vivo on the skull of adult rats as well. Materials and Methods: In present study, nano-hydroxyapatite powder and gelatin were used to provide nano-hydroxyapatite-gelatin scaffold, BMSCs were isolated by Flushing method. Fifteen adult male Wistar rats weighing 250-200 g were used. Studing groups included bone defect with hydroxyapatite-gelatin scaffold, bone defect with hydroxyapatite-gelatin with BMSCs and bone defects without scaffolding as a controlwhich were examined after a week and a month after surgery. MTT assay was used in order to evaluation of biocompatibility of scaffolds. To confirm the healing progress trend and the presence of inflammatory cells we used hematoxylin-eosin and we used Masson's trichrome staining in order to study of synthesis of collagen fibers. Results: The results of MTT showed that the scaffold has no toxic effects on stromal cells. The first signs of ossification in hydroxyapatite-gelatin with BMSCs cells group, appeared in the first week. However, in the fourth week, ossification was completed and the scaffold remaining was found as embedded islands in the spongy bone tissue. The greatest number of lymphocytes was observed in the experimental group after one week of planting scaffold. Conclusion: it seems that Hydroxyapatite-gelatin scaffold coated with BMSCs cells has a potential role in the healing process of bone and it can be suitable as a therapeutic strategy to repair extensive bone lesions.

  16. Actin depolymerization enhances adipogenic differentiation in human stromal stem cells

    DEFF Research Database (Denmark)

    Chen, Li; Hu, Huimin; Qiu, Weimin

    2018-01-01

    Human stromal stem cells (hMSCs) differentiate into adipocytes that play a role in skeletal tissue homeostasis and whole body energy metabolism. During adipocyte differentiation, hMSCs exhibit significant changes in cell morphology suggesting changes in cytoskeletal organization. Here, we examined...... the effect of direct modulation of actin microfilament dynamics on adipocyte differentiation. Stabilizing actin filaments in hMSCs by siRNA-mediated knock down of the two main actin depolymerizing factors (ADFs): Cofilin 1 (CFL1) and Destrin (DSTN) or treating the cells by Phalloidin reduced adipocyte...

  17. Stromal and Hematopoietic Progenitors from C57/BI/6N Murine Bone Marrow After 30-Day "BION-M1" Spaceflight.

    Science.gov (United States)

    Markina, Elena; Andreeva, Elena; Andrianova, Irina; Sotnezova, Elena; Buravkova, Ludmila

    2018-05-02

    Elucidation of the spaceflight (SF) effects on the adult stem and progenitor cells is an important goal in space biology and medicine. A unique opportunity for this was provided by project "BION-M1". The purpose of this study was to evaluate the effects of 30-day SF on biosatellite, 7-day recovery (SFR), and subsequent ground control (GC) experiment on the mononuclear cells (MNCs) from C57/BI/6N murine tibia bone marrow. Also, hematopoietic and stromal precursor functions were characterized ex vivo. There was no significant difference in the total MNC number between experimental groups. After SF, immunophenotyping revealed an increase of large-sized CD45 + MNCs corresponded to committed hematopoietic progenitors. The total hematopoietic colony-forming unit (CFU) number decreased after SF and did not restore after 7 day of recovery due to predominant reduction of bi- and multipotent CFUs and primitive burst-forming units in favor of unipotent CFUs. Functional activity of stromal precursors in vitro was only slightly altered. SF cells displayed the enhanced expression of alkaline phosphatase. The data of the GC experiment demonstrated the preservation of the functional activity of progenitor cells from mice bone marrow. The activation of erythropoiesis in expense of burst-forming units of erythrocytes elevation was detected. After 7 days of recovery, the number of colony-forming units of fibroblast (CFUs-f) was similar to the vivarium control, while the proliferative activity of bone marrow stromal precursors decreased. The present study demonstrated that certain hematopoietic progenitors are susceptible to SF factors, while the stromal precursors displayed a certain degree of resistance. These data indicate mild and reversible alterations of bone marrow progenitors after SF.

  18. In vitro differentiation of bone marrow stromal cells into neurons and glial cells and differential protein expression in a two-compartment bone marrow stromal cell/neuron co-culture system.

    Science.gov (United States)

    Qi, Xu; Shao, Ming; Peng, Haisheng; Bi, Zhenggang; Su, Zhiqiang; Li, Hulun

    2010-07-01

    This study was performed to establish a bone marrow stromal cell (BMSC)/neuron two-compartment co-culture model in which differentiation of BMSCs into neurons could occur without direct contact between the two cell types, and to investigate protein expression changes during differentiation of this entirely BMSC-derived population. Cultured BMSCs isolated from Wistar rats were divided into three groups: BMSC culture, BMSC/neuron co-culture and BMSC/neuron two-compartment co-culture. Cells were examined for neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) expression. The electrophysiological behavior of the BMSCs was examined using patch clamping. Proteins that had significantly different expression levels in BMSCs cultured alone and co-cultured with neurons were studied using a protein chip-mass spectroscopy technique. Expression of NSE and GFAP were significantly higher in co-culture cells than in two-compartment co-culture cells, and significantly higher in both co-culture groups than in BMSCs cultured alone. Five proteins showed significant changes in expression during differentiation: TIP39_RAT and CALC_RAT underwent increases, and INSL6_RAT, PNOC_RAT and PCSK1_RAT underwent decreases in expression. We conclude that BMSCs can differentiate into neurons during both contact co-culture with neurons and two-compartment co-culture with neurons. The rate at which BMSCs differentiated into neurons was higher in contact co-culture than in non-contact co-culture.

  19. Niche matters: The comparison between bone marrow stem cells and endometrial stem cells and stromal fibroblasts reveal distinct migration and cytokine profiles in response to inflammatory stimulus.

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    Masuma Khatun

    Full Text Available Intrinsic inflammatory characteristics play a pivotal role in stem cell recruitment and homing through migration where the subsequent change in niche has been shown to alter these characteristics. The bone marrow mesenchymal stem cells (bmMSCs have been demonstrated to migrate to the endometrium contributing to the stem cell reservoir and regeneration of endometrial tissue. Thus, the aim of the present study was to compare the inflammation-driven migration and cytokine secretion profile of human bmMSCs to endometrial mesenchymal stem cells (eMSCs and endometrial fibroblasts (eSFs.The bmMSCs were isolated from bone marrow aspirates through culturing, whereas eMSCs and eSFs were FACS-isolated. All cell types were tested for their surface marker, proliferation profiles and migration properties towards serum and inflammatory attractants. The cytokine/chemokine secretion profile of 35 targets was analysed in each cell type at basal level along with lipopolysaccharide (LPS-induced state.Both stem cell types, bmMSCs and eMSCs, presented with similar stem cell surface marker profiles as well as possessed high proliferation and migration potential compared to eSFs. In multiplex assays, the secretion of 16 cytokine targets was detected and LPS stimulation expanded the cytokine secretion pattern by triggering the secretion of several targets. The bmMSCs exhibited higher cytokine secretion of vascular endothelial growth factor (VEGF-A, stromal cell-derived factor-1 alpha (SDF-1α, interleukin-1 receptor antagonist (IL-1RA, IL-6, interferon-gamma inducible protein (IP-10, monocyte chemoattractant protein (MCP-1, macrophage inflammatory protein (MIP1α and RANTES compared to eMSCs and/or eSFs after stimulation with LPS. The basal IL-8 secretion was higher in both endometrial cell types compared to bmMSCs.Our results highlight that similar to bmMSCs, the eMSCs possess high migration activity while the differentiation process towards stromal fibroblasts seemed

  20. The tyrosine kinase inhibitor dasatinib induces a marked adipogenic differentiation of human multipotent mesenchymal stromal cells.

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    Adriana Borriello

    Full Text Available BACKGROUND: The introduction of specific BCR-ABL inhibitors in chronic myelogenous leukemia therapy has entirely mutated the prognosis of this hematologic cancer from being a fatal disorder to becoming a chronic disease. Due to the probable long lasting treatment with tyrosine-kinase inhibitors (TKIs, the knowledge of their effects on normal cells is of pivotal importance. DESIGN AND METHODS: We investigated the effects of dasatinib treatment on human bone marrow-derived mesenchymal stromal cells (MSCs. RESULTS: Our findings demonstrate, for the first time, that dasatinib induces MSCs adipocytic differentiation. Particularly, when the TKI is added to the medium inducing osteogenic differentiation, a high MSCs percentage acquires adipocytic morphology and overexpresses adipocytic specific genes, including PPARγ, CEBPα, LPL and SREBP1c. Dasatinib also inhibits the activity of alkaline phosphatase, an osteogenic marker, and remarkably reduces matrix mineralization. The increase of PPARγ is also confirmed at protein level. The component of osteogenic medium required for dasatinib-induced adipogenesis is dexamethasone. Intriguingly, the increase of adipocytic markers is also observed in MSCs treated with dasatinib alone. The TKI effect is phenotype-specific, since fibroblasts do not undergo adipocytic differentiation or PPARγ increase. CONCLUSIONS: Our data demonstrate that dasatinib treatment affects bone marrow MSCs commitment and suggest that TKIs therapy might modify normal phenotypes with potential significant negative consequences.

  1. Intrinsic Sex-Linked Variations in Osteogenic and Adipogenic Differentiation Potential of Bone Marrow Multipotent Stromal Cells.

    Science.gov (United States)

    Bragdon, Beth; Burns, Robert; Baker, Amelia H; Belkina, Anna C; Morgan, Elise F; Denis, Gerald V; Gerstenfeld, Louis C; Schlezinger, Jennifer J

    2015-02-01

    Bone formation and aging are sexually dimorphic. Yet, definition of the intrinsic molecular differences between male and female multipotent mesenchymal stromal cells (MSCs) in bone is lacking. This study assessed sex-linked differences in MSC differentiation in 3-, 6-, and 9-month-old C57BL/6J mice. Analysis of tibiae showed that female mice had lower bone volume fraction and higher adipocyte content in the bone marrow compared to age-matched males. While both males and females lost bone mass in early aging, the rate of loss was higher in males. Similar expression of bone- and adipocyte-related genes was seen in males and females at 3 and 9 months, while at 6 months, females exhibited a twofold greater expression of these genes. Under osteogenic culture conditions, bone marrow MSCs from female 3- and 6-month-old mice expressed similar levels of bone-related genes, but significantly greater levels of adipocyte-related genes, than male MSCs. Female MSCs also responded to rosiglitazone-induced suppression of osteogenesis at a 5-fold lower (10 nM) concentration than male MSCs. Female MSCs grown in estrogen-stripped medium showed similar responses to rosiglitazone as MSCs grown in serum containing estrogen. MSCs from female mice that had undergone ovariectomy before sexual maturity also were sensitive to rosiglitazone-induced effects on osteogenesis. These results suggest that female MSCs are more sensitive to modulation of differentiation by PPARγ and that these differences are intrinsic to the sex of the animal from which the MSCs came. These results also may explain the sensitivity of women to the deleterious effects of rosiglitazone on bone. © 2014 Wiley Periodicals, Inc.

  2. Transfection of CXCR-4 using microbubble-mediated ultrasound irradiation and liposomes improves the migratory ability of bone marrow stromal cells.

    Science.gov (United States)

    Wang, Gong; Zhuo, Zhongxiong; Zhang, Qian; Xu, Yali; Wu, Shengzheng; Li, Lu; Xia, Hongmei; Gao, Yunhua

    2015-01-01

    Bone marrow stromal cells (BMSCs) have proven useful for the treatment of various human diseases and injuries. However, their reparative capacity is limited by their poor migration and homing ability, which are primarily dependent on the SDF-1/CXCR4 axis. Most subcultured BMSCs lack CXCR4 receptor expression on the cell surface and exhibit impaired migratory capacity. To increase responsiveness to SDF-1 and promote cell migration and survival of cultured BMSCs, we used a combination of ultrasound-targeted microbubble destruction (UTMD) and liposomes to increase CXCR4 expression in vitro. We isolated and cultured rat BMSCs to their third passage and transduced them with recombinant plasmid pDsRed-CXCR4 using microbubble-mediated ultrasound irradiation and liposomes. Compared to some viral vectors, the method we employed here resulted in significantly better transfection efficiency, CXCR4 expression, and technical reproducibility. The benefits of this approach are likely due to the combination of "sonoporation" caused by shockwaves and microjet flow resulting from UTMD-generated cavitation. Following transfection, we performed a transwell migration assay and found that the migration ability of CXCR4-modified BMSCs was 9-fold higher than controls. The methods we describe here provide an effective, safe, non-viral means to achieve high levels of CXCR4 expression. This is associated with enhanced migration of subcultured BMSCs and may be useful for clinical application as well.

  3. Chest wall reconstruction in a canine model using polydioxanone mesh, demineralized bone matrix and bone marrow stromal cells.

    Science.gov (United States)

    Tang, Hua; Xu, Zhifei; Qin, Xiong; Wu, Bin; Wu, Lihui; Zhao, XueWei; Li, Yulin

    2009-07-01

    Extensive chest wall defect reconstruction remains a challenging problem for surgeons. In the past several years, little progress has been made in this area. In this study, a biodegradable polydioxanone (PDO) mesh and demineralized bone matrix (DBM) seeded with osteogenically induced bone marrow stromal cells (BMSCs) were used to reconstruct a 6 cm x 5.5 cm chest wall defect. Four experimental groups were evaluated (n=6 per group): polydioxanone (PDO) mesh/DBMs/BMSCs group, polydioxanone (PDO) mesh/DBMs group, polydioxanone (PDO) mesh group, and a blank group (no materials) in a canine model. All the animals survived except those in the blank group. In all groups receiving biomaterial implants, the polydioxanone (PDO) mesh completely degraded at 24 weeks and was replaced by fibrous tissue with thickness close to that of the normal intercostal tissue (P>0.05). In the polydioxanone (PDO) mesh/DBMs/BMSCs group, new bone formation and bone-union were observed by radiographic and histological examination. More importantly, the reconstructed rib could maintain its original radian and achieve satisfactory biomechanics close to normal ribs in terms of bending stress (P>0.05). However, in the other two groups, fibrous tissue was observed in the defect and junctions, and the reconstructed ribs were easily distorted under an outer force. Based on these results, a surgical approach utilizing biodegradable polydioxanone (PDO) mesh in combination with DBMs and BMSCs could repair the chest wall defect not only in function but also in structure.

  4. Conditioned Medium of Bone Marrow-Derived Mesenchymal Stromal Cells as a Therapeutic Approach to Neuropathic Pain: A Preclinical Evaluation

    Directory of Open Access Journals (Sweden)

    Kelly Barbosa Gama

    2018-01-01

    Full Text Available Neuropathic pain is a type of chronic pain caused by injury or dysfunction of the nervous system, without effective therapeutic approaches. Mesenchymal stromal cells (MSCs, through their paracrine action, have great potential in the treatment of this syndrome. In the present study, the therapeutic potential of MSC-derived conditioned medium (CM was investigated in a mouse model of neuropathic pain induced by partial sciatic nerve ligation (PSL. PSL mice were treated by endovenous route with bone marrow-derived MSCs (1 × 106, CM, or vehicle. Gabapentin was the reference drug. Twelve hours after administration, neuropathic mice treated with CM exhibited an antinociceptive effect that was maintained throughout the evaluation period. MSCs also induced nonreversed antinociception, while gabapentin induced short-lasting antinociception. The levels of IL-1β, TNF-α, and IL-6 were reduced, while IL-10 was enhanced on sciatic nerve and spinal cord by treatment with CM and MSCs. Preliminary analysis of the CM secretome revealed the presence of growth factors and cytokines likely involved in the antinociception. In conclusion, the CM, similar to injection of live cells, produces a powerful and long-lasting antinociceptive effect on neuropathic pain, which is related with modulatory properties on peripheral and central levels of cytokines involved with the maintenance of this syndrome.

  5. Effect of Increasing Doses of γ-Radiation on Bone Marrow Stromal Cells Grown on Smooth and Rough Titanium Surfaces

    Directory of Open Access Journals (Sweden)

    Bo Huang

    2015-01-01

    Full Text Available Radiation therapy for oral and maxillofacial tumors could damage bone marrow stromal cells (BMSCs in jaw, which caused dental implant failure. However, how radiation affects BMSCs on SLA (sandblasted with large-grits, acid-etched surfaces is still unknown. The aim of this study was to investigate effect of different dose of γ-radiation on BMSCs on SLA and PT (polished titanium surfaces. Rat BMSCs were radiated with 2, 4, and 8 Gy γ-radiation and then seeded on both surfaces. Cell adhesion, spreading, and proliferation were tested. The osteogenesis and the adipogenesis ability were examined by Alizarin-Red and Oil-Red staining, respectively. Real-time PCR was performed to detect osteogenic (osteocalcin, OCN; runt-related transcription factor 2, Runx2 and adipogenic (peroxisome proliferator-activated receptor gamma, PPARγ gene expression at days 7 and 14 postirradiation. Results showed that γ-radiation reduced cell proliferation, adhesion, spreading, and osteogenic differentiation. 2 Gy radiation promoted adipogenic differentiation, but it was significantly decreased when dosage reached 4 Gy. In conclusion, results suggest that γ-radiation influenced BMSCs behaviors in a dosage-dependent manner except adipogenic differentiation, low dose promoted it, and high dose inhibited it. This effect was influenced by surface characteristics, which may explain the different failure rate of various implants in patients after radiation.

  6. In vitro non-viral murine pro-neurotrophin 3 gene transfer into rat bone marrow stromal cells.

    Science.gov (United States)

    Darabi, Shahram; Tiraihi, Taki; Delshad, AliReza; Sadeghizadeh, Majid; Khalil, Wisam; Taheri, Taher

    2017-04-15

    Neurotrophin 3 (NT-3) is an important factor for promoting prenatal neural development, as well as regeneration, axogenesis and plasticity in postnatal life. Therapy with NT-3 was reported to improve the condition of patients suffering from degenerative diseases and traumatic injuries, however, the disadvantage of NT-3 protein delivery is its short half-life, thus our alternative approach is the use of NT-3 gene therapy. In this study, the bone marrow stromal cells (BMSCs) were isolated from adult rats, cultured for 4 passages and transfected with either pEGFP-N1 or a constructed vector containing murine proNT-3 (pSecTag2/HygroB-murine proNT-3) using Lipofectamine 2000 followed by Hygromycin B (200mg/kg). The transfection efficiency of the transiently transfected BMSCs was evaluated using the green fluorescence protein containing vector (pEGFP-N1). A quantitative evaluation of the NT-3 expression of mRNA using real time qRT-PCR shows that there was double fold increase in NT-3 gene expression compared with non-transfected BMSCs, also, the culture supernatant yielded double fold increase in NT-3 using ELISA technique, the data were supported by immunoblotting technique. This suggests that the use of this transfection technique can be useful for gene therapy in different neurological disorders with neurodegenerative or traumatic origins. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Influence of chitosan-chitin nanofiber composites on cytoskeleton structure and the proliferation of rat bone marrow stromal cells.

    Science.gov (United States)

    Kiroshka, Victoria V; Petrova, Valentina A; Chernyakov, Daniil D; Bozhkova, Yulia O; Kiroshka, Katerina V; Baklagina, Yulia G; Romanov, Dmitry P; Kremnev, Roman V; Skorik, Yury A

    2017-01-01

    Chitosan scaffolds have gained much attention in various tissue engineering applications, but the effect of their microstructure on cell-material spatial interactions remains unclear. Our objective was to evaluate the effect of chitosan-based matrices doping with chitin nano-whiskers (CNW) on adhesion, spreading, cytoskeleton structure, and proliferation of rat bone marrow stromal cells (BMSCs). The behavior of BMSCs during culture on chitosan-CNW films was determined by the molecular mass, hydrophobicity, porosity, crosslinking degree, protonation degree and molecular structure of the composite chitosan-CNW films. The shape, spreading area, cytoskeleton structure, and proliferation of BMSCs on chitosan matrices with a crystalline structure and high porosity were similar to that observed for BMSCs cultured on polystyrene tissue culture plates. The amorphous polymer structure and high swelling led to a decrease in the spreading area and cell proliferation. Thus, we can control the behavior of cells in culture (adhesion, spreading, and proliferation) by changing the physico-chemical properties of the chitosan-CNW films.

  8. Transplantation of autologous bone marrow stromal cells (BMSC for CNS disorders – Strategy and tactics for clinical application

    Directory of Open Access Journals (Sweden)

    Satoshi Kuroda

    2010-01-01

    Full Text Available Background – There is increasing evidence that the transplanted bone marrow stromal cells (BMSC significantly promote functional recovery after central nervous system (CNS damage in the animal models of various kinds of CNS disorders, including cerebral infarct, brain contusion and spinal cord injury. However, there are several shortages of information when considering clinical application of BMSC transplantation for patients with neurological disorders. In this paper, therefore, we discuss what we should clarify to establish cell transplantation therapy in clinical situation and describe our recent works for this purpose.Methods and Results – The BMSC have the ability to alter their gene expression profile and phenotype in response to the surrounding circumstances and to protect the neurons by producing some neurotrophic factors. They also promote neurite extension and rebuild the neural circuits in the injured CNS. Using optical imaging and MRI techniques, the transplanted BMSC can non-invasively be tracked in the living animals for at least 8 weeks after transplantation. Functional imaging such as PET scan may have the potential to assess the beneficial effects of BMSC transplantation. The BMSC can be expanded using the animal protein-free culture medium, which would maintain their potential of proliferation, migration, and neural differentiation.Conclusion – It is urgent issues to develop clinical imaging technique to track the transplanted cells in the CNS and evaluate the therapeutic significance of BMSC transplantation in order to establish it as a definite therapeutic strategy in clinical situation in the future

  9. Mag-seeding of rat bone marrow stromal cells into porous hydroxyapatite scaffolds for bone tissue engineering.

    Science.gov (United States)

    Shimizu, Kazunori; Ito, Akira; Honda, Hiroyuki

    2007-09-01

    Bone tissue engineering has been investigated as an alternative strategy for autograft transplantation. In the process of tissue engineering, cell seeding into three-dimensional (3-D) scaffolds is the first step for constructing 3-D tissues. We have proposed a methodology of cell seeding into 3-D porous scaffolds using magnetic force and magnetite nanoparticles, which we term Mag-seeding. In this study, we applied this Mag-seeding technique to bone tissue engineering using bone marrow stromal cells (BMSCs) and 3-D hydroxyapatite (HA) scaffolds. BMSCs were magnetically labeled with our original magnetite cationic liposomes (MCLs) having a positive surface charge to improve adsorption to cell surface. Magnetically labeled BMSCs were seeded onto a scaffold, and a 1-T magnet was placed under the scaffold. By using Mag-seeding, the cells were successfully seeded into the internal space of scaffolds with a high cell density. The cell seeding efficiency into HA scaffolds by Mag-seeding was approximately threefold larger than that by static-seeding (conventional method, without a magnet). After a 14-d cultivation period using the osteogenic induction medium by Mag-seeding, the level of two representative osteogenic markers (alkaline phosphatase and osteocalcin) were significantly higher than those by static-seeding. These results indicated that Mag-seeding of BMSCs into HA scaffolds is an effective approach to bone tissue engineering.

  10. Cell transplantation for the treatment of spinal cord injury - bone marrow stromal cells and choroid plexus epithelial cells

    Directory of Open Access Journals (Sweden)

    Chizuka Ide

    2016-01-01

    Full Text Available Transplantation of bone marrow stromal cells (BMSCs enhanced the outgrowth of regenerating axons and promoted locomotor improvements of rats with spinal cord injury (SCI. BMSCs did not survive long-term, disappearing from the spinal cord within 2-3 weeks after transplantation. Astrocyte-devoid areas, in which no astrocytes or oligodendrocytes were found, formed at the epicenter of the lesion. It was remarkable that numerous regenerating axons extended through such astrocyte-devoid areas. Regenerating axons were associated with Schwann cells embedded in extracellular matrices. Transplantation of choroid plexus epithelial cells (CPECs also enhanced axonal regeneration and locomotor improvements in rats with SCI. Although CPECs disappeared from the spinal cord shortly after transplantation, an extensive outgrowth of regenerating axons occurred through astrocyte-devoid areas, as in the case of BMSC transplantation. These findings suggest that BMSCs and CPECs secret neurotrophic factors that promote tissue repair of the spinal cord, including axonal regeneration and reduced cavity formation. This means that transplantation of BMSCs and CPECs promotes "intrinsic" ability of the spinal cord to regenerate. The treatment to stimulate the intrinsic regeneration ability of the spinal cord is the safest method of clinical application for SCI. It should be emphasized that the generally anticipated long-term survival, proliferation and differentiation of transplanted cells are not necessarily desirable from the clinical point of view of safety.

  11. Comparison of Adipose-Derived and Bone Marrow Mesenchymal Stromal Cells in a Murine Model of Crohn's Disease.

    Science.gov (United States)

    Xie, Minghao; Qin, Huabo; Luo, Qianxin; He, Xiaosheng; He, Xiaowen; Lan, Ping; Lian, Lei

    2017-01-01

    Mesenchymal stromal cells (MSCs) have been used in the treatment of Crohn's disease (CD) because of the immunomodulatory ability. The aim of this study was to investigate the therapeutic effect of adipose-derived MSCs (AD-MSCs) and to compare the therapeutic effect of AD-MSCs with that of bone marrow MSCs (BM-MSCs) in a murine model of CD. Murine colitis model of CD was created by trinitrobenzene sulfonic acid (TNBS). Twelve hours after treatment with TNBS, the mouse model was injected with MSCs intraperitoneally. Real-time polymerase chain reaction and immunohistochemistry staining were used to measure the expression levels of inflammatory cytokines in colonic tissues to investigate the therapeutic effect of AD-MSCs. The ten-day survival was recorded after infusion of MSCs. Intraperitoneal injection of MSCs alleviated the clinical and histopathologic severity of intestinal inflammation, and improved the survival of the TNBS-induced mouse model of CD. AD-MSCs could effectively increase the expression of interleukin-10 and reduce the secretion of pro-inflammatory cytokines including tumor necrosis factor-α, interleukin-12, and vascular endothelial growth factor. The mucosal injury was repaired by AD-MSCs. These effects were comparable between AD-MSCs and BM-MSCs. The therapeutic effect appears similar between AD-MSCs and BM-MSCs in treating CD. AD-MSCs may be a potential alternative of cell-based therapy for CD.

  12. Expansion of Bone Marrow Mesenchymal Stromal Cells in Perfused 3D Ceramic Scaffolds Enhances In Vivo Bone Formation.

    Science.gov (United States)

    Hoch, Allison I; Duhr, Ralph; Di Maggio, Nunzia; Mehrkens, Arne; Jakob, Marcel; Wendt, David

    2017-12-01

    Bone marrow-derived mesenchymal stromal cells (BMSC), when expanded directly within 3D ceramic scaffolds in perfusion bioreactors, more reproducibly form bone when implanted in vivo as compared to conventional expansion on 2D polystyrene dishes/flasks. Since the bioreactor-based expansion on 3D ceramic scaffolds encompasses multiple aspects that are inherently different from expansion on 2D polystyrene, we aimed to decouple the effects of specific parameters among these two model systems. We assessed the effects of the: 1) 3D scaffold vs. 2D surface; 2) ceramic vs. polystyrene materials; and 3) BMSC niche established within the ceramic pores during in vitro culture, on subsequent in vivo bone formation. While BMSC expanded on 3D polystyrene scaffolds in the bioreactor could maintain their in vivo osteogenic potential, results were similar as BMSC expanded in monolayer on 2D polystyrene, suggesting little influence of the scaffold 3D environment. Bone formation was most reproducible when BMSC are expanded on 3D ceramic, highlighting the influence of the ceramic substrate. The presence of a pre-formed niche within the scaffold pores had negligible effects on the in vivo bone formation. The results of this study allow a greater understanding of the parameters required for perfusion bioreactor-based manufacturing of osteogenic grafts for clinical applications. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Carbon nanotubes functionalized with fibroblast growth factor accelerate proliferation of bone marrow-derived stromal cells and bone formation

    International Nuclear Information System (INIS)

    Hirata, Eri; Takita, Hiroko; Watari, Fumio; Yokoyama, Atsuro; Ménard-Moyon, Cécilia; Venturelli, Enrica; Bianco, Alberto

    2013-01-01

    Multi-walled carbon nanotubes (MWCNTs) were functionalized with fibroblast growth factor (FGF) and the advantages of their use as scaffolds for bone augmentation were evaluated in vitro and in vivo. The activity of FGF was assessed by measuring the effect on the proliferation of rat bone marrow stromal cells (RBMSCs). The presence of FGF enhanced the proliferation of RBMSCs and the FGF covalently conjugated to the nanotubes (FGF–CNT) showed the same effect as FGF alone. In addition, FGF–CNT coated sponges were implanted between the parietal bone and the periosteum of rats and the formation of new bone was investigated. At day 14 after implantation, a larger amount of newly formed bone was clearly observed in most pores of FGF–CNT coated sponges. These findings indicated that MWCNTs accelerated new bone formation in response to FGF, as well as the integration of particles into new bone during its formation. Scaffolds coated with FGF–CNT could be considered as promising novel substituting materials for bone regeneration in future tissue engineering applications. (paper)

  14. Carbon nanotubes functionalized with fibroblast growth factor accelerate proliferation of bone marrow-derived stromal cells and bone formation

    Science.gov (United States)

    Hirata, Eri; Ménard-Moyon, Cécilia; Venturelli, Enrica; Takita, Hiroko; Watari, Fumio; Bianco, Alberto; Yokoyama, Atsuro

    2013-11-01

    Multi-walled carbon nanotubes (MWCNTs) were functionalized with fibroblast growth factor (FGF) and the advantages of their use as scaffolds for bone augmentation were evaluated in vitro and in vivo. The activity of FGF was assessed by measuring the effect on the proliferation of rat bone marrow stromal cells (RBMSCs). The presence of FGF enhanced the proliferation of RBMSCs and the FGF covalently conjugated to the nanotubes (FGF-CNT) showed the same effect as FGF alone. In addition, FGF-CNT coated sponges were implanted between the parietal bone and the periosteum of rats and the formation of new bone was investigated. At day 14 after implantation, a larger amount of newly formed bone was clearly observed in most pores of FGF-CNT coated sponges. These findings indicated that MWCNTs accelerated new bone formation in response to FGF, as well as the integration of particles into new bone during its formation. Scaffolds coated with FGF-CNT could be considered as promising novel substituting materials for bone regeneration in future tissue engineering applications.

  15. Response of murine bone marrow-derived mesenchymal stromal cells to dry-etched porous silicon scaffolds.

    Science.gov (United States)

    Hajj-Hassan, Mohamad; Khayyat-Kholghi, Maedeh; Wang, Huifen; Chodavarapu, Vamsy; Henderson, Janet E

    2011-11-01

    Porous silicon shows great promise as a bio-interface material due to its large surface to volume ratio, its stability in aqueous solutions and to the ability to precisely regulate its pore characteristics. In the current study, porous silicon scaffolds were fabricated from single crystalline silicon wafers by a novel xenon difluoride dry etching technique. This simplified dry etch fabrication process allows selective formation of porous silicon using a standard photoresist as mask material and eliminates the post-formation drying step typically required for the wet etching techniques, thereby reducing the risk of damaging the newly formed porous silicon. The porous silicon scaffolds supported the growth of primary cultures of bone marrow derived mesenchymal stromal cells (MSC) plated at high density for up to 21 days in culture with no significant loss of viability, assessed using Alamar Blue. Scanning electron micrographs confirmed a dense lawn of cells at 9 days of culture and the presence of MSC within the pores of the porous silicon scaffolds. Copyright © 2011 Wiley Periodicals, Inc.

  16. Tissue inhibitor of matrix metalloproteinase-1 suppresses apoptosis of mouse bone marrow stromal cell line MBA-1.

    Science.gov (United States)

    Guo, L-J; Luo, X-H; Xie, H; Zhou, H-D; Yuan, L-Q; Wang, M; Liao, E-Y

    2006-05-01

    We investigated the action of tissue inhibitor of metalloproteinase-1 (TIMP-1) on apoptosis and differentiation of mouse bone marrow stromal cell line MBA-1. TIMP-1 did not affect alkaline phosphatase (ALP) activity, suggesting that it is not involved in osteoblastic differentiation in MBA-1 cells. However, TIMP-1 inhibited MBA-1 apoptosis induced by serum deprivation in a dose-dependent manner. Our study also showed increased Bcl-2 protein expression and decreased Bax protein expression with TIMP-1 treatment. TIMP-1 decreased cytochrome c release and caspase-3 activation in MBA-1 cells. TIMP-1 activated phosphatidylinositol 3-kinase (PI3-kinase) and c-Jun N-terminal kinase (JNK), and the PI3-kinase inhibitor LY294002 or the JNK inhibitor SP600125 abolished its antiapoptotic activity. To investigate whether antiapoptotic action of TIMP-1 was mediated through its inhibition on MMP activities, we constructed mutant TIMP-1 by side-directed mutagenesis, which abolished the inhibitory activity of MMPs by deletion of Cys1 to Ala4. Wild-type TIMP-1 and mutant TIMP-1 expression plasmids were transfected in MBA-1 cells, and results showed that mutant TIMP-1 still protected the induced MBA-1 cell against apoptosis. These data suggest that TIMP-1 antiapoptotic actions are mediated via the PI3-kinase and JNK signaling pathways and independent of TIMP-1 inhibition of MMP activities.

  17. Low physiologic oxygen tensions reduce proliferation and differentiation of human multipotent mesenchymal stromal cells

    Directory of Open Access Journals (Sweden)

    Handgretinger Rupert

    2010-01-01

    Full Text Available Abstract Background Human multipotent mesenchymal stromal cells (MSC can be isolated from various tissues including bone marrow. Here, MSC participate as bone lining cells in the formation of the hematopoietic stem cell niche. In this compartment, the oxygen tension is low and oxygen partial pressure is estimated to range from 1% to 7%. We analyzed the effect of low oxygen tensions on human MSC cultured with platelet-lysate supplemented media and assessed proliferation, morphology, chromosomal stability, immunophenotype and plasticity. Results After transferring MSC from atmospheric oxygen levels of 21% to 1%, HIF-1α expression was induced, indicating efficient oxygen reduction. Simultaneously, MSC exhibited a significantly different morphology with shorter extensions and broader cell bodies. MSC did not proliferate as rapidly as under 21% oxygen and accumulated in G1 phase. The immunophenotype, however, was unaffected. Hypoxic stress as well as free oxygen radicals may affect chromosomal stability. However, no chromosomal abnormalities in human MSC under either culture condition were detected using high-resolution matrix-based comparative genomic hybridization. Reduced oxygen tension severely impaired adipogenic and osteogenic differentiation of human MSC. Elevation of oxygen from 1% to 3% restored osteogenic differentiation. Conclusion Physiologic oxygen tension during in vitro culture of human MSC slows down cell cycle progression and differentiation. Under physiological conditions this may keep a proportion of MSC in a resting state. Further studies are needed to analyze these aspects of MSC in tissue regeneration.

  18. Construction of a human corneal stromal equivalent with non-transfected human corneal stromal cells and acellular porcine corneal stromata.

    Science.gov (United States)

    Diao, Jin-Mei; Pang, Xin; Qiu, Yue; Miao, Ying; Yu, Miao-Miao; Fan, Ting-Jun

    2015-03-01

    A tissue-engineered human corneal stroma (TE-HCS) has been developed as a promising equivalent to the native corneal stroma for replacement therapy. However, there is still a crucial need to improve the current approaches to render the TE-HCS equivalent more favorable for clinical applications. At the present study, we constructed a TE-HCS by incubating non-transfected human corneal stromal (HCS) cells in an acellular porcine corneal stromata (aPCS) scaffold in 20% fetal bovine serum supplemented DMEM/F12 (1:1) medium at 37 °C with 5% CO2in vitro. After 3 days of incubation, the constructed TE-HCS had a suitable tensile strength for transplantation, and a transparency that is comparable to native cornea. The TE-HCS had a normal histological structure which contained regularly aligned collagen fibers and differentiated HCS cells with positive expression of marker and functional proteins, mimicking a native HCS. After transplantation into rabbit models, the TE-HCS reconstructed normal corneal stroma in vivo and function well in maintaining corneal clarity and thickness, indicating that the completely biological TE-HCS could be used as a HCS equivalent. The constructed TE-HCS has promising potentials in regenerative medicine and treatment of diseases caused by corneal stromal disorders. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Potency testing of mesenchymal stromal cell growth expanded in human platelet lysate from different human tissues.

    Science.gov (United States)

    Fazzina, R; Iudicone, P; Fioravanti, D; Bonanno, G; Totta, P; Zizzari, I G; Pierelli, L

    2016-08-25

    Mesenchymal stromal cells (MSCs) have been largely investigated, in the past decade, as potential therapeutic strategies for various acute and chronic pathological conditions. MSCs isolated from different sources, such as bone marrow (BM), umbilical cord tissue (UCT) and adipose tissue (AT), share many biological features, although they may show some differences on cumulative yield, proliferative ability and differentiation potential. The standardization of MSCs growth and their functional amplification is a mandatory objective of cell therapies. The aim of this study was to evaluate the cumulative yield and the ex vivo amplification potential of MSCs obtained from various sources and different subjects, using defined culture conditions with a standardized platelet lysate (PL) as growth stimulus. MSCs isolated from BM, UCT and AT and expanded in human PL were compared in terms of cumulative yield and growth potential per gram of starting tissue. MSCs morphology, phenotype, differentiation potential, and immunomodulatory properties were also investigated to evaluate their biological characteristics. The use of standardized PL-based culture conditions resulted in a very low variability of MSC growth. Our data showed that AT has the greater capacity to generate MSC per gram of initial tissue, compared to BM and UCT. However, UCT-MSCs replicated faster than AT-MSCs and BM-MSCs, revealing a greater proliferation capacity of this source irrespective of its lower MSC yield. All MSCs exhibited the typical MSC phenotype and the ability to differentiate into all mesodermal lineages, while BM-MSCs showed the most prominent immunosuppressive effect in vitro. The adoption of standardized culture conditions may help researchers and clinicians to reveal particular characteristics and inter-individual variability of MSCs sourced from different tissues. These data will be beneficial to set the standards for tissue collection and MSCs clinical-scale expansion both for cell banking

  20. Human adipose stromal cells expanded in human serum promote engraftment of human peripheral blood hematopoietic stem cells in NOD/SCID mice

    International Nuclear Information System (INIS)

    Kim, Su Jin; Cho, Hyun Hwa; Kim, Yeon Jeong; Seo, Su Yeong; Kim, Han Na; Lee, Jae Bong; Kim, Jae Ho; Chung, Joo Seop; Jung, Jin Sup

    2005-01-01

    Human mesenchymal stem cells (hMSC), that have been reported to be present in bone marrow, adipose tissues, dermis, muscles, and peripheral blood, have the potential to differentiate along different lineages including those forming bone, cartilage, fat, muscle, and neuron. Therefore, hMSC are attractive candidates for cell and gene therapy. The optimal conditions for hMSC expansion require medium supplemented with fetal bovine serum (FBS). Some forms of cell therapy will involve multiple doses, raising a concern over immunological reactions caused by medium-derived FBS proteins. In this study, we cultured human adipose stromal cells (hADSC) and bone marrow stroma cells (HBMSC) in human serum (HS) during their isolation and expansion, and demonstrated that they maintain their proliferative capacity and ability for multilineage differentiation and promote engraftment of peripheral blood-derived CD34(+) cells mobilized from bone marrow in NOD/SCID mice. Our results indicate that hADSC and hBMSC cultured in HS can be used for clinical trials of cell and gene therapies, including promotion of engraftment after allogeneic HSC transplantation

  1. Antigen Presenting Cells and Stromal Cells Trigger Human Natural Killer Lymphocytes to Autoreactivity: Evidence for the Involvement of Natural Cytotoxicity Receptors (NCR and NKG2D

    Directory of Open Access Journals (Sweden)

    Alessandro Poggi

    2006-01-01

    Full Text Available Human natural killer (NK lymphocytes should not damage autologous cells due to the engagement of inhibitory receptor superfamily (IRS members by HLA-I. Nevertheless, NK cells kill self cells expressing low levels or lacking HLA-I, as it may occur during viral infections (missing-self hypothesis. Herein, we show that human NK cells can be activated upon binding with self antigen presenting cells or stromal cells despite the expression of HLA-I. Indeed, NK cells can kill and produce pro-inflammatory and regulating cytokines as IFN-γ, TNF-α and IL10 during interaction with autologous dendritic cells or bone marrow stromal cells or skin fibroblasts. The killing of antigen presenting and stromal cells is dependent on LFA1/ICAM1 interaction. Further, the natural cytotoxicity receptors (NCR NKp30 and NKp46 are responsible for the delivery of lethal hit to DC, whereas NKG2D activating receptor, the ligand of the MHC-related molecule MIC-A and the UL16 binding protein, is involved in stromal cell killing. These findings indicate that different activating receptors are involved in cell to self cell interaction. Finally, NK cells can revert the veto effect of stromal cells on mixed lymphocyte reaction further supporting the idea that NK cells may alter the interaction between T lymphocytes and microenvironment leading to autoreactivity.

  2. Actin depolymerization enhances adipogenic differentiation in human stromal stem cells

    DEFF Research Database (Denmark)

    Chen, Li; Hu, Huimin; Qiu, Weimin

    2018-01-01

    Human stromal stem cells (hMSCs) differentiate into adipocytes that play a role in skeletal tissue homeostasis and whole body energy metabolism. During adipocyte differentiation, hMSCs exhibit significant changes in cell morphology suggesting changes in cytoskeletal organization. Here, we examined...... differentiation as evidenced by decreased number of mature adipocytes and decreased adipocyte specific gene expression (ADIPOQ, LPL, PPARG, FABP4). In contrast, disruption of actin cytoskeleton by Cytochalasin D enhanced adipocyte differentiation. Follow up studies revealed that the effects of CFL1 on adipocyte...... differentiation depended on the activity of LIM domain kinase 1 (LIMK1) which is the major upstream kinase of CFL1. Inhibiting LIMK by its specific chemical inhibitor LIMKi inhibited the phosphorylation of CFL1 and actin polymerization, and enhanced the adipocyte differentiation. Moreover, treating h...

  3. 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

  4. Organotypic culture of human bone marrow adipose tissue.

    Science.gov (United States)

    Uchihashi, Kazuyoshi; Aoki, Shigehisa; Shigematsu, Masamori; Kamochi, Noriyuki; Sonoda, Emiko; Soejima, Hidenobu; Fukudome, Kenji; Sugihara, Hajime; Hotokebuchi, Takao; Toda, Shuji

    2010-04-01

    The precise role of bone marrow adipose tissue (BMAT) in the marrow remains unknown. The purpose of the present study was therefore to describe a novel method for studying BMAT using 3-D collagen gel culture of BMAT fragments, immunohistochemistry, ELISA and real-time reverse transcription-polymerase chain reaction. Mature adipocytes and CD45+ leukocytes were retained for >3 weeks. Bone marrow stromal cells (BMSC) including a small number of lipid-laden preadipocytes and CD44+/CD105+ mesenchymal stem cell (MSC)-like cells, developed from BMAT. Dexamethasone (10 micromol/L), but not insulin (20 mU/mL), significantly increased the number of preadipocytes. Dexamethasone and insulin also promoted leptin production and gene expression in BMAT. Adiponectin production by BMAT was BMAT, in which adiponectin protein secretion is normally very low, and that BMAT may exhibit a different phenotype from that of the visceral and subcutaneous adipose tissues. BMAT-osteoblast interactions were also examined, and it was found that osteoblasts inhibited the development of BMSC and reduced leptin production, while BMAT inhibited the growth and differentiation of osteoblasts. The present novel method proved to be useful for the study of BMAT biology.

  5. Actin depolymerization enhances adipogenic differentiation in human stromal stem cells

    Directory of Open Access Journals (Sweden)

    Li Chen

    2018-05-01

    Full Text Available Human stromal stem cells (hMSCs differentiate into adipocytes that play a role in skeletal tissue homeostasis and whole body energy metabolism. During adipocyte differentiation, hMSCs exhibit significant changes in cell morphology suggesting changes in cytoskeletal organization. Here, we examined the effect of direct modulation of actin microfilament dynamics on adipocyte differentiation. Stabilizing actin filaments in hMSCs by siRNA-mediated knock down of the two main actin depolymerizing factors (ADFs: Cofilin 1 (CFL1 and Destrin (DSTN or treating the cells by Phalloidin reduced adipocyte differentiation as evidenced by decreased number of mature adipocytes and decreased adipocyte specific gene expression (ADIPOQ, LPL, PPARG, FABP4. In contrast, disruption of actin cytoskeleton by Cytochalasin D enhanced adipocyte differentiation. Follow up studies revealed that the effects of CFL1 on adipocyte differentiation depended on the activity of LIM domain kinase 1 (LIMK1 which is the major upstream kinase of CFL1. Inhibiting LIMK by its specific chemical inhibitor LIMKi inhibited the phosphorylation of CFL1 and actin polymerization, and enhanced the adipocyte differentiation. Moreover, treating hMSCs by Cytochalasin D inhibited ERK and Smad2 signaling and this was associated with enhanced adipocyte differentiation. On the other hand, Phalloidin enhanced ERK and Smad2 signaling, but inhibited adipocyte differentiation which was rescued by ERK specific chemical inhibitor U0126. Our data provide a link between restructuring of hMSCs cytoskeleton and hMSCs lineage commitment and differentiation. Keywords: Actin cytoskeleton, Actin depolymerizing factors, Adipocyte differentiation, Human stromal stem cells

  6. Increased extracellular and intracellular Ca{sup 2+} lead to adipocyte accumulation in bone marrow stromal cells by different mechanisms

    Energy Technology Data Exchange (ETDEWEB)

    Hashimoto, Ryota, E-mail: hryota@juntendo.ac.jp [Department of Physiology, Juntendo University Faculty of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Katoh, Youichi, E-mail: katoyo@juntendo-urayasu.jp [Juntendo University Faculty of International Liberal Arts, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Department of Cardiology, Juntendo University Faculty of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Miyamoto, Yuki [Juntendo University Faculty of Health Care and Nursing, Takasu 2-5-1, Urayasu-shi, Chiba 279-0023 (Japan); Itoh, Seigo; Daida, Hiroyuki [Department of Cardiology, Juntendo University Faculty of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Nakazato, Yuji [Center for Environmental Research, Department of Cardiology, Juntendo University Faculty of Medicine Urayasu Hospital, Tomioka 2-1-1, Urayasu-shi, Chiba 279-0022 (Japan); Okada, Takao [Department of Physiology, Juntendo University Faculty of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan)

    2015-02-20

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

  7. Interaction between x-irradiated plateau-phase bone marrow stromal cell lines and co-cultivated factor-dependent cell lines leading to leukemogenesis in vitro

    International Nuclear Information System (INIS)

    Naparstek, E.; Anklesaria, P.; FitzGerald, T.J.; Sakakeeny, M.A.; Greenberger, J.S.

    1987-01-01

    Plateau-phase mouse clonal bone marrow stromal cell lines D2XRII and C3H cl 11 produce decreasing levels of M-CSF (CSF-1), a specific macrophage progenitor cell humoral regulator, following X-irradiation in vitro. The decrease did not go below 40% of control levels, even after irradiation doses of 50,000 rad (500 Gy). In contrast, a distinct humoral regulator stimulating growth of GM-CSF/IL-3 factor-dependent (FD) hematopoietic progenitor cell lines was detected following radiation to doses above 2000 rad. This humoral factor was not detectable in conditioned medium from irradiated cells, weakly detected using factor-dependent target cell populations in agar overlay, and was prominently detected by liquid co-cultivation of factor-dependent cells with irradiated stromal cell cultures. Subclonal lines of FD cells, derived after co-cultivation revealed karyotypic abnormalities and induced myeloblastic tumors in syngeneic mice. Five-eight weeks co-cultivation was required for induction of factor independence and malignancy and was associated with dense cell to cell contact between FD cells and stromal cells demonstrated by light and electron microscopy. Increases in hematopoietic to stromal cell surface area, total number of adherent cells per flask, total non-adherent cell colonies per flask, and cumulative non-adherent cell production were observed after irradiation. The present data may prove very relevant to an understanding of the cell to cell interactions during X-irradiation-induced leukemia

  8. Electrospun gelatin/polycaprolactone nanofibrous membranes combined with a coculture of bone marrow stromal cells and chondrocytes for cartilage engineering

    Directory of Open Access Journals (Sweden)

    He X

    2015-03-01

    Full Text Available Xiaomin He,1,* Bei Feng,1,2,* Chuanpei Huang,1 Hao Wang,1 Yang Ge,1 Renjie Hu,1 Meng Yin,1 Zhiwei Xu,1 Wei Wang,1 Wei Fu,1,2 Jinghao Zheng1 1Department of Pediatric Cardiothoracic Surgery, 2Institute of Pediatric Translational Medicine, Shanghai Children’s Medical Center School of Medicine, Shanghai Jiao Tong University, Shanghai, People’s Republic of China *These authors contributed equally to this work Abstract: Electrospinning has recently received considerable attention, showing notable potential as a novel method of scaffold fabrication for cartilage engineering. The aim of this study was to use a coculture strategy of chondrocytes combined with electrospun gelatin/polycaprolactone (GT/PCL membranes, instead of pure chondrocytes, to evaluate the formation of cartilaginous tissue. We prepared the GT/PCL membranes, seeded bone marrow stromal cell (BMSC/chondrocyte cocultures (75% BMSCs and 25% chondrocytes in a sandwich model in vitro, and then implanted the constructs subcutaneously into nude mice for 12 weeks. Gross observation, histological and immunohistological evaluation, glycosaminoglycan analyses, Young’s modulus measurement, and immunofluorescence staining were performed postimplantation. We found that the coculture group formed mature cartilage-like tissue, with no statistically significant difference from the chondrocyte group, and labeled BMSCs could differentiate into chondrocyte-like cells under the chondrogenic niche of chondrocytes. This entire strategy indicates that GT/PCL membranes are also a suitable scaffold for stem cell-based cartilage engineering and may provide a potentially clinically feasible approach for cartilage repairs. Keywords: electrospinning, nanocomposite, cartilage tissue engineering, nanomaterials, stem cells

  9. Effect of growth factors (BMP-4/7 & bFGF on proliferation & osteogenic differentiation of bone marrow stromal cells

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    Shaohui Yuan

    2013-01-01

    Full Text Available Background & objectives: BMP (bone morphogenetic protein-4/7 and bFGF (basic fibroblast growth factor significantly promote the osteogenic activity and the proliferation of rabbit BMSCs (bone marrow stromal cells, respectively. However, their synergistic effects on the proliferation and the differentiation of BMSCs remain unclear. In the present study, the effects of bFGF and BMP-4/7 were investigated on the proliferation and the differentiation of rat BMSCs in vitro. Methods: BMSCs were isolated from New Zealand white rabbits and cultured to the third passage. The samples were divided into five groups according to the material implanted: (A 80 ng/ml BMP-4/7; (B 80 ng/ml bFGF; (C 30 ng/ml BMP-4/7 and 30 ng/ml bFGF; (D 50 ng/ml BMP-4/7 and 50 ng/ml bFGF; and (E 80 ng/ml BMP-4/7 and 80 ng/ml bFGF. Cell proliferation was analyzed using methyl thiazolyl tetrazolium (MTT assay. Alkaline phosphatase activity and osteocalcin (OC dynamics were also measured. Results: BMP-4/7 alone significantly (P<0.05 promoted the proliferation of BMSCs. At the same time, it also promoted or inhibited the osteogenic differentiation of BMSCs. The synergistic effects of BMP-4/7 and bFGF significantly promoted both the proliferation and the osteogenic differentiation of BMSCs. The treatment of the synergistic effects was dose and time dependent. Interpretation & conclusions: A rational combination of BMP-4/7 and bFGF can promote the proliferation and the osteogenic differentiation of BMSCs. In addition, the synergistic functions are effective.

  10. Fibronectin- and collagen-mimetic ligands regulate bone marrow stromal cell chondrogenesis in three-dimensional hydrogels

    Directory of Open Access Journals (Sweden)

    JT Connelly

    2011-09-01

    Full Text Available Modification of tissue engineering scaffolds with bioactive molecules is a potential strategy for modulating cell behavior and guiding tissue regeneration. While adhesion to RGD peptides has been shown to inhibit in vitro chondrogenesis, the effects of extracellular matrix (ECM-mimetic ligands with complex secondary and tertiary structures are unknown. This study aimed to determine whether collagen- and fibronectin-mimetic ligands would retain biologic functionality in three-dimensional (3D hydrogels, whether different ECM-mimetic ligands differentially influence in vitro chondrogenesis, and if effects of ligands on differentiation depend on soluble biochemical stimuli. A linear RGD peptide, a recombinant fibronectin fragment containing the seven to ten Type III repeats (FnIII7-10 and a triple helical, collagen mimetic peptide with the GFOGER motif were covalently coupled to agarose gels using the sulfo-SANPAH crosslinker, and bone marrow stromal cells (BMSCs were cultured within the 3D hydrogels. The ligands retained biologic functionality within the agarose gels and promoted density-dependent BMSC spreading. Interactions with all adhesive ligands inhibited stimulation by chondrogenic factors of collagen Type II and aggrecan mRNA levels and deposition of sulfated glycosaminoglycans. In medium containing fetal bovine serum, interactions with the GFOGER peptide enhanced mRNA expression of the osteogenic gene osteocalcin whereas FnIII7-10 inhibited osteocalcin expression. In conclusion, modification of agarose hydrogels with ECM-mimetic ligands can influence the differentiation of BMSCs in a manner that depends strongly on the presence and nature of soluble biochemical stimuli.

  11. Knockdown of SVCT2 impairs in-vitro cell attachment, migration and wound healing in bone marrow stromal cells

    Directory of Open Access Journals (Sweden)

    Rajnikumar Sangani

    2014-03-01

    Full Text Available Bone marrow stromal cell (BMSC adhesion and migration are fundamental to a number of pathophysiologic processes, including fracture and wound healing. Vitamin C is beneficial for bone formation, fracture repair and wound healing. However, the role of the vitamin C transporter in BMSC adhesion, migration and wound healing is not known. In this study, we knocked-down the sodium-dependent vitamin C transporter, SVCT2, the only known transporter of vitamin C in BMSCs, and performed cell adhesion, migration, in-vitro scratch wound healing and F-actin re-arrangement studies. We also investigated the role of oxidative stress on the above processes. Our results demonstrate that both oxidative stress and down-regulation of SVCT2 decreased cell attachment and spreading. A trans-well cell migration assay showed that vitamin C helped in BMSC migration and that knockdown of SVCT2 decreased cell migration. In the in-vitro scratch wound healing studies, we established that oxidative stress dose-dependently impairs wound healing. Furthermore, the supplementation of vitamin C significantly rescued the BMSCs from oxidative stress and increased wound closing. The knockdown of SVCT2 in BMSCs strikingly decreased wound healing, and supplementing with vitamin C failed to rescue cells efficiently. The knockdown of SVCT2 and induction of oxidative stress in cells produced an alteration in cytoskeletal dynamics. Signaling studies showed that oxidative stress phosphorylated members of the MAP kinase family (p38 and that vitamin C inhibited their phosphorylation. Taken together, these results indicate that both the SVCT2 transporter and oxidative stress play a vital role in BMSC attachment, migration and cytoskeletal re-arrangement. BMSC-based cell therapy and modulation of SVCT2 could lead to a novel therapeutic approach that enhances bone remodeling, fracture repair and wound healing in chronic disease conditions.

  12. Bone Marrow Stromal Cells Contribute to Bone Formation Following Infusion into Femoral Cavities of a Mouse Model of Osteogenesis Imperfecta

    Science.gov (United States)

    Li, Feng; Wang, Xujun; Niyibizi, Christopher

    2010-01-01

    Currently, there are conflicting data in literature regarding contribution of bone marrow stromal cells (BMSCs) to bone formation when the cells are systemically delivered in recipient animals. To understand if BMSCs contribute to bone cell phenotype and bone formation in osteogenesis imperfecta bones (OI), MSCs marked with GFP were directly infused into the femurs of a mouse model of OI (oim). The contribution of the cells to the cell phenotype and bone formation was assessed by histology, immunohistochemistry and biomechanical loading of recipient bones. Two weeks following infusion of BMSCs, histological examination of the recipient femurs demonstrated presence of new bone when compared to femurs injected with saline which showed little or no bone formation. The new bone contained few donor cells as demonstrated by GFP fluorescence. At six weeks following cell injection, new bone was still detectable in the recipient femurs but was enhanced by injection of the cells suspended in pepsin solublized type I collagen. Immunofluorescence and immunohistochemical staining showed that donor GFP positive cells in the new bone were localized with osteocalcin expressing cells suggesting that the cells differentiated into osteoblasts in vivo. Biomechanical loading to failure in thee point bending, revealed that, femurs infused with BMSCs in PBS or in soluble type I collagen were biomechanically stronger than those injected with PBS or type I collagen alone. Taken together, the results indicate that transplanted cells differentiated into osteoblasts in vivo and contributed to bone formation in vivo; we also speculate that donor cells induced differentiation or recruitment of endogenous cells to initiate reparative process at early stages following transplantation. PMID:20570757

  13. Adenoviral Mediated Expression of BMP2 by Bone Marrow Stromal Cells Cultured in 3D Copolymer Scaffolds Enhances Bone Formation.

    Science.gov (United States)

    Sharma, Sunita; Sapkota, Dipak; Xue, Ying; Sun, Yang; Finne-Wistrand, Anna; Bruland, Ove; Mustafa, Kamal

    2016-01-01

    Selection of appropriate osteoinductive growth factors, suitable delivery method and proper supportive scaffold are critical for a successful outcome in bone tissue engineering using bone marrow stromal cells (BMSC). This study examined the molecular and functional effect of a combination of adenoviral mediated expression of bone morphogenetic protein-2 (BMP2) in BMSC and recently developed and characterized, biodegradable Poly(L-lactide-co-є-caprolactone){poly(LLA-co-CL)}scaffolds in osteogenic molecular changes and ectopic bone formation by using in vitro and in vivo approaches. Pathway-focused custom PCR array, validation using TaqMan based quantitative RT-PCR (qRT-PCR) and ALP staining showed significant up-regulation of several osteogenic and angiogenic molecules, including ALPL and RUNX2 in ad-BMP2 BMSC group grown in poly(LLA-co-CL) scaffolds both at 3 and 14 days. Micro CT and histological analyses of the subcutaneously implanted scaffolds in NOD/SCID mice revealed significantly increased radiopaque areas, percentage bone volume and formation of vital bone in ad-BMP2 scaffolds as compared to the control groups both at 2 and 8 weeks. The increased bone formation in the ad-BMP2 group in vivo was paralleled at the molecular level with concomitant over-expression of a number of osteogenic and angiogenic genes including ALPL, RUNX2, SPP1, ANGPT1. The increased bone formation in ad-BMP2 explants was not found to be associated with enhanced endochondral activity as evidenced by qRT-PCR (SOX9 and FGF2) and Safranin O staining. Taken together, combination of adenoviral mediated BMP-2 expression in BMSC grown in the newly developed poly(LLA-co-CL) scaffolds induced expression of osteogenic markers and enhanced bone formation in vivo.

  14. Effects of voluntary exercise on the viability, proliferation and BDNF levels of bone marrow stromal cells in rat pups born from morphine- dependent mothers during pregnancy.

    Science.gov (United States)

    Haydari, Sakineh; Safari, Manouchehr; Zarbakhsh, Sam; Bandegi, Ahmad Reza; Miladi-Gorji, Hossein

    2016-11-10

    This study was designed to investigate whether free access to a running wheel during pregnancy in morphine-dependent mothers would influence the viability, proliferation and BDNF levels of bone marrow stromal cells in rat pups. Pregnant rats were made dependent by chronic administration of morphine in drinking water simultaneously with free access to a running wheel. Male pups are weaned at 21days of birth and their bones marrows were aspirated from the femurs and tibias and also the bone marrow stromal cells (BMSCs) cultured. MTT assay was used to determine cell viability and proliferation rate. The level of BDNF was measured in the supernant of BMSCs culture by ELISA. The sedentary morphine-dependent mothers' pups showed a significant increase in the percentage cell viability and proliferation rate and also a significant decrease in the BDNF protein levels in BMSCs. The rat pups borne from exercising the control and morphine-dependent mothers exhibited an increase in the percentage viability, proliferation rate and BDNF levels of the BMSCs. This study showed that maternal exercise during pregnancy in morphine-dependent and non-dependent mothers, with increasing of BDNF levels increased the proliferation and viability of BMSCs in the rat pups. Also, chronic administration of morphine during pregnancy was able to increase the proliferation and viability of BMSCs in the rat pups. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  15. High Aldehyde Dehydrogenase Activity Identifies a Subset of Human Mesenchymal Stromal Cells with Vascular Regenerative Potential.

    Science.gov (United States)

    Sherman, Stephen E; Kuljanin, Miljan; Cooper, Tyler T; Putman, David M; Lajoie, Gilles A; Hess, David A

    2017-06-01

    During culture expansion, multipotent mesenchymal stromal cells (MSCs) differentially express aldehyde dehydrogenase (ALDH), an intracellular detoxification enzyme that protects long-lived cells against oxidative stress. Thus, MSC selection based on ALDH-activity may be used to reduce heterogeneity and distinguish MSC subsets with improved regenerative potency. After expansion of human bone marrow-derived MSCs, cell progeny was purified based on low versus high ALDH-activity (ALDH hi ) by fluorescence-activated cell sorting, and each subset was compared for multipotent stromal and provascular regenerative functions. Both ALDH l ° and ALDH hi MSC subsets demonstrated similar expression of stromal cell (>95% CD73 + , CD90 + , CD105 + ) and pericyte (>95% CD146 + ) surface markers and showed multipotent differentiation into bone, cartilage, and adipose cells in vitro. Conditioned media (CDM) generated by ALDH hi MSCs demonstrated a potent proliferative and prosurvival effect on human microvascular endothelial cells (HMVECs) under serum-free conditions and augmented HMVEC tube-forming capacity in growth factor-reduced matrices. After subcutaneous transplantation within directed in vivo angiogenesis assay implants into immunodeficient mice, ALDH hi MSC or CDM produced by ALDH hi MSC significantly augmented murine vascular cell recruitment and perfused vessel infiltration compared with ALDH l ° MSC. Although both subsets demonstrated strikingly similar mRNA expression patterns, quantitative proteomic analyses performed on subset-specific CDM revealed the ALDH hi MSC subset uniquely secreted multiple proangiogenic cytokines (vascular endothelial growth factor beta, platelet derived growth factor alpha, and angiogenin) and actively produced multiple factors with chemoattractant (transforming growth factor-β, C-X-C motif chemokine ligand 1, 2, and 3 (GRO), C-C motif chemokine ligand 5 (RANTES), monocyte chemotactic protein 1 (MCP-1), interleukin [IL]-6, IL-8) and matrix

  16. After repeated division, bone marrow stromal cells express inhibitory factors with osteogenic capabilities, and EphA5 is a primary candidate.

    Science.gov (United States)

    Yamada, Tsuyoshi; Yuasa, Masato; Masaoka, Tomokazu; Taniyama, Takashi; Maehara, Hidetsugu; Torigoe, Ichiro; Yoshii, Toshitaka; Shinomiya, Kenichi; Okawa, Atsushi; Sotome, Shinichi

    2013-12-01

    The differentiation capability of human bone marrow stromal cells (hBMSCs) is thought to deteriorate over multiple doubling processes. To clarify the deterioration mechanisms, the multilineage differentiation capabilities of short- and long-term passaged BMSCs were compared. Predictably, long-term passaged BMSCs showed reduced differentiation capacities compared to short-term passaged cells. Furthermore, a non-human primate heterotopic bone formation model demonstrated that long-term passaged BMSCs have bone formation capabilities but also exert inhibitory effects on bone formation. This finding indicated that long-term passaged BMSCs express higher levels of inhibitory factors than short-term passaged BMSCs do. Co-culture assays of short- and long-term passaged BMSCs suggested that the inhibitory signals required cell-cell contact and would therefore be expressed on the cell membrane. A microarray analysis of BMSCs identified ephrin type-A receptor 5 (EphA5) as an inhibitory factor candidate. Quantitative PCR revealed that among all members of the ephrin and Eph receptor families, only the expression of EphA5 was increased by BMSC proliferation. A gene knockdown analysis using siRNAs demonstrated that knockdown of EphA5 gene expression in long-term passaged BMSCs led to an increase in ALP mRNA expression. These results indicate that EphA5 may be a negative regulator of bone formation. A better understanding of the roles of the ephrin and Eph receptor families in hBMSCs may lead to alternative approaches for manipulating hBMSC fate. In addition, this avenue of discovery may provide new therapeutic targets and quality-control markers of the osteogenic differentiation capabilities of hBMSCs. © 2013.

  17. Growth on poly(L-lactic acid) porous scaffold preserves CD73 and CD90 immunophenotype markers of rat bone marrow mesenchymal stromal cells.

    Science.gov (United States)

    Zamparelli, Alessandra; Zini, Nicoletta; Cattini, Luca; Spaletta, Giulia; Dallatana, Davide; Bassi, Elena; Barbaro, Fulvio; Iafisco, Michele; Mosca, Salvatore; Parrilli, Annapaola; Fini, Milena; Giardino, Roberto; Sandri, Monica; Sprio, Simone; Tampieri, Anna; Maraldi, Nadir M; Toni, Roberto

    2014-10-01

    Few data are available on the effect of biomaterials on surface antigens of mammalian bone marrow-derived, adult mesenchymal stromal cells (MSCs). Since poly(L-lactic acid) or PLLA is largely used in tissue engineering of human bones, and we are developing a reverse engineering program to prototype with biomaterials the vascular architecture of bones for their bioartificial reconstruction, both in humans and animal models, we have studied the effect of porous, flat and smooth PLLA scaffolds on the immunophenotype of in vitro grown, rat MSCs in the absence of any coating, co-polymeric enrichment, and differentiation stimuli. Similar to controls on plastic, we show that our PLLA scaffold does not modify the distribution of some surface markers in rat MSCs. In particular, the maintained expression of CD73 and CD90 on two different subpopulations (small and large cells) is consistent with their adhesion to the PLLA scaffold through specialized appendages, and to their prominent content in actin. In addition, our PLLA scaffold favours retention of the intermediate filament desmin, believed a putative marker of undifferentiated state. Finally, it preserves all rat MSCs morphotypes, and allows for their survival, adhesion to the substrate, and replication. Remarkably, a subpopulation of rat MSCs grown on our PLLA scaffold exhibited formation of membrane protrusions of uncertain significance, although in a size range and morphology compatible with either motility blebs or shedding vesicles. In summary, our PLLA scaffold has no detrimental effect on a number of features of rat MSCs, primarily the expression of CD73 and CD90.

  18. Effect of surface modification of nanofibres with glutamic acid peptide on calcium phosphate nucleation and osteogenic differentiation of marrow stromal cells.

    Science.gov (United States)

    Karaman, Ozan; Kumar, Ankur; Moeinzadeh, Seyedsina; He, Xuezhong; Cui, Tong; Jabbari, Esmaiel

    2016-02-01

    Biomineralization is mediated by extracellular matrix (ECM) proteins with amino acid sequences rich in glutamic acid. The objective of this study was to investigate the effect of calcium phosphate deposition on aligned nanofibres surface-modified with a glutamic acid peptide on osteogenic differentiation of rat marrow stromal cells. Blend of EEGGC peptide (GLU) conjugated low molecular weight polylactide (PLA) and high molecular weight poly(lactide-co-glycolide) (PLGA) was electrospun to form aligned nanofibres (GLU-NF). The GLU-NF microsheets were incubated in a modified simulated body fluid for nucleation of calcium phosphate crystals on the fibre surface. To achieve a high calcium phosphate to fibre ratio, a layer-by-layer approach was used to improve diffusion of calcium and phosphate ions inside the microsheets. Based on dissipative particle dynamics simulation of PLGA/PLA-GLU fibres, > 80% of GLU peptide was localized to the fibre surface. Calcium phosphate to fibre ratios as high as 200%, between those of cancellous (160%) and cortical (310%) bone, was obtained with the layer-by-layer approach. The extent of osteogenic differentiation and mineralization of marrow stromal cells seeded on GLU-NF microsheets was directly related to the amount of calcium phosphate deposition on the fibres prior to cell seeding. Expression of osteogenic markers osteopontin, alkaline phosphatase (ALP), osteocalcin and type 1 collagen increased gradually with calcium phosphate deposition on GLU-NF microsheets. Results demonstrate that surface modification of aligned synthetic nanofibres with EEGGC peptide dramatically affects nucleation and growth of calcium phosphate crystals on the fibres leading to increased osteogenic differentiation of marrow stromal cells and mineralization. Copyright © 2013 John Wiley & Sons, Ltd.

  19. Human umbilical cord mesenchymal stromal cells in regenerative medicine.

    Science.gov (United States)

    Detamore, Michael S

    2013-11-25

    Cells of the human umbilical cord offer tremendous potential for improving human health. Cells from the Wharton’s jelly (umbilical cord stroma) in particular, referred to as human umbilical cord mesenchymal stromal cells (HUCMSCs), hold several advantages that make them appealing for translational research. In the previous issue of Stem Cell Research & Therapy, Chon and colleagues made an important contribution to the HUCMSC literature not only by presenting HUCMSCs as an emerging cell source for intervertebral disc regeneration in general and the nucleus pulposus in particular, but also by demonstrating that an extracellular matrix-based strategy might be preferred over the use of growth factors. By culturing HUCMSCs under hypoxia in serum-free conditions in the presence of Matrigel with laminin-111, they were able to achieve intense collagen II staining by 21 days without the addition of exogenous growth factors. There is tremendous translational significance here in that such raw materials may alleviate the need for the use of growth factors in some instances, and this may have important ramifications in reducing product cost and streamlining regulatory approval. Chon and colleagues provide a promising example of the potential of HUCMSCs, demonstrating the ability to guide HUCMSC differentiation even in the absence of serum and growth factors and supporting the use of HUCMSCs as a viable alternative in intervertebral disc regeneration.

  20. Human Platelet Lysate versus Fetal Calf Serum: These Supplements Do Not Select for Different Mesenchymal Stromal Cells.

    Science.gov (United States)

    Fernandez-Rebollo, Eduardo; Mentrup, Birgit; Ebert, Regina; Franzen, Julia; Abagnale, Giulio; Sieben, Torsten; Ostrowska, Alina; Hoffmann, Per; Roux, Pierre-François; Rath, Björn; Goodhardt, Michele; Lemaitre, Jean-Marc; Bischof, Oliver; Jakob, Franz; Wagner, Wolfgang

    2017-07-11

    Culture medium of mesenchymal stromal cells (MSCs) is usually supplemented with either human platelet lysate (HPL) or fetal calf serum (FCS). Many studies have demonstrated that proliferation and cellular morphology are affected by these supplements - it is therefore important to determine if they favor outgrowth of different subpopulations and thereby impact on the heterogeneous composition of MSCs. We have isolated and expanded human bone marrow-derived MSCs in parallel with HPL or FCS and demonstrated that HPL significantly increases proliferation and leads to dramatic differences in cellular morphology. Remarkably, global DNA-methylation profiles did not reveal any significant differences. Even at the transcriptomic level, there were only moderate changes in pairwise comparison. Furthermore, the effects on proliferation, cytoskeletal organization, and focal adhesions were reversible by interchanging to opposite culture conditions. These results indicate that cultivation of MSCs with HPL or FCS has no systematic bias for specific cell types.

  1. Comparison of immunological properties of bone marrow stromal cells and adipose tissue-derived stem cells before and after osteogenic differentiation in vitro

    DEFF Research Database (Denmark)

    Niemeyer, Philipp; Kornacker, Martin; Mehlhorn, Alexander

    2007-01-01

    , the influence of osteogenic differentiation in vitro on the immunological characteristics of BMSCs and ASCs is the subject of this article. Before and after osteogenic induction, the influence of BMSCs and ASCs on the proliferative behavior of resting and activated allogenic peripheral blood mononuclear cells......Mesenchymal stem cells (MSCs) can be isolated from various tissues and represent an attractive cell population for tissue-engineering purposes. MSCs from bone marrow (bone marrow stromal cells [BMSCs]) are negative for immunologically relevant surface markers and inhibit proliferation of allogenic...... T cells in vitro. Therefore, BMSCs are said to be available for allogenic cell therapy. Although the immunological characteristics of BMSCs have been the subject of various investigations, those of stem cells isolated from adipose tissue (ASCs) have not been adequately described. In addition...

  2. The effect of intrathecal delivery of bone marrow stromal cells on hippocampal neurons in rat model of Alzheimer’s disease

    Directory of Open Access Journals (Sweden)

    Mina Eftekharzadeh

    2015-05-01

    Full Text Available Objective(s: Intracerebral injection of bone marrow stromal cells (BMSCs is being investigated as a therapeutic tool to prevent Alzheimer's disease (AD. Our aim was to investigate the effects of BMSCs by intrathecal injection in AD rat model. Materials and Methods: BMSCs were obtained from the bone marrow of Wistar rat and transplanted into AD rat model via intrathecal injection. The rat model had received an injection of β amyloid into the hippocampus for histological and immunohistochemical studies. Results: Histological examination of the brains in transplanted rats compared to controls demonstrated the migration of BrdU-labeled BMSCs from the site of delivery, confirmed the differentiation of BMSCs transplanted cells into the cholinergic neurons, and increased number of healthy and decreased number of dark neurons. Conclusion: Our results showed that BMSCs intratechal administration could be a promising method for treatment ofAlzheimer’s disease in rat model.

  3. HO-1 gene overexpression enhances the beneficial effects of superparamagnetic iron oxide labeled bone marrow stromal cells transplantation in swine hearts underwent ischemia/reperfusion: an MRI study.

    Science.gov (United States)

    Jiang, Yibo; Chen, Lijuan; Tang, Yaoliang; Ma, Genshan; Shen, Chengxing; Qi, Chunmei; Zhu, Qi; Yao, Yuyu; Liu, Naifeng

    2010-05-01

    To determine the effect of intracoronary transfer of superparamagnetic iron oxide (SPIO) labeled heme oxygenase-1 (HO-1) overexpressed bone marrow stromal cells (BMSCs) in a porcine myocardial ischemia/reperfusion model. Cell apoptosis was assayed and supernatant cytokine concentrations were measured in BMSCs that underwent hypoxia/reoxygen in vitro. Female mini-swines that underwent 1 h LAD occlusion followed by 1 h reperfusion were randomly allocated to receive intracoronary saline (control), 1 x 10(7) SPIO-labeled BMSCs transfected with pcDNA3.1-Lacz plasmid (Lacz-BMSCs), pcDNA3.1-human HO-1 (HO-1-BMSCs), pcDNA3.1-hHO-1 pretreated with a HO inhibitor, tin protoporphyrin (SnPP, n = 10 each). MRI and postmortem histological analysis were made at 1 week or 3 months thereafter. Post hypoxia/reoxygen in vitro, apoptosis was significantly reduced, supernatant VEGF significantly increased while TNF-alpha and IL-6 significantly reduced in HO-1-BMSCs group compared with Lacz-BMSCs group (all p < 0.05). Myocardial expression of VEGF was significantly higher in HO-1-BMSCs than in Lacz-BMSCs group at 1 week post transplantation (all p < 0.05). Signal voids induced by the SPIO were detected in the peri-infarction region in all BMSC groups at 1 week but not at 3 months post transplantation and the extent of the hypointense signal was the highest in HO-1-BMSCs group, and histological analysis showed that signal voids represented cardiac macrophages that engulfed the SPIO-labeled BMSCs. Pretreatment with SnPP significantly attenuated the beneficial effects of HO-1-BMSCs. Transplantation of HO-1-overexpressed BMSCs significantly enhanced the beneficial effects of BMSCs on improving cardiac function in this model.

  4. Canine Platelet Lysate Is Inferior to Fetal Bovine Serum for the Isolation and Propagation of Canine Adipose Tissue- and Bone Marrow-Derived Mesenchymal Stromal Cells

    Science.gov (United States)

    Russell, Keith A.; Gibson, Thomas W. G.; Chong, Andrew; Co, Carmon; Koch, Thomas G.

    2015-01-01

    Background Mesenchymal stromal cells (MSC) are increasingly investigated for their clinical utility in dogs. Fetal bovine serum (FBS) is a common culture supplement used for canine MSC expansion. However, FBS content is variable, its clinical use carries risk of an immune response, and its cost is increasing due to global demand. Platelet lysate (PL) has proven to be a suitable alternative to FBS for expansion of human MSC. Hypothesis and Objectives We hypothesized that canine adipose tissue (AT) and bone marrow (BM) MSC could be isolated and expanded equally in PL and FBS at conventionally-used concentrations with differentiation of these MSC unaffected by choice of supplement. Our objectives were to evaluate the use of canine PL in comparison with FBS at four stages: 1) isolation, 2) proliferation, 3) spontaneous differentiation, and 4) directed differentiation. Results 1) Medium with 10% PL was unable to isolate MSC. 2) MSC, initially isolated in FBS-supplemented media, followed a dose-dependent response with no significant difference between PL and FBS cultures at up to 20% (AT) or 30% (BM) enrichment. Beyond these respective peaks, proliferation fell in PL cultures only, while a continued dose-dependent proliferation response was noted in FBS cultures. 3) Further investigation indicated PL expansion culture was inducing spontaneous adipogenesis in concentrations as low as 10% and as early as 4 days in culture. 4) MSC isolated in FBS, but expanded in either FBS or PL, maintained ability to undergo directed adipogenesis and osteogenesis, but not chondrogenesis. Conclusions/Significance Canine PL did not support establishment of MSC colonies from AT and BM, nor expansion of MSC, which appear to undergo spontaneous adipogenesis in response to PL exposure. In vivo studies are warranted to determine if concurrent use of MSC with any platelet-derived products such as platelet-rich plasma are associated with synergistic, neutral or antagonistic effects. PMID:26353112

  5. Canine Platelet Lysate Is Inferior to Fetal Bovine Serum for the Isolation and Propagation of Canine Adipose Tissue- and Bone Marrow-Derived Mesenchymal Stromal Cells.

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    Keith A Russell

    Full Text Available Mesenchymal stromal cells (MSC are increasingly investigated for their clinical utility in dogs. Fetal bovine serum (FBS is a common culture supplement used for canine MSC expansion. However, FBS content is variable, its clinical use carries risk of an immune response, and its cost is increasing due to global demand. Platelet lysate (PL has proven to be a suitable alternative to FBS for expansion of human MSC.We hypothesized that canine adipose tissue (AT and bone marrow (BM MSC could be isolated and expanded equally in PL and FBS at conventionally-used concentrations with differentiation of these MSC unaffected by choice of supplement. Our objectives were to evaluate the use of canine PL in comparison with FBS at four stages: 1 isolation, 2 proliferation, 3 spontaneous differentiation, and 4 directed differentiation.1 Medium with 10% PL was unable to isolate MSC. 2 MSC, initially isolated in FBS-supplemented media, followed a dose-dependent response with no significant difference between PL and FBS cultures at up to 20% (AT or 30% (BM enrichment. Beyond these respective peaks, proliferation fell in PL cultures only, while a continued dose-dependent proliferation response was noted in FBS cultures. 3 Further investigation indicated PL expansion culture was inducing spontaneous adipogenesis in concentrations as low as 10% and as early as 4 days in culture. 4 MSC isolated in FBS, but expanded in either FBS or PL, maintained ability to undergo directed adipogenesis and osteogenesis, but not chondrogenesis.Canine PL did not support establishment of MSC colonies from AT and BM, nor expansion of MSC, which appear to undergo spontaneous adipogenesis in response to PL exposure. In vivo studies are warranted to determine if concurrent use of MSC with any platelet-derived products such as platelet-rich plasma are associated with synergistic, neutral or antagonistic effects.

  6. An In Vivo Characterization of Trophic Factor Production Following Neural Precursor Cell or Bone Marrow Stromal Cell Transplantation for Spinal Cord Injury

    Science.gov (United States)

    Hawryluk, Gregory W.J.; Mothe, Andrea; Wang, Jian; Wang, Shelly; Tator, Charles

    2012-01-01

    Cellular transplantation strategies for repairing the injured spinal cord have shown consistent benefit in preclinical models, and human clinical trials have begun. Interactions between transplanted cells and host tissue remain poorly understood. Trophic factor secretion is postulated a primary or supplementary mechanism of action for many transplanted cells, however, there is little direct evidence to support trophin production by transplanted cells in situ. In the present study, trophic factor expression was characterized in uninjured, injured-untreated, injured-treated with transplanted cells, and corresponding control tissue from the adult rat spinal cord. Candidate trophic factors were identified in a literature search, and primers were designed for these genes. We examined in vivo trophin expression in 3 paradigms involving transplantation of either brain or spinal cord-derived neural precursor cells (NPCs) or bone marrow stromal cells (BMSCs). Injury without further treatment led to a significant elevation of nerve growth factor (NGF), leukemia inhibitory factor (LIF), insulin-like growth factor-1 (IGF-1), and transforming growth factor-β1 (TGF-β1), and lower expression of vascular endothelial growth factor isoform A (VEGF-A) and platelet-derived growth factor-A (PDGF-A). Transplantation of NPCs led to modest changes in trophin expression, and the co-administration of intrathecal trophins resulted in significant elevation of the neurotrophins, glial-derived neurotrophic factor (GDNF), LIF, and basic fibroblast growth factor (bFGF). BMSCs transplantation upregulated NGF, LIF, and IGF-1. NPCs isolated after transplantation into the injured spinal cord expressed the neurotrophins, ciliary neurotrophic factor (CNTF), epidermal growth factor (EGF), and bFGF at higher levels than host cord. These data show that trophin expression in the spinal cord is influenced by injury and cell transplantation, particularly when combined with intrathecal trophin infusion

  7. In vitro expression of erythropoietin by transfected human mesenchymal stromal cells.

    Science.gov (United States)

    Mok, P-L; Cheong, S-K; Leong, C-F; Othman, A

    2008-01-01

    Mesenchymal stromal cells (MSC) are pluripotent progenitor cells that can be found in human bone marrow (BM). These cells have low immunogenicity and could suppress alloreactive T-cell responses. In the current study, MSC were tested for their capacity to carry and deliver the erythropoietin (EPO) gene in vitro. Expanded BM MSC was transfected with EPO-encoded plasmid pMCV1.2 and EPO-encoded MIDGE (minimalistic immunologically defined gene expression) vector by electroporation. The expressed EPO was used to induce hematopoietic stem cells (HSC) into erythroid colonies. The results showed that the MIDGE vector was more effective and stable than the plasmid (pMCV1.2) in delivering EPO gene into MSC. The supernatants containing EPO obtained from the transfected cell culture were able to induce the differentiation of HSC into erythroid colonies. MSC hold promise as a cell factory for the production of biologic molecules, and MIDGE vector is more effective and stable than the plasmid in nucleofection involving the EPO gene.

  8. Quantitative proteomics and transcriptomics reveals metabolic differences in attracting and non-attracting human-in-mouse glioma stem cell xenografts and stromal cells

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    Norelle C. Wildburger

    2015-09-01

    Full Text Available Bone marrow-derived human mesenchymal stem cells (BM-hMSCs show promise as cell-based delivery vehicles for anti-glioma therapeutics, due to innate tropism for gliomas. However, in clinically relevant human-in-mouse glioma stem cell xenograft models, BM-hMSCs tropism is variable. We compared the proteomic profile of cancer and stromal cells in GSCXs that attract BM-hMSCs (“attractors” with those to do not (“non-attractors” to identify pathways that may modulate BM-hMSC homing, followed by targeted transcriptomics. The results provide the first link between fatty acid metabolism, glucose metabolism, ROS, and N-glycosylation patterns in attractors. Reciprocal expression of these pathways in the stromal cells suggests microenvironmental cross-talk.

  9. Recipient bone marrow-derived stromal cells prolong graft survival in a rat hind limb allotransplantation model.

    Science.gov (United States)

    Ikeguchi, Ryosuke; Kakinoki, Ryosuke; Ohta, Souichi; Oda, Hiroki; Yurie, Hirofumi; Kaizawa, Yukitoshi; Mitsui, Hiroto; Aoyama, Tomoki; Toguchida, Junya; Matsuda, Shuichi

    2017-09-01

    Recent studies have indicated that bone marrow-derived stromal cells (BMSCs) have immunomodulatory properties that suppress the T cell responses that cause graft rejection. The purpose of this study is to evaluate the effect of recipient BMSCs intravenous infusion for immunomodulation in a rat vascularized composite allotransplantation model. A total of nine Wistar (WIS) rats and thirty Lewis (LEW) rats were used. BMSCs were harvested from three LEW rats. Twenty-four LEW rats were used as recipients and divided randomly into four groups: BMSC group, FK group, UT group, and Iso group. In the BMSC group, orthotopic rat hind limb transplantation was performed between WIS donor and LEW recipient rats. Recipient rats were injected intravenously with 2 × 10 6 recipient BMSCs on day 6, and with 0.2 mg/kg/day tacrolimus administered over 7 days (n = 6). In the FK group, recipient rats were treated with tacrolimus alone (n = 6). Rats in the UT group received no immunosuppressive treatment (n = 6). In the Iso group, transplantation was performed from three LEW donor rats to six LEW recipient rats without any immunosuppressive treatment (n = 6). Graft survival was assessed by daily inspection and histology. The immunological reactions of recipients were also evaluated. The graft survival of recipient rats in the BMSC group (24.5 days) was significantly prolonged in comparison with that of the FK group (18 days) (P Recipient rats in the BMSC group had significantly reduced serum IFN-γ cytokine levels (1.571 ± 0.779 pg/ml) in comparison with that of the FK group (7.059 ± 1.522 pg/ml) (P = .001). In in vitro study, BMSCs induce T cell hyporesponsiveness in a mixed lymphocyte reaction. BMSCs induce T cell hyporesponsiveness and prolong graft survival in the rat vascularized composite allotransplantation model. BMSCs exhibit immunomodulatory properties against acute rejection that can be realized without the need for significant recipient

  10. Effects of bone marrow stromal cell transplantation through CSF on the subacute and chronic spinal cord injury in rats.

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    Norihiko Nakano

    Full Text Available It has been demonstrated that the infusion of bone marrow stromal cells (BMSCs through the cerebrospinal fluid (CSF has beneficial effects on acute spinal cord injury (SCI in rats. The present study examined whether BMSC infusion into the CSF is effective for subacute (1- and 2-week post-injury, and/or chronic (4-week post-injury SCI in rats. The spinal cord was contused by dropping a weight at the thoracic 8-9 levels. BMSCs cultured from GFP-transgenic rats of the same strain were injected three times (once weekly into the CSF through the fourth ventricle, beginning at 1, 2 and 4 weeks post-injury. At 4 weeks after initial injection, the average BBB score for locomotor assessment increased from 1.0-3.5 points before injection to 9.0-10.9 points in the BMSC-injection subgroups, while, in the PBS (vehicle-injection subgroups, it increased only from 0.5-4.0 points before injection to 3.0-5.1 points. Numerous axons associated with Schwann cells extended longitudinally through the connective tissue matrices in the astrocyte-devoid lesion without being blocked at either the rostral or the caudal borders in the BMSC-injection subgroups. A small number of BMSCs were found to survive within the spinal cord lesion in SCI of the 1-week post-injury at 2 days of injection, but none at 7 days. No BMSCs were found in the spinal cord lesion at 2 days or at 7 days in the SCI of the 2-week and the 4-week post-injury groups. In an in vitro experiment, BMSC-injected CSF promoted the survival and the neurite extension of cultured neurons more effectively than did the PBS-injected CSF. These results indicate that BMSCs had beneficial effects on locomotor improvement as well as on axonal regeneration in both subacute and chronic SCI rats, and the results also suggest that BMSCs might function as neurotrophic sources via the CSF.

  11. Effects of Bone Marrow Stromal Cell Transplantation through CSF on the Subacute and Chronic Spinal Cord Injury in Rats

    Science.gov (United States)

    Nakano, Norihiko; Nakai, Yoshiyasu; Seo, Tae-Beom; Homma, Tamami; Yamada, Yoshihiro; Ohta, Masayoshi; Suzuki, Yoshihisa; Nakatani, Toshio; Fukushima, Masanori; Hayashibe, Miki; Ide, Chizuka

    2013-01-01

    It has been demonstrated that the infusion of bone marrow stromal cells (BMSCs) through the cerebrospinal fluid (CSF) has beneficial effects on acute spinal cord injury (SCI) in rats. The present study examined whether BMSC infusion into the CSF is effective for subacute (1- and 2-week post-injury), and/or chronic (4-week post-injury) SCI in rats. The spinal cord was contused by dropping a weight at the thoracic 8-9 levels. BMSCs cultured from GFP-transgenic rats of the same strain were injected three times (once weekly) into the CSF through the fourth ventricle, beginning at 1, 2 and 4 weeks post-injury. At 4 weeks after initial injection, the average BBB score for locomotor assessment increased from 1.0–3.5 points before injection to 9.0-10.9 points in the BMSC-injection subgroups, while, in the PBS (vehicle)-injection subgroups, it increased only from 0.5–4.0 points before injection to 3.0-5.1 points. Numerous axons associated with Schwann cells extended longitudinally through the connective tissue matrices in the astrocyte-devoid lesion without being blocked at either the rostral or the caudal borders in the BMSC-injection subgroups. A small number of BMSCs were found to survive within the spinal cord lesion in SCI of the 1-week post-injury at 2 days of injection, but none at 7 days. No BMSCs were found in the spinal cord lesion at 2 days or at 7 days in the SCI of the 2-week and the 4-week post-injury groups. In an in vitro experiment, BMSC-injected CSF promoted the survival and the neurite extension of cultured neurons more effectively than did the PBS-injected CSF. These results indicate that BMSCs had beneficial effects on locomotor improvement as well as on axonal regeneration in both subacute and chronic SCI rats, and the results also suggest that BMSCs might function as neurotrophic sources via the CSF. PMID:24039961

  12. The effects of dynamic and three-dimensional environments on chondrogenic differentiation of bone marrow stromal cells

    Energy Technology Data Exchange (ETDEWEB)

    Jung, Youngmee; Kim, Sang-Heon; Kim, Soo Hyun [Biomaterials Research Center, Korea Institute of Science and Technology, PO Box 131, Cheonryang, Seoul, 130-650 (Korea, Republic of); Kim, Young Ha, E-mail: soohkim@kist.re.k [Department of Materials Science and Engineering, Gwangju Institute of Science and Technology, 261 Cheomdan-gwagiro, Buk-gu, Gwangju 500-712 (Korea, Republic of)

    2009-10-15

    Articular cartilage is subjected to complex loading, which plays a major role in its growth, development and maintenance. Previously, we found that mechanical stimuli enhanced the development and function of engineered cartilage tissues in elastic mechano-active poly(lactide-co-caprolactone) (PLCL) scaffolds. In addition, it is well known that the three-dimensional spatial organization of cells and extracellular matrices in hydrogels is crucial to chondrogenesis. This study was conducted to enhance the chondrogenic differentiation of bone marrow stromal cells (BMSCs) in the hybrid scaffolds of fibrin gels and PLCL scaffolds in dynamic environments by compression. A highly elastic scaffold was fabricated from very elastic PLCL with 85% porosity and a 300-500{mu}m pore size using a gel-pressing method. A mixture of rabbit BMSCs and fibrin gels was then seeded onto the PLCL scaffolds and subjected to continuous compressive deformation of 5% strain at 0.1 Hz for 10 days in a chondrogenic medium containing 10 ng ml{sup -1} TGF-beta{sub 1}. The BMSCs-seeded scaffold constructs were then implanted subcutaneously into nude mice. As a control, the cell-PLCL scaffold constructs were cultured under dynamic conditions or the cell-PLCL/fibrin hybrid scaffold constructs and the cell-PLCL scaffold constructs were cultured under static conditions for 10 days in vitro. The results revealed that cells adhered onto the hybrid scaffolds of fibrin gels and PLCL scaffolds cultured under dynamic conditions. In addition, the accumulation of the extracellular matrix of cell-scaffold constructs, which was increased through mechanical stimulation, showed that chondrogenic differentiation was sustained and enhanced significantly in the stimulated hybrid scaffold constructs. Overall, the results of this study indicate that the proper periodic application of dynamic compression and the three-dimensional environments of the hybrid scaffolds composed of fibrin gels and elastic PLCL can encourage

  13. Genetically engineered mesenchymal stromal cells produce IL-3 and TPO to further improve human scaffold-based xenograft models.

    Science.gov (United States)

    Carretta, Marco; de Boer, Bauke; Jaques, Jenny; Antonelli, Antonella; Horton, Sarah J; Yuan, Huipin; de Bruijn, Joost D; Groen, Richard W J; Vellenga, Edo; Schuringa, Jan Jacob

    2017-07-01

    Recently, NOD-SCID IL2Rγ -/- (NSG) mice were implanted with human mesenchymal stromal cells (MSCs) in the presence of ceramic scaffolds or Matrigel to mimic the human bone marrow (BM) microenvironment. This approach allowed the engraftment of leukemic samples that failed to engraft in NSG mice without humanized niches and resulted in a better preservation of leukemic stem cell self-renewal properties. To further improve our humanized niche scaffold model, we genetically engineered human MSCs to secrete human interleukin-3 (IL-3) and thrombopoietin (TPO). In vitro, these IL-3- and TPO-producing MSCs were superior in expanding human cord blood (CB) CD34 + hematopoietic stem/progenitor cells. MLL-AF9-transduced CB CD34 + cells could be transformed efficiently along myeloid or lymphoid lineages on IL-3- and TPO-producing MSCs. In vivo, these genetically engineered MSCs maintained their ability to differentiate into bone, adipocytes, and other stromal components. Upon transplantation of MLL-AF9-transduced CB CD34 + cells, acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) developed in engineered scaffolds, in which a significantly higher percentage of myeloid clones was observed in the mouse compartments compared with previous models. Engraftment of primary AML, B-cell ALL, and biphenotypic acute leukemia (BAL) patient samples was also evaluated, and all patient samples could engraft efficiently; the myeloid compartment of the BAL samples was better preserved in the human cytokine scaffold model. In conclusion, we show that we can genetically engineer the ectopic human BM microenvironment in a humanized scaffold xenograft model. This approach will be useful for functional study of the importance of niche factors in normal and malignant human hematopoiesis. Copyright © 2017 ISEH - International Society for Experimental Hematology. All rights reserved.

  14. 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

  15. Effects of intravenous administration of bone marrow stromal stem cells on cognitive impairment of the whole-brain irradiated rat models

    International Nuclear Information System (INIS)

    Ding Weijun; Wang Jianhua; Zhu Min; Chen Baoguo; Wang Yang

    2007-01-01

    Objective: To explore the effect of intravenous infusion of bone marrow stromal stem cells(MSCs) on cognitive function of rats after whole brain irradiation. Methods: MSCs were isolated and cultured from adult rats. After Sprague-Dawly female rats were anaesthetized with chloral hydrate, their whole cerebrum was irradiated with a single dose of 20 Gy by 6 MV X-ray. Seven days after irradiation, 4 x 106 Hoechst33342-1abelled MSCs were intravenously injected into the tail vein of these rats. Four and 8 weeks after transplantation, the learning and memorizing ability was measured with the Y maze test. Immunohistochemical method was used to identify MSCs or ceils derived from MSCs in the brain. Results: The learning and memorizing ability of irradiation groups were significantly different from that of normal control group (P < 0.01). Significant improvement of cognitive impairment was observed in rats treated with MSCs at 4 and 8 weeks after transplantation as compared with the controll groups (P<0.05). This showed that the MSCs survived and were localized to the brain tissue. The number of Hoechst33342 immunohistofluorescence positive cells and double-immunostaining cells significantly decreased in 8 weeks group as compared with the 4 weeks group. Conclusion: Marrow stromal stem cells delivered to the irradiation brain tissue through intravenous route improve the cognitive impairment after whole brain irradiation. These cells may survive and differentiate in the brain tissue of irradiated rats. (authors)

  16. Endogenous collagen influences differentiation of human multipotent mesenchymal stromal cells.

    Science.gov (United States)

    Fernandes, Hugo; Mentink, Anouk; Bank, Ruud; Stoop, Reinout; van Blitterswijk, Clemens; de Boer, Jan

    2010-05-01

    Human multipotent mesenchymal stromal cells (hMSCs) are multipotent cells that, in the presence of appropriate stimuli, can differentiate into different lineages such as the osteogenic, chondrogenic, and adipogenic lineages. In the presence of ascorbic acid, MSCs secrete an extracellular matrix mainly composed of collagen type I. Here we assessed the potential role of endogenous collagen synthesis in hMSC differentiation and stem cell maintenance. We observed a sharp reduction in proliferation rate of hMSCs in the absence of ascorbic acid, concomitant with a reduction in osteogenesis in vitro and bone formation in vivo. In line with a positive role for collagen type I in osteogenesis, gene expression profiling of hMSCs cultured in the absence of ascorbic acid demonstrated increased expression of genes involved in adipogenesis and chondrogenesis and a reduction in expression of osteogenic genes. We also observed that matrix remodeling and anti-osteoclastogenic signals were high in the presence of ascorbic acid. The presence of collagen type I during the expansion phase of hMSCs did not affect their osteogenic and adipogenic differentiation potential. In conclusion, the collagenous matrix supports both proliferation and differentiation of osteogenic hMSCs but, on the other hand, presents signals stimulating matrix remodeling and inhibiting osteoclastogenesis.

  17. Actin depolymerization enhances adipogenic differentiation in human stromal stem cells.

    Science.gov (United States)

    Chen, Li; Hu, Huimin; Qiu, Weimin; Shi, Kaikai; Kassem, Moustapha

    2018-05-01

    Human stromal stem cells (hMSCs) differentiate into adipocytes that play a role in skeletal tissue homeostasis and whole body energy metabolism. During adipocyte differentiation, hMSCs exhibit significant changes in cell morphology suggesting changes in cytoskeletal organization. Here, we examined the effect of direct modulation of actin microfilament dynamics on adipocyte differentiation. Stabilizing actin filaments in hMSCs by siRNA-mediated knock down of the two main actin depolymerizing factors (ADFs): Cofilin 1 (CFL1) and Destrin (DSTN) or treating the cells by Phalloidin reduced adipocyte differentiation as evidenced by decreased number of mature adipocytes and decreased adipocyte specific gene expression (ADIPOQ, LPL, PPARG, FABP4). In contrast, disruption of actin cytoskeleton by Cytochalasin D enhanced adipocyte differentiation. Follow up studies revealed that the effects of CFL1 on adipocyte differentiation depended on the activity of LIM domain kinase 1 (LIMK1) which is the major upstream kinase of CFL1. Inhibiting LIMK by its specific chemical inhibitor LIMKi inhibited the phosphorylation of CFL1 and actin polymerization, and enhanced the adipocyte differentiation. Moreover, treating hMSCs by Cytochalasin D inhibited ERK and Smad2 signaling and this was associated with enhanced adipocyte differentiation. On the other hand, Phalloidin enhanced ERK and Smad2 signaling, but inhibited adipocyte differentiation which was rescued by ERK specific chemical inhibitor U0126. Our data provide a link between restructuring of hMSCs cytoskeleton and hMSCs lineage commitment and differentiation. Copyright © 2018 Elsevier B.V. All rights reserved.

  18. Multipotent human stromal cells improve cardiac function after myocardial infarction in mice without long-term engraftment

    International Nuclear Information System (INIS)

    Iso, Yoshitaka; Spees, Jeffrey L.; Serrano, Claudia; Bakondi, Benjamin; Pochampally, Radhika; Song, Yao-Hua; Sobel, Burton E.; Delafontaine, Patrick; Prockop, Darwin J.

    2007-01-01

    The aim of this study was to determine whether intravenously administered multipotent stromal cells from human bone marrow (hMSCs) can improve cardiac function after myocardial infarction (MI) without long-term engraftment and therefore whether transitory paracrine effects or secreted factors are responsible for the benefit conferred. hMSCs were injected systemically into immunodeficient mice with acute MI. Cardiac function and fibrosis after MI in the hMSC-treated group were significantly improved compared with controls. However, despite the cardiac improvement, there was no evident hMSC engraftment in the heart 3 weeks after MI. Microarray assays and ELISAs demonstrated that multiple protective factors were expressed and secreted from the hMSCs in culture. Factors secreted by hMSCs prevented cell death of cultured cardiomyocytes and endothelial cells under conditions that mimicked tissue ischemia. The favorable effects of hMSCs appear to reflect the impact of secreted factors rather than engraftment, differentiation, or cell fusion

  19. Manufacturing of Human Umbilical Cord Mesenchymal Stromal Cells on Microcarriers in a Dynamic System for Clinical Use

    Directory of Open Access Journals (Sweden)

    Florian Petry

    2016-01-01

    Full Text Available The great properties of human mesenchymal stromal cells (hMSCs make these cells an important tool in regenerative medicine. Because of the limitations of hMSCs derived from the bone marrow during isolation and expansion, hMSCs derived from the umbilical cord stroma are a great alternative to overcome these issues. For a large expansion of these cells, we performed a process transfer from static culture to a dynamic system. For this reason, a microcarrier selection out of five microcarrier types was made to achieve a suitable growth surface for the cells. The growth characteristics and metabolite consumption and production were used to compare the cells growth in 12-well plate and spinner flask. The goal to determine relevant process parameters to transfer the expansion process into a stirred tank bioreactor was achieved.

  20. Microarray Analysis on Gene Regulation by Estrogen, Progesterone and Tamoxifen in Human Endometrial Stromal Cells

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    Chun-E Ren

    2015-03-01

    Full Text Available Epithelial stromal cells represent a major cellular component of human uterine endometrium that is subject to tight hormonal regulation. Through cell-cell contacts and/or paracrine mechanisms, stromal cells play a significant role in the malignant transformation of epithelial cells. We isolated stromal cells from normal human endometrium and investigated the morphological and transcriptional changes induced by estrogen, progesterone and tamoxifen. We demonstrated that stromal cells express appreciable levels of estrogen and progesterone receptors and undergo different morphological changes upon hormonal stimulation. Microarray analysis indicated that both estrogen and progesterone induced dramatic alterations in a variety of genes associated with cell structure, transcription, cell cycle, and signaling. However, divergent patterns of changes, and in some genes opposite effects, were observed for the two hormones. A large number of genes are identified as novel targets for hormonal regulation. These hormone-responsive genes may be involved in normal uterine function and the development of endometrial malignancies.

  1. Specific depletion of mature T lymphocytes from human bone marrow

    DEFF Research Database (Denmark)

    Geisler, C; Møller, J; Plesner, T

    1989-01-01

    An effective method for specific depletion of mature T lymphocytes from human bone marrow mononuclear cells (BMMC) with preservation of prethymic T cells and natural killer (NK) cells is presented. The BMMC were incubated with F101.01, a monoclonal antibody recognizing an epitope of the T...

  2. Evaluation of mRNA expression levels and electrophysiological function of neuron-like cells derived from canine bone marrow stromal cells.

    Science.gov (United States)

    Nakano, Rei; Edamura, Kazuya; Sugiya, Hiroshi; Narita, Takanori; Okabayashi, Ken; Moritomo, Tadaaki; Teshima, Kenji; Asano, Kazushi; Nakayama, Tomohiro

    2013-10-01

    To investigate the in vitro differentiation of canine bone marrow stromal cells (BMSCs) into functional, mature neurons. Bone marrow from 6 adult dogs. BMSCs were isolated from bone marrow and chemically induced to develop into neurons. The morphology of the BMSCs during neuronal induction was monitored, and immunocytochemical analyses for neuron markers were performed after the induction. Real-time PCR methods were used to evaluate the mRNA expression levels of markers for neural stem or progenitor cells, neurons, and ion channels, and western blotting was used to assess the expression of neuronal proteins before and after neuronal induction. The electrophysiological properties of the neuron-like cells induced from canine BMSCs were evaluated with fluorescent dye to monitor Ca(2)+ influx. Canine BMSCs developed a neuron-like morphology after neuronal induction. Immunocytochemical analysis revealed that these neuron-like cells were positive for neuron markers. After induction, the cells' mRNA expression levels of almost all neuron and ion channel markers increased, and the protein expression levels of nestin and neurofilament-L increased significantly. However, the neuron-like cells derived from canine BMSCs did not have the Ca(2)+ influx characteristic of spiking neurons. Although canine BMSCs had neuron-like morphological and biochemical properties after induction, they did not develop the electrophysiological characteristics of neurons. Thus, these results have suggested that canine BMSCs could have the capacity to differentiate into a neuronal lineage, but the differentiation protocol used may have been insufficient to induce development into functional neurons.

  3. Tissue non-specific alkaline phosphatase production by human dental pulp stromal cells is enhanced by high density cell culture.

    Science.gov (United States)

    Tomlinson, Matthew J; Dennis, Caitriona; Yang, Xuebin B; Kirkham, Jennifer

    2015-08-01

    The cell surface hydrolase tissue non-specific alkaline phosphatase (TNAP) (also known as MSCA-1) is used to identify a sub-population of bone marrow stromal cells (BMSCs) with high mineralising potential and is found on subsets of cells within the dental pulp. We aim to determine whether TNAP is co-expressed by human dental pulp stromal cells (hDPSCs) alongside a range of BMSC markers, whether this is an active form of the enzyme and the effects of culture duration and cell density on its expression. Cells from primary dental pulp and culture expanded hDPSCs expressed TNAP. Subsequent analyses revealed persistent TNAP expression and co-expression with BMSC markers such as CD73 and CD90. Flow cytometry and biochemical assays showed that increased culture durations and cell densities enhanced TNAP expression by hDPSCs. Arresting the hDPSC cell cycle also increased TNAP expression. These data confirm that TNAP is co-expressed by hDPSCs together with other BMSC markers and show that cell density affects TNAP expression levels. We conclude that TNAP is a potentially useful marker for hDPSC selection especially for uses in mineralised tissue regenerative therapies.

  4. 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

    Directory of Open Access Journals (Sweden)

    Jensen Jonas

    2016-01-01

    Full Text Available 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 marrow and the molar teeth of each pig, respectively. BMSCs and DPSCs were cultured in monolayer and on a three-dimensional (3D polycaprolactone (PCL – hyaluronic acid – tricalcium phosphate (HT-PCL scaffold. Population doubling (PD, alkaline phosphatase (ALP activity, and calcium deposition were measured in monolayer. In the 3D culture ALP activity, DNA content, and calcium deposition were evaluated. Six non-penetrating critical-size defects were made in each calvarium of 14 pigs. Three paired sub-studies were conducted: (1 empty defects vs. HT-PCL scaffolds; (2 PCL scaffolds vs. HT-PCL scaffolds; and (3 autologous BMSCs on HT-PCL scaffolds vs. autologous DPSCs on HT-PCL scaffolds. The observation time was five weeks. Bone volume fractions (BV/TV were assessed with micro-computed tomography (μCT and histomorphometry. Results and discussion: The results from the in vitro study revealed a higher ALP activity and calcium deposition of the DPSC cultures compared with BMSC cultures. Significantly more bone was present in the HT-PCL group than in both the pure PCL scaffold group and the empty defect group in vivo. DPSCs generated more bone than BMSCs when seeded on HT-PCL. In conclusion, DPSCs exhibited a higher osteogenic potential compared with BMSCs both in vitro and in vivo, making it a potential cell source for future bone tissue engineering.

  5. Mesenchymal Stromal Cells Implantation in Combination with Platelet Lysate Product Is Safe for Reconstruction of Human Long Bone Nonunion.

    Science.gov (United States)

    Labibzadeh, Narges; Emadedin, Mohsen; Fazeli, Roghayeh; Mohseni, Fatemeh; Hosseini, Seyedeh Esmat; Moghadasali, Reza; Mardpour, Soura; Azimian, Vajiheh; Ghorbani Liastani, Maede; Mirazimi Bafghi, Ali; Baghaban Eslaminejad, Mohamadreza; Aghdami, Nasser

    2016-01-01

    Nonunion is defined as a minimum of 9 months since injury without any visible progressive signs of healing for 3 months. Recent literature has shown that the application of mesenchymal stromal cells is safe, in vitro and in vivo, for treating long bone nonunion. The present study was performed to investigate the safety of mesenchymal stromal cell (MSC) implantation in combination with platelet lysate (PL) product for treating human long bone nonunion. In this case series clinical trial, orthopedic surgeons visited eighteen patients with long bone nonunion, of whom 7 complied with the eligibility criteria. These patients received mesenchymal stromal cells (20 million cells implanted once into the nonunion site using a fluoroscopic guide) in combination with PL product. For evaluation of the effects of this intervention all the patients were followed up by taking anterior-posterior and lateral X-rays of the affected limb before and 1, 3, 6, and 12 months after the implantation. All side effects (local or systemic, serious or non-serious, related or unrelated) were observed during this time period. From a safety perspective the MSC implantation in combination with PL was very well tolerated during the 12 months of the trial. Four patients were healed; based on the control Xray evidence, bony union had occurred. Results from the present study suggest that the implantation of bone marrow-derived MSCs in combination with PL is safe for the treatment of nonunion. A double blind, controlled clinical trial is required to assess the efficacy of this treatment (Registration Number: NCT01206179).

  6. Wound-healing potential of human umbilical cord blood-derived mesenchymal stromal cells in vitro--a pilot study.

    Science.gov (United States)

    You, Hi-Jin; Namgoong, Sik; Han, Seung-Kyu; Jeong, Seong-Ho; Dhong, Eun-Sang; Kim, Woo-Kyung

    2015-11-01

    Our previous studies demonstrated that human bone marrow-derived mesenchymal stromal cells have great potential for wound healing. However, it is difficult to clinically utilize cultured stem cells. Recently, human umbilical cord blood-derived mesenchymal stromal cells (hUCB-MSCs) have been commercialized for cartilage repair as a first cell therapy product that uses allogeneic stem cells. Should hUCB-MSCs have a superior effect on wound healing as compared with fibroblasts, which are the main cell source in current cell therapy products for wound healing, they may possibly replace fibroblasts. The purpose of this in vitro study was to compare the wound-healing activity of hUCB-MSCs with that of fibroblasts. This study was particularly designed to compare the effect of hUCB-MSCs on diabetic wound healing with those of allogeneic and autologous fibroblasts. Healthy (n = 5) and diabetic (n = 5) fibroblasts were used as the representatives of allogeneic and autologous fibroblasts for diabetic patients in the control group. Human UCB-MSCs (n = 5) were used in the experimental group. Cell proliferation, collagen synthesis and growth factor (basic fibroblast growth factor, vascular endothelial growth factor and transforming growth factor-β) production were compared among the three cell groups. Human UCB-MSCs produced significantly higher amounts of vascular endothelial growth factor and basic fibroblast growth factor when compared with both fibroblast groups. Human UCB-MSCs were superior to diabetic fibroblasts but not to healthy fibroblasts in collagen synthesis. There were no significant differences in cell proliferation and transforming growth factor-β production. Human UCB-MSCs may have greater capacity for diabetic wound healing than allogeneic or autologous fibroblasts, especially in angiogenesis. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  7. In vitro differentiation of human skin-derived multipotent stromal cells into putative endothelial-like cells

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    Vishnubalaji Radhakrishnan

    2012-01-01

    Full Text Available Abstract Background Multipotent stem cells have been successfully isolated from various tissues and are currently utilized for tissue-engineering and cell-based therapies. Among the many sources, skin has recently emerged as an attractive source for multipotent cells because of its abundance. Recent literature showed that skin stromal cells (SSCs possess mesoderm lineage differentiation potential; however, the endothelial differentiation and angiogenic potential of SSC remains elusive. In our study, SSCs were isolated from human neonatal foreskin (hNFSSCs and adult dermal skin (hADSSCs using explants cultures and were compared with bone marrow (hMSC-TERT and adipose tissue-derived mesenchymal stem cells (hADMSCs for their potential differentiation into osteoblasts, adipocytes, and endothelial cells. Results Concordant with previous studies, both MSCs and SSCs showed similar morphology, surface protein expression, and were able to differentiate into osteoblasts and adipocytes. Using an endothelial induction culture system combined with an in vitro matrigel angiogenesis assay, hNFSSCs and hADSSCs exhibited the highest tube-forming capability, which was similar to those formed by human umbilical vein endothelial cells (HUVEC, with hNFSSCs forming the most tightly packed, longest, and largest diameter tubules among the three cell types. CD146 was highly expressed on hNFSSCs and HUVEC followed by hADSSCs, and hMSC-TERT, while its expression was almost absent on hADMSCs. Similarly, higher vascular density (based on the expression of CD31, CD34, vWF, CD146 and SMA was observed in neonatal skin, followed by adult dermal skin and adipose tissue. Thus, our preliminary data indicated a plausible relationship between vascular densities, and the expression of CD146 on multipotent cells derived from those tissues. Conclusions Our data is the first to demonstrate that human dermal skin stromal cells can be differentiated into endothelial lineage. Hence, SSCs

  8. Knitted poly-lactide-co-glycolide scaffold loaded with bone marrow stromal cells in repair and regeneration of rabbit Achilles tendon.

    Science.gov (United States)

    Ouyang, Hong Wei; Goh, James C H; Thambyah, Ashvin; Teoh, Swee Hin; Lee, Eng Hin

    2003-06-01

    The objectives of this study were to evaluate the morphology and biomechanical function of Achilles tendons regenerated using knitted poly-lactide-co-glycolide (PLGA) loaded with bone marrow stromal cells (bMSCs). The animal model used was that of an adult female New Zealand White rabbit with a 10-mm gap defect of the Achilles tendon. In group I, 19 hind legs with the created defects were treated with allogeneic bMSCs seeded on knitted PLGA scaffold. In group II, the Achilles tendon defects in 19 hind legs were repaired using the knitted PLGA scaffold alone, and in group III, 6 hind legs were used as normal control. The tendon-implant constructs of groups I and II were evaluated postoperatively at 2, 4, 8, and 12 weeks using macroscopic, histological, and immunohistochemical techniques. In addition, specimens from group I (n = 7), group II (n = 7), and group III (n = 6) were harvested for biomechanical test 12 weeks after surgery. Postoperatively, at 2 and 4 weeks, the histology of group I specimens exhibited a higher rate of tissue formation and remodeling as compared with group II, whereas at 8 and 12 weeks postoperation, the histology of both group I and group II was similar to that of native tendon tissue. The wound sites of group I healed well and there was no apparent lymphocyte infiltration. Immunohistochemical analysis showed that the regenerated tendons were composed of collagen types I and type III fibers. The tensile stiffness and modulus of group I were 87 and 62.6% of normal tendon, respectively, whereas those of group II were about 56.4 and 52.9% of normal tendon, respectively. These results suggest that the knitted PLGA biodegradable scaffold loaded with allogeneic bone marrow stromal cells has the potential to regenerate and repair gap defect of Achilles tendon and to effectively restore structure and function.

  9. Kaempferol-immobilized titanium dioxide promotes formation of new bone: effects of loading methods on bone marrow stromal cell differentiation in vivo and in vitro.

    Science.gov (United States)

    Tsuchiya, Shuhei; Sugimoto, Keisuke; Kamio, Hisanobu; Okabe, Kazuto; Kuroda, Kensuke; Okido, Masazumi; Hibi, Hideharu

    2018-01-01

    Surface modification of titanium dioxide (TiO 2 ) implants promotes bone formation and shortens the osseointegration period. Kaempferol is a flavonoid that has the capacity to promote osteogenic differentiation in bone marrow stromal cells. The aim of this study was to promote bone formation around kaempferol immobilized on TiO 2 implants. There were four experimental groups. Alkali-treated TiO 2 samples (implants and discs) were used as a control and immersed in Dulbecco's phosphate-buffered saline (DPBS) (Al-Ti). For the coprecipitation sample (Al-cK), the control samples were immersed in DPBS containing 50 µg kaempferol/100% ethanol. For the adsorption sample (Al-aK), 50 µg kaempferol/100% ethanol was dropped onto control samples. The surface topography of the TiO 2 implants was observed by scanning electron microscopy with energy-dispersive X-ray spectroscopy, and a release assay was performed. For in vitro experiments, rat bone marrow stromal cells (rBMSCs) were cultured on each of the TiO 2 samples to analyze cell proliferation, alkaline phosphatase activity, calcium deposition, and osteogenic differentiation. For in vivo experiments, TiO 2 implants placed on rat femur bones were analyzed for bone-implant contact by histological methods. Kaempferol was detected on the surface of Al-cK and Al-aK. The results of the in vitro study showed that rBMSCs cultured on Al-cK and Al-aK promoted alkaline phosphatase activity, calcium deposition, and osteogenic differentiation. The in vivo histological analysis revealed that Al-cK and Al-aK stimulated new bone formation around implants. TiO 2 implant-immobilized kaempferol may be an effective tool for bone regeneration around dental implants.

  10. Nitrogen-containing bisphosphonate, YM529/ONO-5920 (a novel minodronic acid), inhibits RANKL expression in a cultured bone marrow stromal cell line ST2

    International Nuclear Information System (INIS)

    Nishida, Shozo; Tsubaki, Masanobu; Hoshino, Mayumi; Namimatsu, Ayumi; Uji, Hiromi; Yoshioka, Shohei; Tanimori, Yoshihiro; Yanae, Masashi; Iwaki, Masahiro; Irimajiri, Kiyohiro

    2005-01-01

    Increase in bone resorption by osteoclasts can cause metabolic bone diseases, such as osteoporosis. Recent attention has been paid to the receptor activator of the NF-κB ligand (RANKL), an accelerator of osteoclast differentiation. RANKL is expressed on the bone marrow-derived stromal cell membrane and induces the differentiation of osteoclasts by binding to RANK expressed on the osteoclast precursor cell membrane. Since the inhibition of RANKL expression can lead to the inhibition of osteoclastic bone resorption, the clinical application of RANKL inhibition could be expected to have a major effect on metabolic bone disease therapy. In this study, we investigated whether or not YM529/ONO-5920, a nitrogen-containing bisphosphonate (a novel minodronic acid), inhibits RANKL expression in a bone marrow-derived stromal cell line (ST2 cells). Reverse transcription-polymerase chain reaction revealed that the administration of YM529/ONO-5920 to ST2 cells inhibited RANKL mRNA expression and reduced RANKL proteins as assessed by Western blot analysis. The inhibition of RANKL mRNA expression was reversed when geranylgeranyl pyrophosphate (GGPP), an intermediate in the mevalonate pathway, was used in combination. Furthermore, YM529/ONO-5920 reduced phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2), and similarly, U0126, a mitogen-activated protein kinase kinase 1/2 inhibitor, inhibited RANKL expression. Pretreatment with GGPP reversed the YM529/ONO-5920-induced decrease in phosphorylation of ERK. Furthermore, YM529/ONO-5920 decreased TRAP-positive cells in co-culture of ST2 cells and an osteoclast cell line, C7 cells, and this decrease was inhibited by pretreatment with GGPP. This indicates that YM529/ONO-5920 inhibits GGPP biosynthesis in the mevalonate pathway and then signal transduction in the Ras-mitogen-activated protein kinase pathway, thereby inhibiting RANKL expression on ST2 cells. These results suggest a newly elucidated action of bisphosphonates in

  11. Encapsulation of rat bone marrow stromal cells using a poly-ion complex gel of chitosan and succinylated poly(Pro-Hyp-Gly).

    Science.gov (United States)

    Kusumastuti, Yuni; Shibasaki, Yoshiaki; Hirohara, Shiho; Kobayashi, Mime; Terada, Kayo; Ando, Tsuyoshi; Tanihara, Masao

    2017-03-01

    Encapsulation of stem cells into a three-dimensional (3D) scaffold is necessary to achieve tissue regeneration. Prefabricated 3D scaffolds, such as fibres or porous sponges, have limitations regarding homogeneous cell distribution. Hydrogels that can encapsulate cells such as animal-derived collagen gels need adjustment of the pH and/or temperature upon cell mixing. In this report, we fabricated a poly-ion complex (PIC) hydrogel of chitosan and succinylated poly(Pro-Hyp-Gly) and assessed its effect on cell viability after encapsulation of rat bone marrow stromal cells. PIC hydrogels were obtained successfully with a concentration of each precursor as low as 3.0-3.8 mg/ml. The maximum gelation and swelling ratios were achieved with an equal molar ratio (1:1) of anionic and cationic groups. Using chitosan acetate as a cationic precursor produced a PIC hydrogel with both a significantly greater gelation ratio and a better swelling ratio than chitosan chloride. Ammonium succinylated poly(Pro-Hyp-Gly) as an anionic precursor gave similar gelation and swelling ratios to those of sodium succinylated poly(Pro-Hyp-Gly). Cell encapsulation was also achieved successfully by mixing rat bone marrow stromal cells with the PIC hydrogel simultaneously during its formation. The PIC hydrogel was maintained in the culture medium for 7 days at 37°C and the encapsulated cells survived and proliferated in it. Although it is necessary to improve its functionality, this PIC hydrogel has the potential to act as a 3D scaffold for cell encapsulation and tissue regeneration. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  12. Differentiation of human mesenchymal stromal cells cultured on collagen sponges for cartilage repair.

    Science.gov (United States)

    Sanjurjo-Rodríguez, Clara; Martínez-Sánchez, Adela Helvia; Hermida-Gómez, Tamara; Fuentes-Boquete, Isaac; Díaz-Prado, Silvia; Blanco, Francisco J

    2016-11-01

    The aim of this study was to evaluate proliferation and chondrogenic differentiation of human bone-marrow mesenchymal stromal cells (hBMSCs) cultured on collagen biomaterials. hBMSCs were seeded on five different collagen (Col) sponges: C1C2 (types I and II Col), C1C2HS (types I and II Col plus heparan sulphate (HS)), C1C2CHS (types I and II Col plus chondroitin sulphate (CHS)), C1-OLH3 (type I Col plus low molecular weight heparin) and C1CHS (type I Col plus CHS). The resulting constructs were analyzed by histological and immunohistochemical staining, molecular biology and electron microscopy. Col released into culture media was measured by a dye-binding method Results: hBMSCs on biomaterials C1C2, C1C2HS and C1C2CHS had more capacity to attach, proliferate and synthesize Col II and proteoglycans in the extracellular matrix (ECM) than on C1-OLH3 and C1CHS. The presence of aggrecan was detected only at the gene level. Total Col liberated by the cells in the supernatants in all scaffold cultures was detected. The level of Col I in the ECM was lower in C1-OLH3 and that of Col II was highest in C1C2 and C1C2HS. Electron microscopy showed differently shaped cells, from rounded to flattened, in all constructs. Col fibers in bundles were observed in C1C2CHS by transmission electron microscopy. The results show that Col I and Col II (C1C2, C1C2HS and C1C2CHS) biomaterials allowed cell proliferation and chondrogenic-like differentiation of hBMSCs at an early stage. Constructs cultured on C1C2HS and C1C2CHS showed better cartilage-like phenotype than the other ones.

  13. Priming 3D cultures of human mesenchymal stromal cells toward cartilage formation via developmental pathways.

    Science.gov (United States)

    Centola, Matteo; Tonnarelli, Beatrice; Schären, Stefan; Glaser, Nicolas; Barbero, Andrea; Martin, Ivan

    2013-11-01

    The field of regenerative medicine has increasingly recognized the importance to be inspired by developmental processes to identify signaling pathways crucial for 3D organogenesis and tissue regeneration. Here, we aimed at recapitulating the first events occurring during limb development (ie, cell condensation and expansion of an undifferentiated mesenchymal cell population) to prime 3D cultures of human bone marrow-derived mesenchymal stromal/stem cells (hBM-MSC) toward the chondrogenic route. Based on embryonic development studies, we hypothesized that Wnt3a and fibroblast growth factor 2 (FGF2) induce hBM-MSC to proliferate in 3D culture as an undifferentiated pool of progenitors (defined by clonogenic capacity and expression of typical markers), retaining chondrogenic potential upon induction by suitable morphogens. hBM-MSC were responsive to Wnt signaling in 3D pellet culture, as assessed by significant upregulation of main target genes and increase of unphosphorylated β-catenin levels. Wnt3a was able to induce a five-fold increase in the number of proliferating hBM-MSC (6.4% vs. 1.3% in the vehicle condition), although total DNA content of the 3D construct was decreasing over time. Preconditioning with Wnt3a improved transforming growth factor-β1 mediated chondrogenesis (30% more glycosaminoglycans/cell in average). In contrast to developmental and 2D MSC culture models, FGF2 antagonized the Wnt-mediated effects. Interestingly, the CD146⁺ subpopulation was found to be more responsive to Wnt3a. The presented data indicate a possible strategy to prime 3D cultures of hBM-MSC by invoking a "developmental engineering" approach. The study also identifies some opportunities and challenges to cross-fertilize skeletal development models and 3D hBM-MSC culture systems.

  14. Developmentally inspired programming of adult human mesenchymal stromal cells toward stable chondrogenesis.

    Science.gov (United States)

    Occhetta, Paola; Pigeot, Sebastien; Rasponi, Marco; Dasen, Boris; Mehrkens, Arne; Ullrich, Thomas; Kramer, Ina; Guth-Gundel, Sabine; Barbero, Andrea; Martin, Ivan

    2018-05-01

    It is generally accepted that adult human bone marrow-derived mesenchymal stromal cells (hMSCs) are default committed toward osteogenesis. Even when induced to chondrogenesis, hMSCs typically form hypertrophic cartilage that undergoes endochondral ossification. Because embryonic mesenchyme is obviously competent to generate phenotypically stable cartilage, it is questioned whether there is a correspondence between mesenchymal progenitor compartments during development and in adulthood. Here we tested whether forcing specific early events of articular cartilage development can program hMSC fate toward stable chondrogenesis. Inspired by recent findings that spatial restriction of bone morphogenetic protein (BMP) signaling guides embryonic progenitors toward articular cartilage formation, we hypothesized that selective inhibition of BMP drives the phenotypic stability of hMSC-derived chondrocytes. Two BMP type I receptor-biased kinase inhibitors were screened in a microfluidic platform for their time- and dose-dependent effect on hMSC chondrogenesis. The different receptor selectivity profile of tested compounds allowed demonstration that transient blockade of both ALK2 and ALK3 receptors, while permissive to hMSC cartilage formation, is necessary and sufficient to maintain a stable chondrocyte phenotype. Remarkably, even upon compound removal, hMSCs were no longer competent to undergo hypertrophy in vitro and endochondral ossification in vivo, indicating the onset of a constitutive change. Our findings demonstrate that adult hMSCs effectively share properties of embryonic mesenchyme in the formation of transient but also of stable cartilage. This opens potential pharmacological strategies to articular cartilage regeneration and more broadly indicates the relevance of developmentally inspired protocols to control the fate of adult progenitor cell systems.

  15. Decreased hypertrophic differentiation accompanies enhanced matrix formation in co-cultures of outer meniscus cells with bone marrow mesenchymal stromal cells

    Science.gov (United States)

    2012-01-01

    Introduction The main objective of this study was to determine whether meniscus cells from the outer (MCO) and inner (MCI) regions of the meniscus interact similarly to or differently with mesenchymal stromal stem cells (MSCs). Previous study had shown that co-culture of meniscus cells with bone marrow-derived MSCs result in enhanced matrix formation relative to mono-cultures of meniscus cells and MSCs. However, the study did not examine if cells from the different regions of the meniscus interacted similarly to or differently with MSCs. Methods Human menisci were harvested from four patients undergoing total knee replacements. Tissue from the outer and inner regions represented pieces taken from one third and two thirds of the radial distance of the meniscus, respectively. Meniscus cells were released from the menisci after collagenase treatment. Bone marrow MSCs were obtained from the iliac crest of two patients after plastic adherence and in vitro culture until passage 2. Primary meniscus cells from the outer (MCO) or inner (MCI) regions of the meniscus were co-cultured with MSCs in three-dimensional (3D) pellet cultures at 1:3 ratio, respectively, for 3 weeks in the presence of serum-free chondrogenic medium containing TGF-β1. Mono-cultures of MCO, MCI and MSCs served as experimental control groups. The tissue formed after 3 weeks was assessed biochemically, histochemically and by quantitative RT-PCR. Results Co-culture of inner (MCI) or outer (MCO) meniscus cells with MSCs resulted in neo-tissue with increased (up to 2.2-fold) proteoglycan (GAG) matrix content relative to tissues formed from mono-cultures of MSCs, MCI and MCO. Co-cultures of MCI or MCO with MSCs produced the same amount of matrix in the tissue formed. However, the expression level of aggrecan was highest in mono-cultures of MSCs but similar in the other four groups. The DNA content of the tissues from co-cultured cells was not statistically different from tissues formed from mono-cultures of

  16. Posttransplant Intramuscular Injection of PLX-R18 Mesenchymal-Like Adherent Stromal Cells Improves Human Hematopoietic Engraftment in A Murine Transplant Model

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    Leland Metheny

    2018-02-01

    Full Text Available Late-term complications of hematopoietic cell transplantation (HCT are numerous and include incomplete engraftment. One possible mechanism of incomplete engraftment after HCT is cytokine-mediated suppression or dysfunction of the bone marrow microenvironment. Mesenchymal stromal cells (MSCs elaborate cytokines that nurture or stimulate the marrow microenvironment by several mechanisms. We hypothesize that the administration of exogenous MSCs may modulate the bone marrow milieu and improve peripheral blood count recovery in the setting of incomplete engraftment. In the current study, we demonstrated that posttransplant intramuscular administration of human placental derived mesenchymal-like adherent stromal cells [PLacental eXpanded (PLX-R18] harvested from a three-dimensional in vitro culture system improved posttransplant engraftment of human immune compartment in an immune-deficient murine transplantation model. As measured by the percentage of CD45+ cell recovery, we observed improvement in the peripheral blood counts at weeks 6 (8.4 vs. 24.1%, p < 0.001 and 8 (7.3 vs. 13.1%, p < 0.05 and in the bone marrow at week 8 (28 vs. 40.0%, p < 0.01 in the PLX-R18 cohort. As measured by percentage of CD19+ cell recovery, there was improvement at weeks 6 (12.6 vs. 3.8% and 8 (10.1 vs. 4.1%. These results suggest that PLX-R18 may have a therapeutic role in improving incomplete engraftment after HCT.

  17. Exosomes Derived from Human Bone Marrow Mesenchymal Stem Cells Promote Tumor Growth Through Hedgehog Signaling Pathway

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    Jin Qi

    2017-08-01

    Full Text Available Background/Aims: Mesenchymal stem/stromal cells (MSCs are known to home to sites of tumor microenvironments where they participate in the formation of the tumor microenvironment and to interplay with tumor cells. However, the potential functional effects of MSCs on tumor cell growth are controversial. Here, we, from the view of bone marrow MSC-derived exosomes, study the molecular mechanism of MSCs on the growth of human osteosarcoma and human gastric cancer cells. Methods: MSCs derived from human bone marrow (hBMSCs were isolated and cultured in complete DMEM/F12 supplemented with 10% exosome-depleted fetal bovine serum and 1% penicillin-streptomycin, cell culture supernatants containing exosomes were harvested and exosome purification was performed by ultracentrifugation. Osteosarcoma (MG63 and gastric cancer (SGC7901 cells, respectively, were treated with hBMSC-derived exosomes in the presence or absence of a small molecule inhibitor of Hedgehog pathway. Cell viability was measured by transwell invasion assay, scratch migration assay and CCK-8 test. The expression of the signaling molecules Smoothened, Patched-1, Gli1 and the ligand Shh were tested by western blot and RT-PCR. Results: In this study, we found that hBMSC-derived exosomes promoted MG63 and SGC7901 cell growth through the activation of Hedgehog signaling pathway. Inhibition of Hedgehog signaling pathway significantly suppressed the process of hBMSC-derived exosomes on tumor growth. Conclusion: Our findings demonstrated the new roles of hedgehog signaling pathway in the hBMSCs-derived exosomes induced tumor progression.

  18. Expression of chemokine receptor-4 in bone marrow mesenchymal stem cells on experimental rat abdominal aortic aneurysms and the migration of bone marrow mesenchymal stem cells with stromal-derived factor-1

    Directory of Open Access Journals (Sweden)

    Miao-Yun Long

    2014-05-01

    Full Text Available This study investigated the expression and role of chemokine receptor-4 (CXCR4 in bone marrow mesenchymal stem cells (BMSCs from experimental rats with abdominal aortic aneurysms (AAA for migration of BMSCs. Sprague–Dawley rats were divided into an experimental group and control group (n = 18 each. AAA was induced with 0.75 M solution infiltrate for 30 minutes, after which the abdomen was rinsed and closed. Saline was used in place of CaCl2 in the control group. CD34 and CD29 were detected by flow cytometry, the gene and protein expression of CXCR4 were detected by real-time polymerase chain reaction and western blot, respectively. The migration of BMSCs with stromal-derived factor-1 was detected by Transwell chamber. CD34 expression was negative and CD29 expression was positive. The gene and protein expression of CXCR4 were significantly higher in experimental group than them in control group (p < 0.05, the migration ability of BMSCs from the experimental group was significantly higher than that from the control group (p < 0.05. Stromal-derived factor -1/CXCR4 can enhance the migration of BMSCs in vitro in a rat AAA model.

  19. Role of Stromal Paracrine Signals in Proliferative Diseases of the Aging Human Prostate

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    Kenichiro Ishii

    2018-04-01

    Full Text Available Androgens are essential for the development, differentiation, growth, and function of the prostate through epithelial–stromal interactions. However, androgen concentrations in the hypertrophic human prostate decrease significantly with age, suggesting an inverse correlation between androgen levels and proliferative diseases of the aging prostate. In elderly males, age- and/or androgen-related stromal remodeling is spontaneously induced, i.e., increased fibroblast and myofibroblast numbers, but decreased smooth muscle cell numbers in the prostatic stroma. These fibroblasts produce not only growth factors, cytokines, and extracellular matrix proteins, but also microRNAs as stromal paracrine signals that stimulate prostate epithelial cell proliferation. Surgical or chemical castration is the standard systemic therapy for patients with advanced prostate cancer. Androgen deprivation therapy induces temporary remission, but the majority of patients eventually progress to castration-resistant prostate cancer, which is associated with a high mortality rate. Androgen deprivation therapy-induced stromal remodeling may be involved in the development and progression of castration-resistant prostate cancer. In the tumor microenvironment, activated fibroblasts stimulating prostate cancer cell proliferation are called carcinoma-associated fibroblasts. In this review, we summarize the role of stromal paracrine signals in proliferative diseases of the aging human prostate and discuss the potential clinical applications of carcinoma-associated fibroblast-derived exosomal microRNAs as promising biomarkers.

  20. TBTC induces adipocyte differentiation in human bone marrow long term culture

    International Nuclear Information System (INIS)

    Carfi, M.; Croera, C.; Ferrario, D.; Campi, V.; Bowe, G.; Pieters, R.; Gribaldo, L.

    2008-01-01

    Organotins are widely used in agriculture and the chemical industry, causing persistent and widespread pollution. Organotins may affect the brain, liver and immune system and eventually human health. Recently, it has been shown that tri-butyltin (TBT) interacts with nuclear receptors PPARγ (peroxisome proliferator-activated receptor γ) and RXR (retinoid x receptor) leading to adipocyte differentiation in the 3T3 cell line. Since adipocytes are known to influence haematopoiesis, for instance through the expression of cytokines and adhesion molecules, it was considered of interest to further study the adipocyte-stimulating effect of TBTC in human bone marrow cultures. Nile Red spectrofluorimetric analysis showed a significant increase of adipocytes in TBTC-treated cultures after 14 days of long term culture. Real-time PCR and Western blot analysis confirmed the high expression of the specific adipocyte differentiation marker aP2 (adipocyte-specific fatty acid binding protein). PPARγ, but not RXR, mRNA was increased after 24 h and 48 h exposure. TBTC also induced a decrease in a number of chemokines, interleukins, and growth factors. Also the expression of leptin, a hormone involved in haematopoiesis, was down regulated by TBTC treatment. It therefore appears that TBTC induced adipocyte differentiation, whilst reducing a number of haematopoietic factors. This study indicates that TBTC may interfere in the haematopoietic process through an alteration of the stromal layer and cytokine homeostasis

  1. The Origin of Human Mesenchymal Stromal Cells Dictates Their Reparative Properties

    DEFF Research Database (Denmark)

    Naftali-Shani, Nili; Itzhaki-Alfia, Ayelet; Landa-Rouben, Natalie

    2013-01-01

    Human mesenchymal stromal cells (hMSCs) from adipose cardiac tissue have attracted considerable interest in regard to cell-based therapies. We aimed to test the hypothesis that hMSCs from the heart and epicardial fat would be better cells for infarct repair....

  2. What Makes Umbilical Cord Tissue-Derived Mesenchymal Stromal Cells Superior Immunomodulators When Compared to Bone Marrow Derived Mesenchymal Stromal Cells?

    Directory of Open Access Journals (Sweden)

    R. N. Bárcia

    2015-01-01

    Full Text Available MSCs derived from the umbilical cord tissue, termed UCX, were investigated for their immunomodulatory properties and compared to bone marrow-derived MSCs (BM-MSCs, the gold-standard in immunotherapy. Immunogenicity and immunosuppression were assessed by mixed lymphocyte reactions, suppression of lymphocyte proliferation and induction of regulatory T cells. Results showed that UCX were less immunogenic and showed higher immunosuppression activity than BM-MSCs. Further, UCX did not need prior activation or priming to exert their immunomodulatory effects. This was further corroborated in vivo in a model of acute inflammation. To elucidate the potency differences observed between UCX and BM-MSCs, gene expression related to immune modulation was analysed in both cell types. Several gene expression profile differences were found between UCX and BM-MSCs, namely decreased expression of HLA-DRA, HO-1, IGFBP1, 4 and 6, ILR1, IL6R and PTGES and increased expression of CD200, CD273, CD274, IL1B, IL-8, LIF and TGFB2. The latter were confirmed at the protein expression level. Overall, these results show that UCX seem to be naturally more potent immunosuppressors and less immunogenic than BM-MSCs. We propose that these differences may be due to increased levels of immunomodulatory surface proteins such as CD200, CD273, CD274 and cytokines such as IL1β, IL-8, LIF and TGFβ2.

  3. Bone-marrow densitometry: Assessment of marrow space of human vertebrae by single energy high resolution-quantitative computed tomography

    International Nuclear Information System (INIS)

    Peña, Jaime A.; Damm, Timo; Bastgen, Jan; Barkmann, Reinhard; Glüer, Claus C.; Thomsen, Felix; Campbell, Graeme M.

    2016-01-01

    Purpose: Accurate noninvasive assessment of vertebral bone marrow fat fraction is important for diagnostic assessment of a variety of disorders and therapies known to affect marrow composition. Moreover, it provides a means to correct fat-induced bias of single energy quantitative computed tomography (QCT) based bone mineral density (BMD) measurements. The authors developed new segmentation and calibration methods to obtain quantitative surrogate measures of marrow-fat density in the axial skeleton. Methods: The authors developed and tested two high resolution-QCT (HR-QCT) based methods which permit segmentation of bone voids in between trabeculae hypothesizing that they are representative of bone marrow space. The methods permit calculation of marrow content in units of mineral equivalent marrow density (MeMD). The first method is based on global thresholding and peeling (GTP) to define a volume of interest away from the transition between trabecular bone and marrow. The second method, morphological filtering (MF), uses spherical elements of different radii (0.1–1.2 mm) and automatically places them in between trabeculae to identify regions with large trabecular interspace, the bone-void space. To determine their performance, data were compared ex vivo to high-resolution peripheral CT (HR-pQCT) images as the gold-standard. The performance of the methods was tested on a set of excised human vertebrae with intact bone marrow tissue representative of an elderly population with low BMD. Results: 86% (GTP) and 87% (MF) of the voxels identified as true marrow space on HR-pQCT images were correctly identified on HR-QCT images and thus these volumes of interest can be considered to be representative of true marrow space. Within this volume, MeMD was estimated with residual errors of 4.8 mg/cm 3 corresponding to accuracy errors in fat fraction on the order of 5% both for GTP and MF methods. Conclusions: The GTP and MF methods on HR-QCT images permit noninvasive

  4. Bone-marrow densitometry: Assessment of marrow space of human vertebrae by single energy high resolution-quantitative computed tomography

    Energy Technology Data Exchange (ETDEWEB)

    Peña, Jaime A.; Damm, Timo; Bastgen, Jan; Barkmann, Reinhard; Glüer, Claus C., E-mail: glueer@rad.uni-kiel.de [Sektion Biomedizinische Bildgebung, Klinik für Radiologie und Neuroradiologie, Christian-Albrechts-Universität zu Kiel, Campus Kiel, Kiel 24118 (Germany); Thomsen, Felix [Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad Nacional del Sur, Bahía Blanca 8000 (Argentina); Campbell, Graeme M. [Sektion Biomedizinische Bildgebung, Klinik für Radiologie und Neuroradiologie, Christian-Albrechts-Universität zu Kiel, Campus Kiel, Kiel 24118, Germany and Institut für Biomechanik, Technische Universität Hamburg-Harburg (TUHH), Hamburg 21073 (Germany)

    2016-07-15

    Purpose: Accurate noninvasive assessment of vertebral bone marrow fat fraction is important for diagnostic assessment of a variety of disorders and therapies known to affect marrow composition. Moreover, it provides a means to correct fat-induced bias of single energy quantitative computed tomography (QCT) based bone mineral density (BMD) measurements. The authors developed new segmentation and calibration methods to obtain quantitative surrogate measures of marrow-fat density in the axial skeleton. Methods: The authors developed and tested two high resolution-QCT (HR-QCT) based methods which permit segmentation of bone voids in between trabeculae hypothesizing that they are representative of bone marrow space. The methods permit calculation of marrow content in units of mineral equivalent marrow density (MeMD). The first method is based on global thresholding and peeling (GTP) to define a volume of interest away from the transition between trabecular bone and marrow. The second method, morphological filtering (MF), uses spherical elements of different radii (0.1–1.2 mm) and automatically places them in between trabeculae to identify regions with large trabecular interspace, the bone-void space. To determine their performance, data were compared ex vivo to high-resolution peripheral CT (HR-pQCT) images as the gold-standard. The performance of the methods was tested on a set of excised human vertebrae with intact bone marrow tissue representative of an elderly population with low BMD. Results: 86% (GTP) and 87% (MF) of the voxels identified as true marrow space on HR-pQCT images were correctly identified on HR-QCT images and thus these volumes of interest can be considered to be representative of true marrow space. Within this volume, MeMD was estimated with residual errors of 4.8 mg/cm{sup 3} corresponding to accuracy errors in fat fraction on the order of 5% both for GTP and MF methods. Conclusions: The GTP and MF methods on HR-QCT images permit noninvasive

  5. IL-8 and MCP Gene Expression and Production by LPS-Stimulated Human Corneal Stromal Cells

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    Roni M. Shtein

    2012-01-01

    Full Text Available Purpose. To determine time course of effect of lipopolysaccharide (LPS on production of interleukin-8 (IL-8 and monocyte chemotactic protein (MCP by cultured human corneal stromal cells. Methods. Human corneal stromal cells were harvested from donor corneal specimens, and fourth to sixth passaged cells were used. Cell cultures were stimulated with LPS for 2, 4, 8, and 24 hours. Northern blot analysis of IL-8 and MCP gene expression and ELISA for IL-8 and MCP secretion were performed. ELISA results were analyzed for statistical significance using two-tailed Student's t-test. Results. Northern blot analysis demonstrated significantly increased IL-8 and MCP gene expression after 4 and 8 hours of exposure to LPS. ELISA for secreted IL-8 and MCP demonstrated statistically significant increases (P<0.05 after corneal stromal cell stimulation with LPS. Conclusions. This paper suggests that human corneal stromal cells may participate in corneal inflammation by secreting potent leukocyte chemotactic and activating proteins in a time-dependent manner when exposed to LPS.

  6. In vitro mesenchymal trilineage differentiation and extracellular matrix production by adipose and bone marrow derived adult equine multipotent stromal cells on a collagen scaffold.

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    Xie, Lin; Zhang, Nan; Marsano, Anna; Vunjak-Novakovic, Gordana; Zhang, Yanru; Lopez, Mandi J

    2013-12-01

    Directed differentiation of adult multipotent stromal cells (MSC) is critical for effective treatment strategies. This study was designed to evaluate the capability of equine MSC from bone marrow (BMSC) and adipose tissue (ASC) on a type I collagen (COLI) scaffold to undergo chondrogenic, osteogenic and adipogenic differentiation and form extracellular matrix (ECM) in vitro. Following determination of surface antigen expression, MSC were loaded into scaffolds in a perfusion bioreactor and loading efficiency was quantified. Cell-scaffold constructs were assessed after loading and 7, 14 and 21 days of culture in stromal or induction medium. Cell number was determined with DNA content, cell viability and spatial uniformity with confocal laser microscopy and cell phenotype and matrix production with light and scanning electron microscopy and mRNA levels. The MSC were positive for CD29 (>90 %), CD44 (>99 %), and CD105 (>60 %). Loading efficiencies were >70 %. The ASC and BMSC cell numbers on scaffolds were affected by culture in induction medium differently. Viable cells remained uniformly distributed in scaffolds for up to 21 days and could be directed to differentiate or to maintain an MSC phenotype. Micro- and ultrastructure showed lineage-specific cell and ECM changes. Lineage-specific mRNA levels differed between ASC and BMSC with induction and changed with time. Based on these results, equine ASC and BMSC differentiate into chondrogenic, osteogenic and adipogenic lineages and form ECM similarly on COLI scaffolds. The collected data supports the potential for equine MSC-COLI constructs to support diverse equine tissue formation for controlled biological studies.

  7. Reconstruction of hematopoietic inductive microenvironment after transplantation of VCAM-1-modified human umbilical cord blood stromal cells.

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    Yao Liu

    Full Text Available The hematopoietic inductive microenvironment (HIM is where hematopoietic stem/progenitor cells grow and develop. Hematopoietic stromal cells were the key components of the HIM. In our previous study, we had successfully cultured and isolated human cord blood-derived stromal cells (HUCBSCs and demonstrated that they could secret hemopoietic growth factors such as GM-CSF, TPO, and SCF. However, it is still controversial whether HUCBSCs can be used for reconstruction of HIM. In this study, we first established a co-culture system of HUCBSCs and cord blood CD34(+ cells and then determined that using HUCBSCs as the adherent layer had significantly more newly formed colonies of each hematopoietic lineage than the control group, indicating that HUCBSCs had the ability to promote the proliferation of hematopoietic stem cells/progenitor cells. Furthermore, the number of colonies was significantly higher in vascular cell adhesion molecule-1 (VCAM-1-modified HUCBSCs, suggesting that the ability of HUCBSCs in promoting the proliferation of hematopoietic stem cells/progenitor cells was further enhanced after having been modified with VCAM-1. Next, HUCBSCs were infused into a radiation-damaged animal model, in which the recovery of hematopoiesis was observed. The results demonstrate that the transplanted HUCBSCs were "homed in" to bone marrow and played roles in promoting the recovery of irradiation-induced hematopoietic damage and repairing HIM. Compared with the control group, the HUCBSC group had significantly superior effectiveness in terms of the recovery time for hemogram and myelogram, CFU-F, CFU-GM, BFU-E, and CFU-Meg. Such differences were even more significant in VCAM-1-modified HUCBSCs group. We suggest that HUCBSCs are able to restore the functions of HIM and promote the recovery of radiation-induced hematopoietic damage. VCAM-1 plays an important role in supporting the repair of HIM damage.

  8. Evaluation of Human Adipose Tissue Stromal Heterogeneity in Metabolic Disease Using Single Cell RNA-Seq

    Science.gov (United States)

    2017-09-01

    AWARD NUMBER: W81XWH-15-1-0251 TITLE: “Evaluation of Human Adipose Tissue Stromal Heterogeneity in Metabolic Disease Using Single Cell RNA...Heterogeneity in Metabolic Disease Using Single- Cell RNA-Seq 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER Linus Tzu-Yen...ABSTRACT We have developed a robust protocol to generate single cell transcriptional profiles from subcutaneous adipose tissue samples of both human

  9. Fetal mesenchymal stromal cells differentiating towards chondrocytes acquire a gene expression profile resembling human growth plate cartilage.

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    Sandy A van Gool

    Full Text Available We used human fetal bone marrow-derived mesenchymal stromal cells (hfMSCs differentiating towards chondrocytes as an alternative model for the human growth plate (GP. Our aims were to study gene expression patterns associated with chondrogenic differentiation to assess whether chondrocytes derived from hfMSCs are a suitable model for studying the development and maturation of the GP. hfMSCs efficiently formed hyaline cartilage in a pellet culture in the presence of TGFβ3 and BMP6. Microarray and principal component analysis were applied to study gene expression profiles during chondrogenic differentiation. A set of 232 genes was found to correlate with in vitro cartilage formation. Several identified genes are known to be involved in cartilage formation and validate the robustness of the differentiating hfMSC model. KEGG pathway analysis using the 232 genes revealed 9 significant signaling pathways correlated with cartilage formation. To determine the progression of growth plate cartilage formation, we compared the gene expression profile of differentiating hfMSCs with previously established expression profiles of epiphyseal GP cartilage. As differentiation towards chondrocytes proceeds, hfMSCs gradually obtain a gene expression profile resembling epiphyseal GP cartilage. We visualized the differences in gene expression profiles as protein interaction clusters and identified many protein clusters that are activated during the early chondrogenic differentiation of hfMSCs showing the potential of this system to study GP development.

  10. Biological, functional and genetic characterization of bone marrow-derived mesenchymal stromal cells from pediatric patients affected by acute lymphoblastic leukemia.

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    Antonella Conforti

    Full Text Available Alterations in hematopoietic microenvironment of acute lymphoblastic leukemia patients have been claimed to occur, but little is known about the components of marrow stroma in these patients. In this study, we characterized mesenchymal stromal cells (MSCs isolated from bone marrow (BM of 45 pediatric patients with acute lymphoblastic leukemia (ALL-MSCs at diagnosis (day+0 and during chemotherapy treatment (days: +15; +33; +78, the time points being chosen according to the schedule of BM aspirates required by the AIEOP-BFM ALL 2009 treatment protocol. Morphology, proliferative capacity, immunophenotype, differentiation potential, immunomodulatory properties and ability to support long-term hematopoiesis of ALL-MSCs were analysed and compared with those from 41 healthy donors (HD-MSCs. ALL-MSCs were also genetically characterized through array-CGH, conventional karyotyping and FISH analysis. Moreover, we compared ALL-MSCs generated at day+0 with those isolated during chemotherapy. Morphology, immunophenotype, differentiation potential and in vitro life-span did not differ between ALL-MSCs and HD-MSCs. ALL-MSCs showed significantly lower proliferative capacity (p<0.001 and ability to support in vitro hematopoiesis (p = 0.04 as compared with HD-MSCs, while they had similar capacity to inhibit in vitro mitogen-induced T-cell proliferation (p = N.S.. ALL-MSCs showed neither the typical translocations carried by the leukemic clone (when present, nor other genetic abnormalities acquired during ex vivo culture. Our findings indicate that ALL-MSCs display reduced ability to proliferate and to support long-term hematopoiesis in vitro. ALL-MSCs isolated at diagnosis do not differ from those obtained during treatment.

  11. Electroacupuncture modulates stromal cell-derived factor-1α expression and mobilization of bone marrow endothelial progenitor cells in focal cerebral ischemia/reperfusion model rats.

    Science.gov (United States)

    Xie, Chenchen; Gao, Xiang; Luo, Yong; Pang, Yueshan; Li, Man

    2016-10-01

    Stromal cell-derived factor-1α(SDF-1α) plays a crucial role in regulating the mobilization, migration and homing of endothelial progenitor cells(EPCs). Electroacupuncture(EA), a modern version of Traditional Chinese Medicine, can improve neurological recovery and angiogenesis in cerebral ischemic area. This study aimed to investigate the effects of electroacupuncture(EA) on the mobilization and migration of bone marrow EPCs and neurological functional recovery in rats model after focal cerebral ischemia/reperfusion and the potentially involved mechanisms. Sprague-Dawley rats received filament occlusion of the right middle cerebral artery for 2h followed by reperfusion for 12h, 1d, 2d, 3d, 7d respectively. Rats were randomly divided into sham group, model group and EA group. After 2h of the reperfusion, EA was given at the "Baihui" (GV 20)/Siguan ("Hegu" (LI 4)/"Taichong" (LR 3)) acupoints in the EA group. Modified neurological severity score (mNSS) was used to assess the neurological functional recovery. EPCs number and SDF-1α level in bone marrow(BM) and peripheral blood(PB) were detected by using fluorescence-activated cell sorting (FACS) analysis and quantitative real time polymerase chain reaction (qRT-PCR) respectively. An mNSS test showed that EA treatment significantly improved the neurological functional outcome. EPCs number in PB and BM were obviously increased in the EA group. After cerebral ischemia, the SDF-1α level was decreased in BM while it was increased in PB, which implied a gradient of SDF-1α among BM and PB after ischemia. It suggested that the forming of SDF-1α concentration gradient can induce the mobilization and homing of EPCs. Eletroacupuncture as a treatment can accelerate and increase the forming of SDF-1α concentration gradient to further induce the mobilization of EPCs and angiogenesis in ischemic brain and improve the neurological function recovery. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. A Stromal Cell Niche for Human and Mouse Type 3 Innate Lymphoid Cells.

    Science.gov (United States)

    Hoorweg, Kerim; Narang, Priyanka; Li, Zhi; Thuery, Anne; Papazian, Natalie; Withers, David R; Coles, Mark C; Cupedo, Tom

    2015-11-01

    Adaptive immunity critically depends on the functional compartmentalization of secondary lymphoid organs. Mesenchymal stromal cells create and maintain specialized niches that support survival, activation, and expansion of T and B cells, and integrated analysis of lymphocytes and their niche has been instrumental in understanding adaptive immunity. Lymphoid organs are also home to type 3 innate lymphoid cells (ILC3), innate effector cells essential for barrier immunity. However, a specialized stromal niche for ILC3 has not been identified. A novel lineage-tracing approach now identifies a subset of murine fetal lymphoid tissue organizer cells that gives rise exclusively to adult marginal reticular cells. Moreover, both cell types are conserved from mice to humans and colocalize with ILC3 in secondary lymphoid tissues throughout life. In sum, we provide evidence that fetal stromal organizers give rise to adult marginal reticular cells and form a dedicated stromal niche for innate ILC3 in adaptive lymphoid organs. Copyright © 2015 by The American Association of Immunologists, Inc.

  13. Decidualized Human Endometrial Stromal Cells Mediate Hemostasis, Angiogenesis, and Abnormal Uterine Bleeding

    Science.gov (United States)

    Lockwood, Charles J.; Krikun, Graciela; Hickey, Martha; Huang, S. Joseph; Schatz, Frederick

    2011-01-01

    Factor VII binds trans-membrane tissue factor to initiate hemostasis by forming thrombin. Tissue factor expression is enhanced in decidualized human endometrial stromal cells during the luteal phase. Long-term progestin only contraceptives elicit: 1) abnormal uterine bleeding from fragile vessels at focal bleeding sites, 2) paradoxically high tissue factor expression at bleeding sites; 3) reduced endometrial blood flow promoting local hypoxia and enhancing reactive oxygen species levels; and 4) aberrant angiogenesis reflecting increased stromal cell-expressed vascular endothelial growth factor, decreased Angiopoietin-1 and increased endothelial cell-expressed Angiopoietin-2. Aberrantly high local vascular permeability enhances circulating factor VII to decidualized stromal cell-expressed tissue factor to generate excess thrombin. Hypoxia-thrombin interactions augment expression of vascular endothelial growth factor and interleukin-8 by stromal cells. Thrombin, vascular endothelial growth factor and interlerukin-8 synergis-tically augment angiogenesis in a milieu of reactive oxygen species-induced endothelial cell activation. The resulting enhanced vessel fragility promotes abnormal uterine bleeding. PMID:19208784

  14. Feasibility and safety of treating non-unions in tibia, femur and humerus with autologous, expanded, bone marrow-derived mesenchymal stromal cells associated with biphasic calcium phosphate biomaterials in a multicentric, non-comparative trial.

    Science.gov (United States)

    Gómez-Barrena, Enrique; Rosset, Philippe; Gebhard, Florian; Hernigou, Philippe; Baldini, Nicola; Rouard, Helène; Sensebé, Luc; Gonzalo-Daganzo, Rosa M; Giordano, Rosaria; Padilla-Eguiluz, Norma; García-Rey, Eduardo; Cordero-Ampuero, José; Rubio-Suárez, Juan Carlos; Stanovici, Julien; Ehrnthaller, Christian; Huber-Lang, Markus; Flouzat-Lachaniette, Charles Henri; Chevallier, Nathalie; Donati, Davide Maria; Ciapetti, Gabriela; Fleury, Sandrine; Fernandez, Manuel-Nicolás; Cabrera, José-Rafael; Avendaño-Solá, Cristina; Montemurro, Tiziana; Panaitescu, Carmen; Veronesi, Elena; Rojewski, Markus Thomas; Lotfi, Ramin; Dominici, Massimo; Schrezenmeier, Hubert; Layrolle, Pierre

    2018-03-19

    ORTHO-1 is a European, multicentric, first in human clinical trial to prove safety and feasibility after surgical implantation of commercially available biphasic calcium phosphate bioceramic granules associated during surgery with autologous mesenchymal stromal cells expanded from bone marrow (BM-hMSC) under good manufacturing practices, in patients with long bone pseudarthrosis. Twenty-eight patients with femur, tibia or humerus diaphyseal or metaphyso-diaphyseal non-unions were recruited and surgically treated in France, Germany, Italy and Spain with 100 or 200 million BM-hMSC/mL associated with 5-10 cc of bioceramic granules. Patients were followed up during one year. The investigational advanced therapy medicinal product (ATMP) was expanded under the same protocol in all four countries, and approved by each National Competent Authority. With safety as primary end-point, no severe adverse event was reported as related to the BM-hMSC. With feasibility as secondary end-point, the participating production centres manufactured the BM-hMSC as planned. The ATMP combined to the bioceramic was surgically delivered to the non-unions, and 26/28 treated patients were found radiologically healed at one year (3 out of 4 cortices with bone bridging). Safety and feasibility were clinically proven for surgical implantation of expanded autologous BM-hMSC with bioceramic. EU-FP7-HEALTH-2009, REBORNE Project (GA: 241876). Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

  15. Bone Marrow Stromal Antigen 2 Is a Novel Plasma Biomarker and Prognosticator for Colorectal Carcinoma: A Secretome-Based Verification Study

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    Sum-Fu Chiang

    2015-01-01

    Full Text Available Background. The cancer cell secretome has been recognized as a valuable reservoir for identifying novel serum/plasma biomarkers for different cancers, including colorectal cancer (CRC. This study aimed to verify four CRC cell-secreted proteins (tumor-associated calcium signal transducer 2/trophoblast cell surface antigen 2 (TACSTD2/TROP2, tetraspanin-6 (TSPAN6, bone marrow stromal antigen 2 (BST2, and tumor necrosis factor receptor superfamily member 16 (NGFR as potential plasma CRC biomarkers. Methods. The study population comprises 152 CRC patients and 152 controls. Target protein levels in plasma and tissue samples were assessed by ELISA and immunohistochemistry, respectively. Results. Among the four candidate proteins examined by ELISA in a small sample set, only BST2 showed significantly elevated plasma levels in CRC patients versus controls. Immunohistochemical analysis revealed the overexpression of BST2 in CRC tissues, and higher BST2 expression levels correlated with poorer 5-year survival (46.47% versus 65.57%; p=0.044. Further verification confirmed the elevated plasma BST2 levels in CRC patients (2.35 ± 0.13 ng/mL versus controls (1.04 ± 0.03 ng/mL (p<0.01, with an area under the ROC curve (AUC being 0.858 comparable to that of CEA (0.867. Conclusion. BST2, a membrane protein selectively detected in CRC cell secretome, may be a novel plasma biomarker and prognosticator for CRC.

  16. Response of bone marrow stromal cells to graded co-electrospun scaffolds and its implications for engineering the ligament-bone interface.

    Science.gov (United States)

    Samavedi, Satyavrata; Guelcher, Scott A; Goldstein, Aaron S; Whittington, Abby R

    2012-11-01

    Biomaterial scaffolds with gradients in architecture, mechanical and chemical properties have the potential to improve the osseointegration of ligament grafts by recapitulating phenotypic gradients that exist at the natural ligament-bone (L-B) interface. Towards the larger goal of regenerating the L-B interface, this in vitro study was performed to investigate the potential of two scaffolds with mineral gradients in promoting a spatial gradient of osteoblastic differentiation. Specifically, the first graded scaffold was fabricated by co-electrospinning two polymer solutions (one doped with nano-hydroxyapatite particles) from offset spinnerets, while the second was created by immersing the first scaffold in a 5 × simulated body fluid. Rat bone marrow stromal cells, cultured in the presence of osteogenic supplements, were found to be metabolically active on all regions of both scaffolds after 1 and 7 days of culture. Gene expression of bone morphogenic protein-2 and osteopontin was elevated on mineral-containing regions as compared to regions without mineral, while the expression of alkaline phosphatase mRNA revealed the opposite trend. Finally, the presence of osteopontin and bone sialoprotein confirmed osteoblastic phenotypic maturation by day 28. This study indicates that co-electrospun scaffolds with gradients in mineral content can guide the formation of phenotypic gradients and may thus promote the regeneration of the L-B interface. Copyright © 2012 Elsevier Ltd. All rights reserved.

  17. Optimal delivery route of bone marrow stromal cells for rat infarct brain – A study using non-invasive optical imaging

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    Tamaki N

    2010-01-01

    Full Text Available BACKGROUND - Recent studies have indicated that bone marrow stromal cells (BMSC have the potential to improve neurological function when transplanted into animal model of central nervous system (CNS disorders. However, there still exist several questions to solved prior to clinical application. In this study, therefore, we aimed to clarify the optimal delivery route of BMSC transplantation over a reasonable time window.MATERIALS AND METHODS - The rats were subjected to permanent middle cerebral artery occlusion. The BMSC were labeled with quantum dot (QD 800. The labeled BMSC were transplanted into the infarct brain directly or intravenously at 7 days after the insult. Motor function was serially assessed. The BMSC were also tracked using near infrared (NIR fluorescence imaging technique every week. The fate of the transplanted BMSC was examined at 5 weeks after transplantation, using Immunohistochemistry. RESULTS - Direct, but not intravenous, transplantation of BMSC significantly enhanced functional recovery. NIR fluorescence imaging could visualize their migration towards cerebral infarct in directly, but not intravenously, injected animals. The findings were supported on histological analysis. Thus, the BMSC were widely engrafted in the infarct brain in the directly injected animals, but few BMSC were observed in the intravenously injected ones. CONCLUSION - This study strongly suggests that direct transplantation of BMSC may be more beneficial in treating patients with ischemic stroke than their intravenous transplantation. Therapeutic time window must be called into account when considering the route of BMSC transplantation.

  18. 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.

  19. 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.

  20. Intravenous administration of bone marrow-derived multipotent mesenchymal stromal cells has a neutral effect on obesity-induced diabetic cardiomyopathy

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    Sebastián D Calligaris

    2013-01-01

    Full Text Available Obesity is a major global health issue. Obese patients develop metabolic syndrome, which is a cluster of clinical features characterized by insulin resistance and dyslipidemia. Its cardiac manifestation, diabetic cardiomyopathy, leads to heart failure. Bone marrow-derived multipotent mesenchymal stromal cells, also referred to as mesenchymal stem cells (MSC are envisioned as a therapeutic tool not only for cardiovascular diseases but also for other degenerative conditions. Our aim was to evaluate whether the intravenous administration of MSC modifies cardiac dysfunction in obese mice. To this end, C57BL/6 mice were fed a regular (normal or high-fat diet (obese. Obese animals received the vehicle (obese, a single dose (obese + 1x MSC or three doses (obese + 3x MSC of 0.5x10(6 syngeneic MSC. Two to three months following MSC administration, cardiac function was assessed by cardiac catheterization, at basal condition and after a pharmacological stress. Compared to normal mice, obese mice presented hyperglycemia, hyperinsulinemia, hypercholesterolemia and cardiac dysfunction after stress condition. Exogenous MSC neither improved nor impaired this cardiac dysfunction. Thus, intravenous administration of MSC has neutral effect on obesity-induced diabetic cardiomyopathy

  1. Comparisons of phenotype and immunomodulatory capacity among rhesus bone-marrow-derived mesenchymal stem/stromal cells, multipotent adult progenitor cells, and dermal fibroblasts

    Science.gov (United States)

    Wang, Qi; Clarkson, Christina; Graham, Melanie; Donahue, Robert; Hering, Bernhard J.; Verfaillie, Catherine M.; Bansal-Pakala, Pratima; O'Brien, Timothy D.

    2015-01-01

    Background Potent immunomodulatory effects have been reported for mesenchymal stem/stromal cells (MSCs), multipotent adult progenitor cells (MAPCs), and fibroblasts. However, side-by-side comparisons of these cells specifically regarding immunophenotype, gene expression, and suppression of proliferation of CD4+ and CD8+ lymphocyte populations have not been reported. Methods We developed MAPC and MSC lines from rhesus macaque bone marrow and fibroblast cell lines from rhesus dermis and assessed phenotypes based upon differentiation potential, flow cytometric analysis of immunophenotype, and quantitative RT-PCR analysis of gene expression. Using allogeneic lymphocyte proliferation assays, we compared the in vitro immunomodulatory potency of each cell type. Results and Conclusions Extensive phenotypic similarities exist among each cell type, although immunosuppressive potencies are distinct. MAPCs are most potent, and fibroblasts are the least potent cell type. All three cell types demonstrated immunomodulatory capacity such that each may have potential therapeutic applications such as in organ transplantation, where reduced local immune response is desirable. PMID:24825538

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

    Science.gov (United States)

    Akhoundzadeh, Kobar; Vakili, Abedin; Sameni, Hamid Reza; Vafaei, Abbas Ali; Rashidy-Pour, Ali; Safari, Manouchehr; Mohammadkhani, Razieh

    2017-08-01

    This study examined whether post-stroke bone marrow stromal cells (BMSCs) therapy combined with exercise (EX) and/or thyroid hormone (TH) could reduce brain damage in an experimental ischemic stroke in mice. Focal cerebral ischemia was induced under Laser Doppler Flowmetry (LDF) guide by 45 min of middle cerebral artery occlusion (MCAO), followed by 7 days of reperfusion in albino mice. BMSCs were injected into the right cerebral ventricle 24 h after MCAO, followed by daily injection of T3 (20 μg/100 g weight S.C) and 6 days of running on a treadmill. Infarct size, neurobehavioral test, TUNEL and BrdU positive cells were evaluated at 7 days after MCAO. Treatment with BMSCs and mild EX alone significantly reduced the infarct volume by 23% and 44%, respectively (both, p cells (a marker of apoptosis) was significantly reduced in the EX, BMSCs, BMSCs + EX, BMSCs + TH, and BMSCs + EX + TH groups (all, p cells in the subventricular zone (SVZ) (p cells and the attenuation of apoptosis in ischemia stroke in young mice.

  3. Anti-angiogenesis therapy based on the bone marrow-derived stromal cells genetically engineered to express sFlt-1 in mouse tumor model

    Directory of Open Access Journals (Sweden)

    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.

  4. Loss of Asxl1 Alters Self-Renewal and Cell Fate of Bone Marrow Stromal Cell, Leading to Bohring-Opitz-like Syndrome in Mice

    Directory of Open Access Journals (Sweden)

    Peng Zhang

    2016-06-01

    Full Text Available De novo ASXL1 mutations are found in patients with Bohring-Opitz syndrome, a disease with severe developmental defects and early childhood mortality. The underlying pathologic mechanisms remain largely unknown. Using Asxl1-targeted murine models, we found that Asxl1 global loss as well as conditional deletion in osteoblasts and their progenitors led to significant bone loss and a markedly decreased number of bone marrow stromal cells (BMSCs compared with wild-type littermates. Asxl1−/− BMSCs displayed impaired self-renewal and skewed differentiation, away from osteoblasts and favoring adipocytes. RNA-sequencing analysis revealed altered expression of genes involved in cell proliferation, skeletal development, and morphogenesis. Furthermore, gene set enrichment analysis showed decreased expression of stem cell self-renewal gene signature, suggesting a role of Asxl1 in regulating the stemness of BMSCs. Importantly, re-introduction of Asxl1 normalized NANOG and OCT4 expression and restored the self-renewal capacity of Asxl1−/− BMSCs. Our study unveils a pivotal role of ASXL1 in the maintenance of BMSC functions and skeletal development.

  5. Loss of Asxl1 Alters Self-Renewal and Cell Fate of Bone Marrow Stromal Cell, Leading to Bohring-Opitz-like Syndrome in Mice.

    Science.gov (United States)

    Zhang, Peng; Xing, Caihong; Rhodes, Steven D; He, Yongzheng; Deng, Kai; Li, Zhaomin; He, Fuhong; Zhu, Caiying; Nguyen, Lihn; Zhou, Yuan; Chen, Shi; Mohammad, Khalid S; Guise, Theresa A; Abdel-Wahab, Omar; Xu, Mingjiang; Wang, Qian-Fei; Yang, Feng-Chun

    2016-06-14

    De novo ASXL1 mutations are found in patients with Bohring-Opitz syndrome, a disease with severe developmental defects and early childhood mortality. The underlying pathologic mechanisms remain largely unknown. Using Asxl1-targeted murine models, we found that Asxl1 global loss as well as conditional deletion in osteoblasts and their progenitors led to significant bone loss and a markedly decreased number of bone marrow stromal cells (BMSCs) compared with wild-type littermates. Asxl1(-/-) BMSCs displayed impaired self-renewal and skewed differentiation, away from osteoblasts and favoring adipocytes. RNA-sequencing analysis revealed altered expression of genes involved in cell proliferation, skeletal development, and morphogenesis. Furthermore, gene set enrichment analysis showed decreased expression of stem cell self-renewal gene signature, suggesting a role of Asxl1 in regulating the stemness of BMSCs. Importantly, re-introduction of Asxl1 normalized NANOG and OCT4 expression and restored the self-renewal capacity of Asxl1(-/-) BMSCs. Our study unveils a pivotal role of ASXL1 in the maintenance of BMSC functions and skeletal development. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  6. Ex vivo expansion of bone marrow stromal cells by platelet-rich plasma: a promising strategy in maxillo-facial surgery.

    Science.gov (United States)

    Oliva, A; Passaro, I; Di Pasquale, R; Di Feo, A; Criscuolo, M; Zappia, V; Della Ragione, F; D'Amato, S; Annunziata, M; Guida, L

    2005-01-01

    The aim of our study is to evaluate in vitro the response of bone marrow stromal cells (BMSCs) to platelet-rich plasma (PRP), in order to clarify the potential role of their combined use in a preclinical phase preceding BMSCs transplantation for bone repair and regeneration procedures. The incubation of BMSCs with PRP promoted a remarkable, dose- and time- dependent, growth stimulation, that was paralleled to a strong increase in the quantity of type I collagen and to a significant decrease in the activity of the early osteoblastic differentiation marker, alkaline phosphatase (AP). Once PRP was removed and osteogenic inducers were added, AP returned to levels comparable to the control, while the late phenotypic markers, osteocalcin and matrix calcification, were enhanced to higher levels than in controls. Our data demonstrate that PRP induces a remarkable ex vivo enrichment of BMSCs maintaining their differentiative potential. Thus PRP represents a valid preclinical tool for obtaining an effective, rapid and safe ex vivo expansion of BMSCs prior to their clinical utilization in bone engineering.

  7. Tantalum coating of porous carbon scaffold supplemented with autologous bone marrow stromal stem cells for bone regeneration in vitro and in vivo.

    Science.gov (United States)

    Wei, Xiaowei; Zhao, Dewei; Wang, Benjie; Wang, Wei; Kang, Kai; Xie, Hui; Liu, Baoyi; Zhang, Xiuzhi; Zhang, Jinsong; Yang, Zhenming

    2016-03-01

    Porous tantalum metal with low elastic modulus is similar to cancellous bone. Reticulated vitreous carbon (RVC) can provide three-dimensional pore structure and serves as the ideal scaffold of tantalum coating. In this study, the biocompatibility of domestic porous tantalum was first successfully tested with bone marrow stromal stem cells (BMSCs) in vitro and for bone tissue repair in vivo. We evaluated cytotoxicity of RVC scaffold and tantalum coating using BMSCs. The morphology, adhesion, and proliferation of BMSCs were observed via laser scanning confocal microscope and scanning electron microscopy. In addition, porous tantalum rods with or without autologous BMSCs were implanted on hind legs in dogs, respectively. The osteogenic potential was observed by hard tissue slice examination. At three weeks and six weeks following implantation, new osteoblasts and new bone were observed at the tantalum-host bone interface and pores. At 12 weeks postporous tantalum with autologous BMSCs implantation, regenerated trabecular equivalent to mature bone was found in the pore of tantalum rods. Our results suggested that domestic porous tantalum had excellent biocompatibility and could promote new bone formation in vivo. Meanwhile, the osteogenesis of porous tantalum associated with autologous BMSCs was more excellent than only tantalum implantation. Future clinical studies are warranted to verify the clinical efficacy of combined implantation of this domestic porous tantalum associated with autologous BMSCs implantation and compare their efficacy with conventional autologous bone grafting carrying blood vessel in patients needing bone repairing. © 2016 by the Society for Experimental Biology and Medicine.

  8. Use of long-term human marrow cultures to demonstrate progenitor cell precursors in marrow treated with 4-hydroperoxycyclophosphamide

    International Nuclear Information System (INIS)

    Winton, E.F.; Colenda, K.W.

    1987-01-01

    The continued retrieval of progenitor cells (CFU-GEMM, BFU-E, CFU-E, CFU-GM) from human long-term marrow cultures (LTMC) is not uncommonly used as evidence that proliferation and differentiation are occurring in more primitive hematopoietic stem cells (HSC) in these cultures. Alternatively, the continued presence of progenitors in LTMC could be the result of survival and/or limited self-renewal of progenitor cells present when the culture was initiated, and such progenitors would have little relevance to the parent HSC. The following studies were designed to determine the relative contributions of precursors of progenitor cells to the total progenitor cells present in LTMC using a two-stage regeneration model. The adherent layer in LTMC was established over 3 weeks, irradiated (875 rad) to permanently eliminate resident hematopoietic cells, and recharged with autologous cryo-preserved marrow that was either treated or not treated (control) with 4-hydroperoxycyclophosphamide (4-HC, 100 micrograms/ml for 30 min). The 4-HC-treated marrow contained no progenitor cells, yet based on clinical autologous bone marrow transplant experience, has intact HSC. Within 1-3 weeks, progenitor cells reappeared in the irradiated LTMC recharged with 4-HC-treated marrow, and were preferentially located in the adherent layer. By 2-6 weeks, the number of progenitor cells in the adherent layer of LTMC recharged with 4-HC marrow was equivalent to control LTMC. The progenitors regenerating in the irradiated LTMC recharged with 4-HC-treated marrow appear to originate from precursors of progenitor cells, perhaps HSC. We propose this model may be useful in elucidating cellular and molecular correlates of progenitor cell regeneration from precursors

  9. Honokiol, a constituent of Magnolia species, inhibits adrenergic contraction of human prostate strips and induces stromal cell death

    Directory of Open Access Journals (Sweden)

    Daniel Herrmann

    2014-09-01

    Conclusions: Honokiol inhibits smooth muscle contraction in the human prostate, and induces cell death in cultured stromal cells. Because prostate smooth muscle tone and prostate growth may cause LUTS, it appears possible that honokiol improves voiding symptoms.

  10. Stimulation of host bone marrow stromal cells by sympathetic nerves promotes breast cancer bone metastasis in mice.

    Science.gov (United States)

    Campbell, J Preston; Karolak, Matthew R; Ma, Yun; Perrien, Daniel S; Masood-Campbell, S Kathryn; Penner, Niki L; Munoz, Steve A; Zijlstra, Andries; Yang, Xiangli; Sterling, Julie A; Elefteriou, Florent

    2012-07-01

    Bone and lung metastases are responsible for the majority of deaths in patients with breast cancer. Following treatment of the primary cancer, emotional and psychosocial factors within this population precipitate time to recurrence and death, however the underlying mechanism(s) remain unclear. Using a mouse model of bone metastasis, we provide experimental evidence that activation of the sympathetic nervous system, which is one of many pathophysiological consequences of severe stress and depression, promotes MDA-231 breast cancer cell colonization of bone via a neurohormonal effect on the host bone marrow stroma. We demonstrate that induction of RANKL expression in bone marrow osteoblasts, following β2AR stimulation, increases the migration of metastatic MDA-231 cells in vitro, independently of SDF1-CXCR4 signaling. We also show that the stimulatory effect of endogenous (chronic stress) or pharmacologic sympathetic activation on breast cancer bone metastasis in vivo can be blocked with the β-blocker propranolol, and by knockdown of RANK expression in MDA-231 cells. These findings indicate that RANKL promotes breast cancer cell metastasis to bone via its pro-migratory effect on breast cancer cells, independently of its effect on bone turnover. The emerging clinical implication, supported by recent epidemiological studies, is that βAR-blockers and drugs interfering with RANKL signaling, such as Denosumab, could increase patient survival if used as adjuvant therapy to inhibit both the early colonization of bone by metastatic breast cancer cells and the initiation of the "vicious cycle" of bone destruction induced by these cells.

  11. Bone marrow contributes to epithelial cancers in mice and humans as developmental mimicry.

    Science.gov (United States)

    Cogle, Christopher R; Theise, Neil D; Fu, Dongtao; Ucar, Deniz; Lee, Sean; Guthrie, Steven M; Lonergan, Jean; Rybka, Witold; Krause, Diane S; Scott, Edward W

    2007-08-01

    Bone marrow cells have the capacity to contribute to distant organs. We show that marrow also contributes to epithelial neoplasias of the small bowel, colon, and lung, but not the skin. In particular, epithelial neoplasias found in patients after hematopoietic cell transplantations demonstrate that human marrow incorporates into neoplasias by adopting the phenotype of the surrounding neoplastic environment. To more rigorously evaluate marrow contribution to epithelial cancer, we employed mouse models of intestinal and lung neoplasias, which revealed specifically that the hematopoietic stem cell and its progeny incorporate within cancer. Furthermore, this marrow involvement in epithelial cancer does not appear to occur by induction of stable fusion. Whereas previous claims have been made that marrow can serve as a direct source of epithelial neoplasia, our results indicate a more cautionary note, that marrow contributes to cancer as a means of developmental mimicry. Disclosure of Potential Conflicts of Interest is found at the end of this article.

  12. CULTIVATION OF HUMAN LIVER CELLS AND ADIPOSE-DERIVED MESENCHYMAL STROMAL CELLS IN PERFUSION BIOREACTOR

    Directory of Open Access Journals (Sweden)

    Yu. В. Basok

    2018-01-01

    Full Text Available Aim: to show the progress of the experiment of cultivation of human liver cells and adipose-derived mesenchymal stromal cells in perfusion bioreactor.Materials and methods. The cultivation of a cell-engineered construct, consisting of a biopolymer microstructured collagen-containing hydrogel, human liver cells, adipose-derived mesenchymal stromal cells, and William’s E Medium, was performed in a perfusion bioreactor.Results. On the 7th day large cells with hepatocyte morphology – of a polygonal shape and a centrally located round nucleus, – were present in the culture chambers of the bioreactor. The metabolic activity of hepatocytes in cell-engineered constructs was confi rmed by the presence of urea in the culture medium on the seventh day of cultivation in the bioreactor and by the resorption of a biopolymer microstructured collagen-containing hydrogel.

  13. Prospectively Isolated Human Bone Marrow Cell-Derived MSCs Support Primitive Human CD34-Negative Hematopoietic Stem Cells.

    Science.gov (United States)

    Matsuoka, Yoshikazu; Nakatsuka, Ryusuke; Sumide, Keisuke; Kawamura, Hiroshi; Takahashi, Masaya; Fujioka, Tatsuya; Uemura, Yasushi; Asano, Hiroaki; Sasaki, Yutaka; Inoue, Masami; Ogawa, Hiroyasu; Takahashi, Takayuki; Hino, Masayuki; Sonoda, Yoshiaki

    2015-05-01

    Hematopoietic stem cells (HSCs) are maintained in a specialized bone marrow (BM) niche, which consists of osteoblasts, endothelial cells, and a variety of mesenchymal stem/stromal cells (MSCs). However, precisely what types of MSCs support human HSCs in the BM remain to be elucidated because of their heterogeneity. In this study, we succeeded in prospectively isolating/establishing three types of MSCs from human BM-derived lineage- and CD45-negative cells, according to their cell surface expression of CD271 and stage-specific embryonic antigen (SSEA)-4. Among them, the MSCs established from the Lineage(-) CD45(-) CD271(+) SSEA-4(+) fraction (DP MSC) could differentiate into osteoblasts and chondrocytes, but they lacked adipogenic differentiation potential. The DP MSCs expressed significantly higher levels of well-characterized HSC-supportive genes, including IGF-2, Wnt3a, Jagged1, TGFβ3, nestin, CXCL12, and Foxc1, compared with other MSCs. Interestingly, these osteo-chondrogenic DP MSCs possessed the ability to support cord blood-derived primitive human CD34-negative severe combined immunodeficiency-repopulating cells. The HSC-supportive actions of DP MSCs were partially carried out by soluble factors, including IGF-2, Wnt3a, and Jagged1. Moreover, contact between DP MSCs and CD34-positive (CD34(+) ) as well as CD34-negative (CD34(-) ) HSCs was important for the support/maintenance of the CD34(+/-) HSCs in vitro. These data suggest that DP MSCs might play an important role in the maintenance of human primitive HSCs in the BM niche. Therefore, the establishment of DP MSCs provides a new tool for the elucidation of the human HSC/niche interaction in vitro as well as in vivo. © 2014 AlphaMed Press.

  14. Intrathecal administration of autologous bone marrow stromal cells improves neuropathic pain in patients with spinal cord injury.

    Science.gov (United States)

    Vaquero, J; Zurita, M; Rico, M A; Aguayo, C; Fernández, C; Gutiérrez, R; Rodríguez-Boto, G; Saab, A; Hassan, R; Ortega, C

    2018-03-23

    Neuropathic pain (NP) is highly disabling, responds poorly to pharmacological treatment, and represents a significant cause of decreased quality of life in patients suffering from spinal cord injury (SCI). In recent years, cell therapy with autologous mesenchymal stromal cells (MSCs) has been considered as a potential therapeutic weapon in this entity. Ten patients suffering chronic SCI received 100 million MSCs into subarachnoid space by lumbar puncture (month 1 of the study) and this procedure was repeated at months 4 and 7 until reaching a total doses of 300 million MSCs. Intensity of NP was measured by standard numerical rating scale (VAS) from 0 to 10, recording scores previous to the first MSCs administration and monthly, until month 10 of follow-up. Months 1, 4, 7 and 10 of the study were selected as time points in order to a statistical analysis by the nonparametric Wilcoxon rank test. Our results showed significant and progressive improvement in NP intensity after the first administration of MSCs (p: 0.003). This study supports the benefit of intrathecal administration of autologous MSCs for the treatment of NP in patients with SCI. Copyright © 2018 Elsevier B.V. All rights reserved.

  15. Human mesenchymal stromal cell-secreted lactate induces M2-macrophage differentiation by metabolic reprogramming

    OpenAIRE

    Selleri, Silvia; Bifsha, Panojot; Civini, Sara; Pacelli, Consiglia; Dieng, Mame Massar; Lemieux, William; Jin, Ping; Bazin, Ren?e; Patey, Natacha; Marincola, Francesco M.; Moldovan, Florina; Zaouter, Charlotte; Trudeau, Louis-Eric; Benabdhalla, Basma; Louis, Isabelle

    2016-01-01

    Human mesenchymal stromal cells (MSC) have been shown to dampen immune response and promote tissue repair, but the underlying mechanisms are still under investigation. Herein, we demonstrate that umbilical cord-derived MSC (UC-MSC) alter the phenotype and function of monocyte-derived dendritic cells (DC) through lactate-mediated metabolic reprogramming. UC-MSC can secrete large quantities of lactate and, when present during monocyte-to-DC differentiation, induce instead the acquisition of M2-...

  16. Stimulation of host bone marrow stromal cells by sympathetic nerves promotes breast cancer bone metastasis in mice.

    Directory of Open Access Journals (Sweden)

    J Preston Campbell

    2012-07-01

    Full Text Available Bone and lung metastases are responsible for the majority of deaths in patients with breast cancer. Following treatment of the primary cancer, emotional and psychosocial factors within this population precipitate time to recurrence and death, however the underlying mechanism(s remain unclear. Using a mouse model of bone metastasis, we provide experimental evidence that activation of the sympathetic nervous system, which is one of many pathophysiological consequences of severe stress and depression, promotes MDA-231 breast cancer cell colonization of bone via a neurohormonal effect on the host bone marrow stroma. We demonstrate that induction of RANKL expression in bone marrow osteoblasts, following β2AR stimulation, increases the migration of metastatic MDA-231 cells in vitro, independently of SDF1-CXCR4 signaling. We also show that the stimulatory effect of endogenous (chronic stress or pharmacologic sympathetic activation on breast cancer bone metastasis in vivo can be blocked with the β-blocker propranolol, and by knockdown of RANK expression in MDA-231 cells. These findings indicate that RANKL promotes breast cancer cell metastasis to bone via its pro-migratory effect on breast cancer cells, independently of its effect on bone turnover. The emerging clinical implication, supported by recent epidemiological studies, is that βAR-blockers and drugs interfering with RANKL signaling, such as Denosumab, could increase patient survival if used as adjuvant therapy to inhibit both the early colonization of bone by metastatic breast cancer cells and the initiation of the "vicious cycle" of bone destruction induced by these cells.

  17. Identification of a candidate proteomic signature to discriminate multipotent and non-multipotent stromal cells.

    Science.gov (United States)

    Rosu-Myles, Michael; She, Yi-Min; Fair, Joel; Muradia, Gauri; Mehic, Jelica; Menendez, Pablo; Prasad, Shiv S; Cyr, Terry D

    2012-01-01

    Bone marrow stromal cell cultures contain multipotent cells that may have therapeutic utility for tissue restoration; however, the identity of the cell that maintains this function remains poorly characterized. We have utilized a unique model of murine bone marrow stroma in combination with liquid chromatography mass spectrometry to compare the nuclear, cytoplasmic and membrane associated proteomes of multipotent (MSC) (CD105+) and non-multipotent (CD105-) stromal cells. Among the 25 most reliably identified proteins, 10 were verified by both real-time PCR and Western Blot to be highly enriched, in CD105+ cells and were members of distinct biological pathways and functional networks. Five of these proteins were also identified as potentially expressed in human MSC derived from both standard and serum free human stromal cultures. The quantitative amount of each protein identified in human stromal cells was only minimally affected by media conditions but varied highly between bone marrow donors. This study provides further evidence of heterogeneity among cultured bone marrow stromal cells and identifies potential candidate proteins that may prove useful for identifying and quantifying both murine and human MSC in vitro.

  18. Identification of a candidate proteomic signature to discriminate multipotent and non-multipotent stromal cells.

    Directory of Open Access Journals (Sweden)

    Michael Rosu-Myles

    Full Text Available Bone marrow stromal cell cultures contain multipotent cells that may have therapeutic utility for tissue restoration; however, the identity of the cell that maintains this function remains poorly characterized. We have utilized a unique model of murine bone marrow stroma in combination with liquid chromatography mass spectrometry to compare the nuclear, cytoplasmic and membrane associated proteomes of multipotent (MSC (CD105+ and non-multipotent (CD105- stromal cells. Among the 25 most reliably identified proteins, 10 were verified by both real-time PCR and Western Blot to be highly enriched, in CD105+ cells and were members of distinct biological pathways and functional networks. Five of these proteins were also identified as potentially expressed in human MSC derived from both standard and serum free human stromal cultures. The quantitative amount of each protein identified in human stromal cells was only minimally affected by media conditions but varied highly between bone marrow donors. This study provides further evidence of heterogeneity among cultured bone marrow stromal cells and identifies potential candidate proteins that may prove useful for identifying and quantifying both murine and human MSC in vitro.

  19. The Effects of Photobiomodulation of 808 nm Diode Laser Therapy at Higher Fluence on the in Vitro Osteogenic Differentiation of Bone Marrow Stromal Cells

    Directory of Open Access Journals (Sweden)

    Andrea Amaroli

    2018-02-01

    Full Text Available The literature has supported the concept of mesenchymal stromal cells (MSCs in bone regeneration as one of the most important applications in oro-maxillofacial reconstructions. However, the fate of the transplanted cells and their effects on the clinical outcome is still uncertain. Photobiomodulation (PBM plays an important role in the acceleration of tissue regeneration and potential repair. The aim of this in vitro study is to evaluate the effectiveness of PBM with 808 nm diode laser therapy, using a flat-top hand-piece delivery system at a higher-fluence (64 J/cm2 irradiation (1 W, continuous-wave on bone marrow stromal cells (BMSCs. The BMSCs of 3 old female Balb-c mice were analyzed. The cells were divided into two groups: irradiated group and control group. In the former the cells were irradiated every 24 h during 0 day (T0, 5 (T1, 10 (T2, and 15 (T3 days, whereas the control group was non-irradiated. The results have shown that the 64 J/cm2 laser irradiation has increased the Runt-related transcription factor 2 (Runx2. Runx2 is the most important early marker of osteoblast differentiation. The higher-fluence suppressed the synthesis of adipogenic transcription factor (PPARγ, the pivotal transcription factor in adipogenic differentiation. Also, the osteogenic markers such as Osterix (Osx and alkaline phosphatase (ALP were upregulated with an increase in the matrix mineralization. Furthermore, western blotting data demonstrated that the laser therapy has induced a statistically valid increase in the synthesis of transforming growth factor β1 (TGF-β1 but had no effects on the tumor necrosis factor α (TNFα production. The data has statistically validated the down-regulation of the important pro-inflammatory cytokines such as interleukin IL-6, and IL-17 after 808 nm PBM exposition. An increase in anti-inflammatory cytokines such as IL-1rα and IL-10 was observed. These in vitro studies provide for first time the initial proof that the PBM

  20. In vivo MR imaging of nanometer magnetically labeled bone marrow stromal cells transplanted via portal vein in rat liver

    International Nuclear Information System (INIS)

    Wang Ping; Wang Jianhua; Yan Zhiping; Hu Meiyu; Xu Pengju; Zhou Meiling; Ya Fuhua; Fan Sheung-tat; Luk John-m

    2006-01-01

    Objective: To evaluate in vivo magnetic resonance imaging with a conventional 1.5-T system for tracking of intra-portal vein transplantation nanometer magnetically labeled BMSCs in rat liver. Methods: BMSCs were isolated from 5 SD rats bone marrow with the density gradient centrifugation method. Then BMSCs were labeled with nanometer superpara-magnetic iron oxide and transfection agent. Cell labeling efficiency was assessed with determination of the percentage of Peris Prussian blue stain. Then BMSCs transplanted into normal rats' livers via portal vein. The receipts were divided into 5 groups ,including sham control,2 h ,3 d,7 d and 2 w after transplantation. Follow-up serial T 1 WI,T 2 WI and T 2 * -weighted gradient- echo MR imaging were performed at 1.5 T MRI system. MR imaging findings were compared with histology. Results: Cell labeling efficiency was more than 95% by Perls Prussian blue stain. After transplantation of labeled BMSCs via portal vein, liver's had diffuse granular signal intensity appearance in T 2 * WI MRI. Cells were detected for up to 2 w in receipts' liver's. At histologic analysis, signal intensity loss correlated with iron-loaded cells. Conclusion: MR imaging could aid in monitoring of magnetically labeled BMSCs administered via portal vein in vivo. (authors)

  1. Skeletal (stromal) stem cells

    DEFF Research Database (Denmark)

    Abdallah, Basem M; Kermani, Abbas Jafari; Zaher, Walid

    2015-01-01

    Skeletal (marrow stromal) stem cells (BMSCs) are a group of multipotent cells that reside in the bone marrow stroma and can differentiate into osteoblasts, chondrocytes and adipocytes. Studying signaling pathways that regulate BMSC differentiation into osteoblastic cells is a strategy....../preadipocyte factor 1 (Dlk1/Pref-1), the Wnt co-receptor Lrp5 and intracellular kinases. This article is part of a Special Issue entitled: Stem Cells and Bone....

  2. Mineral trioxide aggregate upregulates odonto/osteogenic capacity of bone marrow stromal cells from craniofacial bones via JNK and ERK MAPK signalling pathways.

    Science.gov (United States)

    Wang, Y; Li, J; Song, W; Yu, J

    2014-06-01

    The aim of this study was to investigate effects of mineral trioxide aggregate (MTA) on odonto/osteogenic differentiation of bone marrow stromal cells (BMSCs) from craniofacial bones. Craniofacial BMSCs were isolated from rat mandible and effects of MTA on their proliferation, differentiation and MAPK pathway involvement were subsequently investigated, in vitro. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2,5-tetrazoliumbromide) assay was performed to evaluate proliferation of the MTA-treated cells. Alkaline phosphatase (ALP) activity, alizarin red staining, real-time reverse transcription polymerase chain reaction and western blot assays were used to assess differentiation capacity as well as MAPK pathway involvement. 0.02 mg/ml MTA-treated BMSCs had significantly higher ALP activity and formed more mineralized nodules than the untreated group. Odonto/osteoblastic marker genes/proteins (Alp, Runx2/RUNX2, Osx/OSX, Ocn/OCN and Dspp/DSP respectively) in MTA-treated cells were remarkably upregulated compared to untreated ones. Mechanistically, phosphorylated Jun N-terminal kinase (P-JNK) and phosphorylated extracellular regulated protein kinases (P-ERK) in MTA-treated BMSCs increased significantly in a time-dependent manner, while inhibition of JNK and ERK MAPK pathways dramatically blocked MTA-induced odonto/osteoblastic differentiation, as indicated by reduced ALP levels, weakened mineralization capacity and downregulated levels of odonto/osteoblastic marker genes (Alp, Runx2, Osx, Ocn and Dspp). Mineral trioxide aggregate promoted odonto/osteogenic capacity of craniofacial BMSCs via JNK and ERK MAPK signalling pathways. © 2014 John Wiley & Sons Ltd.

  3. Naringin promotes osteogenic differentiation of bone marrow stromal cells by up-regulating Foxc2 expression via the IHH signaling pathway.

    Science.gov (United States)

    Lin, Fei-Xiang; Du, Shi-Xin; Liu, De-Zhong; Hu, Qin-Xiao; Yu, Guo-Yong; Wu, Chu-Cheng; Zheng, Gui-Zhou; Xie, Da; Li, Xue-Dong; Chang, Bo

    2016-01-01

    Naringin is an active compound extracted from Rhizoma Drynariae, and studies have revealed that naringin can promote proliferation and osteogenic differentiation of bone marrow stromal cells (BMSCs). In this study, we explored whether naringin could promote osteogenic differentiation of BMSCs by upregulating Foxc2 expression via the Indian hedgehog (IHH) signaling pathway. BMSCs were cultured in basal medium, basal medium with naringin, osteogenic induction medium, osteogenic induction medium with naringin and osteogenic induction medium with naringin in the presence of the IHH inhibitor cyclopamine (CPE). We examined cell proliferation by using a WST-8 assay, and differentiation by Alizarin Red S staining (for mineralization) and alkaline phosphatase (ALP) activity. In addition, we detected core-binding factor α1 (Cbfα1), osteocalcin (OCN), bone sialoprotein (BSP), peroxisome proliferation-activated receptor gamma 2 (PPARγ2) and Foxc2 expression by using RT-PCR. We also determined Foxc2 and IHH protein levels by western blotting. Naringin increased the mineralization of BMSCs, as shown by Alizarin red S assays, and induced ALP activity. In addition, naringin significantly increased the mRNA levels of Foxc2, Cbfα1, OCN, and BSP, while decreasing PPARγ2 mRNA levels. Furthermore, the IHH inhibitor CPE inhibited the osteogenesis-potentiating effects of naringin. Naringin increased Foxc2 and stimulated the activation of IHH, as evidenced by increased expression of proteins that were inhibited by CPE. Our findings indicate that naringin promotes osteogenic differentiation of BMSCs by up-regulating Foxc2 expression via the IHH signaling pathway.

  4. Presentation of a novel model of chitosan- polyethylene oxide-nanohydroxyapatite nanofibers together with bone marrow stromal cells to repair and improve minor bone defects

    Directory of Open Access Journals (Sweden)

    Asgar Emamgholi

    2015-09-01

    Full Text Available Objective(s:Various methods for repairing bone defects are presented. Cell therapy is one of these methods. Bone marrow stromal cells (BMSCs seem to be suitable for this purpose. On the other hand, lots of biomaterials are used to improve and repair the defect in the body, so in this study we tried to produce a similar structure to the bone by the chitosan and hydroxyapatite. Materials and Methods: In this study, the solution of chitosan-nanohydroxyapatite-polyethylene oxide (PEO Nanofibers was produced by electrospinning method, and then the BMSCs were cultured on this solution. A piece of chitosan-nanohydroxyapatite Nanofibers with BMSCs was placed in a hole with the diameter of 1 mm at the distal epiphysis of the rat femur. Then the biomechanical and radiographic studies were performed. Results: Biomechanical testing results showed that bone strength was significantly higher in the Nanofiber/BMSCs group in comparison with control group. Also the bone strength in nanofiber/BMSCs group was significant, but in nanofiber group was nearly significant. Radiographic studies also showed that the average amount of callus formation (radio opacity in nanofiber and control group was not significantly different. The callus formation in nanofiber/BMSCs group was increased compared to the control group, and it was not significant in the nanofiber group. Conclusion: Since chitosan-nanohydroxyapatite nanofibers with BMSCs increases the rate of bone repair, the obtained cell-nanoscaffold shell can be used in tissue engineering and cell therapy, especially for bone defects.

  5. Interleukin-6: a bone marrow stromal cell paracrine signal that induces neuroendocrine differentiation and modulates autophagy in bone metastatic PCa cells.

    Science.gov (United States)

    Delk, Nikki A; Farach-Carson, Mary C

    2012-04-01

    Autophagy reallocates nutrients and clears normal cells of damaged proteins and organelles. In the context of metastatic disease, invading cancer cells hijack autophagic processes to survive and adapt in the host microenvironment. We sought to understand how autophagy is regulated in the metastatic niche for prostate cancer (PCa) cells where bone marrow stromal cell (BMSC) paracrine signaling induces PCa neuroendocrine differentiation (NED). In PCa, this transdifferentiation of metastatic PCa cells to neuronal-like cells correlates with advanced disease. Because autophagy provides a survival advantage for cancer cells and promotes cell differentiation, we hypothesized that autophagy mediates PCa NED in the bone. Thus, we determined the ability of paracrine factors in conditioned media (CM) from two separate BMSC subtypes, HS5 and HS27a, to induce autophagy in C4-2 and C4-2B bone metastatic PCa cells by characterizing the autophagy marker, LC3. Unlike HS27a CM, HS5 CM induced LC3 accumulation in PCa cells, suggesting autophagy was induced and indicating that HS5 and HS27a secrete a different milieu of paracrine factors that influence PCa autophagy. We identified interleukin-6 (IL-6), a cytokine more highly expressed in HS5 cells than in HS27a cells, as a paracrine factor that regulates PCa autophagy. Pharmacological inhibition of STAT3 activity did not attenuate LC3 accumulation, implying that IL-6 regulates NED and autophagy through different pathways. Finally, chloroquine inhibition of autophagic flux blocked PCa NED; hence autophagic flux maintains NED. Our studies imply that autophagy is cytoprotective for PCa cells in the bone, thus targeting autophagy is a potential therapeutic strategy.

  6. Dual delivery of rhPDGF-BB and bone marrow mesenchymal stromal cells expressing the BMP2 gene enhance bone formation in a critical-sized defect model.

    Science.gov (United States)

    Park, Shin-Young; Kim, Kyoung-Hwa; Shin, Seung-Yun; Koo, Ki-Tae; Lee, Yong-Moo; Seol, Yang-Jo

    2013-11-01

    Bone tissue healing is a dynamic, orchestrated process that relies on multiple growth factors and cell types. Platelet-derived growth factor-BB (PDGF-BB) is released from platelets at wound sites and induces cellular migration and proliferation necessary for bone regeneration in the early healing process. Bone morphogenetic protein-2 (BMP-2), the most potent osteogenic differentiation inducer, directs new bone formation at the sites of bone defects. This study evaluated a combinatorial treatment protocol of PDGF-BB and BMP-2 on bone healing in a critical-sized defect model. To mimic the bone tissue healing process, a dual delivery approach was designed to deliver the rhPDGF-BB protein transiently during the early healing phase, whereas BMP-2 was supplied by rat bone marrow stromal cells (BMSCs) transfected with an adenoviral vector containing the BMP2 gene (AdBMP2) for prolonged release throughout the healing process. In in vitro experiments, the dual delivery of rhPDGF-BB and BMP2 significantly enhanced cell proliferation. However, the osteogenic differentiation of BMSCs was significantly suppressed even though the amount of BMP-2 secreted by the AdBMP2-transfected BMSCs was not significantly affected by the rhPDGF-BB treatment. In addition, dual delivery inhibited the mRNA expression of BMP receptor type II and Noggin in BMSCs. In in vivo experiments, critical-sized calvarial defects in rats showed enhanced bone regeneration by dual delivery of autologous AdBMP2-transfected BMSCs and rhPDGF-BB in both the amount of new bone formed and the bone mineral density. These enhancements in bone regeneration were greater than those observed in the group treated with AdBMP2-transfected BMSCs alone. In conclusion, the dual delivery of rhPDGF-BB and AdBMP2-transfected BMSCs improved the quality of the regenerated bone, possibly due to the modulation of PDGF-BB on BMP-2-induced osteogenesis.

  7. Cell tracking and therapy evaluation of bone marrow monocytes and stromal cells using SPECT and CMR in a canine model of myocardial infarction

    Directory of Open Access Journals (Sweden)

    Merrifield Peter

    2009-04-01

    Full Text Available Abstract Background The clinical application of stem cell therapy for myocardial infarction will require the development of methods to monitor treatment and pre-clinical assessment in a large animal model, to determine its effectiveness and the optimum cell population, route of delivery, timing, and flow milieu. Objectives To establish a model for a in vivo tracking to monitor cell engraftment after autologous transplantation and b concurrent measurement of infarct evolution and remodeling. Methods We evaluated 22 dogs (8 sham controls, 7 treated with autologous bone marrow monocytes, and 7 with stromal cells using both imaging of 111Indium-tropolone labeled cells and late gadolinium enhancement CMR for up to12 weeks after a 3 hour coronary occlusion. Hearts were also examined using immunohistochemistry for capillary density and presence of PKH26 labeled cells. Results In vivo Indium imaging demonstrated an effective biological clearance half-life from the injection site of ~5 days. CMR demonstrated a pattern of progressive infarct shrinkage over 12 weeks, ranging from 67–88% of baseline values with monocytes producing a significant treatment effect. Relative infarct shrinkage was similar through to 6 weeks in all groups, following which the treatment effect was manifest. There was a trend towards an increase in capillary density with cell treatment. Conclusion This multi-modality approach will allow determination of the success and persistence of engraftment, and a correlation of this with infarct size shrinkage, regional function, and left ventricular remodeling. There were overall no major treatment effects with this particular model of transplantation immediately post-infarct.

  8. Creatine Enhances Transdifferentiation of Bone Marrow Stromal Cell-Derived Neural Stem Cell Into GABAergic Neuron-Like Cells Characterized With Differential Gene Expression.

    Science.gov (United States)

    Darabi, Shahram; Tiraihi, Taki; Delshad, AliReza; Sadeghizadeh, Majid; Taheri, Taher; Hassoun, Hayder K

    2017-04-01

    Creatine was reported to induce bone marrow stromal cells (BMSC) into GABAergic neuron-like cells (GNLC). In a previous study, creatine was used as a single inducer for BMSC into GNLC with low yield. In this study, BMSC-derived neurospheres (NS) have been used in generating GABAergic phenotype. The BMSC were isolated from adult rats and used in generating neurospheres and used for producing neural stem cells (NSC). A combination of all-trans-retinoic acid (RA), the ciliary neurotrophic factor (CNTF), and creatine was used in order to improve the yield of GNLC. We also used other protocols for the transdifferentiation including RA alone; RA and creatine; RA and CNTF; and RA, CNTF, and creatine. The BMSC, NSC, and GNLC were characterized by specific markers. The activity of the GNLC was evaluated using FM1-43. The isolated BMSC expressed Oct4, fibronectin, and CD44. The NS were immunoreactive to nestin and SOX2, the NSC were immunoreactive to nestin, NF68 and NF160, while the GNLC were immunoreactive to GAD1/2, VGAT, GABA, and synaptophysin. Oct4 and c-MYC, pluripotency genes, were expressed in the BMSC, while SOX2 and c-MYC were expressed in the NSC. The activity of GNLC indicates that the synaptic vesicles were released upon stimulation. The conclusion is that the combination of RA, CNTF, and creatine induced differentiation of neurosphere-derived NSC into GNLC within 1 week. This protocol gives higher yield than the other protocols used in this study. The mechanism of induction was clearly associated with several differential pluripotent genes.

  9. Bone marrow stromal cells on a three-dimensional bioactive fiber mesh undergo osteogenic differentiation in the absence of osteogenic media supplements: the effect of silanol groups.

    Science.gov (United States)

    Rodrigues, Márcia T; Leonor, Isabel B; Gröen, Nathalie; Viegas, Carlos A; Dias, Isabel R; Caridade, Sofia G; Mano, João F; Gomes, Manuela E; Reis, Rui L

    2014-10-01

    Osteogenic differentiation is a tightly regulated process dependent on the stimuli provided by the micro-environment. Silicon-substituted materials are known to have an influence on the osteogenic phenotype of undifferentiated and bone-derived cells. This study aims to investigate the bioactivity profile as well as the mechanical properties of a blend of starch and poly-caprolactone (SPCL) polymeric fiber mesh scaffolds functionalized with silanol (Si-OH) groups as key features for bone tissue engineering strategies. The scaffolds were made from SPCL by a wet spinning technique. A calcium silicate solution was used as a non-solvent to develop an in situ functionalization with Si-OH groups in a single-step approach. We also explored the relevance of silicon incorporated in SPCL-Si scaffolds to the in vitro osteogenic process of goat bone marrow stromal cells (gBMSCs) with and without osteogenic supplements in the culture medium. We hypothesized that SPCL-Si scaffolds could act as physical and chemical millieus to induce per se the osteogenic differentiation of gBMSCs. Results show that osteogenic differentiation of gBMSCs and the production of a mineralized extracellular matrix on bioactive SPCL-Si scaffolds occur for up to 2weeks, even in the absence of osteogenic supplements in the culture medium. The omission of media supplements to induce osteogenic differentiation is a promising feature towards simplified and cost-effective cell culturing procedures of a potential bioengineered product, and concomitant translation into the clinical field. Thus, the present work demonstrates that SPCL-Si scaffolds and their intrinsic properties sustain gBMSC osteogenic features in vitro, even in the absence of osteogenic supplements to the culture medium, and show great potential for bone regeneration strategies. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  10. Transplantation of osteoporotic bone marrow stromal cells rejuvenated by the overexpression of SATB2 prevents alveolar bone loss in ovariectomized rats.

    Science.gov (United States)

    Xu, Rongyao; Fu, Zongyun; Liu, Xue; Xiao, Tao; Zhang, Ping; Du, Yifei; Yuan, Hua; Cheng, Jie; Jiang, Hongbing

    2016-11-01

    Estrogen-deficient osteoporosis is an aging-related disease with high morbidity that not only significantly increases a woman's risk of fragility fracture but is also associated with tooth and bone loss in the supporting alveolar bone of the jaw. Emerging evidence suggests that the aging of bone marrow stromal cells (BMSCs) contributes to the development of osteoporosis. In this study, we aimed to investigate the role of the special AT-rich sequence-binding protein 2 (SATB2), a stemness and senescence regulator of craniofacial BMSCs, in rat ovariectomy-induced alveolar osteoporosis. We also sought to determine whether transplantation of SATB2-modified BMSCs could ameliorate estrogen deficient alveolar bone loss. Our data revealed that BMSCs from ovariectomy-induced alveolar bone exhibited typical senescence phenotypes such as diminished stemness and osteogenic capacity, increased expression of senescence or osteoclastic markers and enhanced adipogenic potential. These phenotypic changes are a result of SATB2-mediated senescence dysregulation as evidenced by nuclear γH2AX foci formation. Moreover, overexpression of SATB2 significantly alleviated the senescence of osteoporotic BMSCs in vitro. Importantly, transplantation of SATB2-modified BMSCs significantly attenuated ovariectomy-induced alveolar bone loss in vivo. Together, our results revealed that SATB2 is a critical regulator of alveolar BMSC senescence, and its overexpression decreases these senescent changes both in vitro and in vivo. SATB2-modified BMSC delivery could be a viable and promising therapeutic strategy for alveolar bone loss induced by estrogen-deficient osteoporosis. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Bone marrow-derived mesenchymal stromal cells regress aortic aneurysm via the NF-kB, Smad3 and Akt signaling pathways.

    Science.gov (United States)

    Yamawaki-Ogata, Aika; Oshima, Hideki; Usui, Akihiko; Narita, Yuji

    2017-10-01

    We have confirmed that aortic aneurysm (AA) can be regressed by the administration of bone marrow-derived mesenchymal stromal cells (BM-MSCs). We investigated the kinetics of signaling pathways in AA following treatment with BM-MSCs. Angiotensin II-infused apolipoprotein E-deficient mice were treated by intravenous injection of 1 × 10 6 BM-MSCs in 0.2 mL saline (BM-MSCs group, n = 5) or 0.2 mL saline (saline group, n = 5). Mice were sacrificed 2 weeks after injection and subjected to measurements of the incidence of AA and levels of phosphorylated proteins. Levels of proteins in conditioned media of BM-MSCs were also measured. The incidence of AA in the BM-MSCs group was reduced (BM-MSC 40% versus saline 100%, P kB and pSTAT1 were reduced (pNF-kB: 0.28 versus 0.45 unit/mL, P kB, pAkt, and pSmad3 were correlated with aortic diameters. Trophic factors including IGFPB-3, NRF, Activin A and PDGF-AA were secreted from BM-MSCs (IGFBP-3: 35.2 pg/mL, NRF: 3.1 pg/mL, Activin A: 3.1 pg/mL, PDGF-AA: 0.45 pg/mL). Our findings suggested that the therapeutic mechanism of BM-MSC-mediated AA regression could contribute to regulation of the NF-kB, Smad3 and Akt signaling pathways. In addition, paracrine actions by factors including NRF, IGFBP-3, Activin A and PDGF-AA might have affected these signaling pathways. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  12. Autologous Pancreatic Islet Transplantation in Human Bone Marrow

    Science.gov (United States)

    Maffi, Paola; Balzano, Gianpaolo; Ponzoni, Maurilio; Nano, Rita; Sordi, Valeria; Melzi, Raffaella; Mercalli, Alessia; Scavini, Marina; Esposito, Antonio; Peccatori, Jacopo; Cantarelli, Elisa; Messina, Carlo; Bernardi, Massimo; Del Maschio, Alessandro; Staudacher, Carlo; Doglioni, Claudio; Ciceri, Fabio; Secchi, Antonio; Piemonti, Lorenzo

    2013-01-01

    The liver is the current site of choice for pancreatic islet transplantation, even though it is far from being ideal. We recently have shown in mice that the bone marrow (BM) may be a valid alternative to the liver, and here we report a pilot study to test feasibility and safety of BM as a site for islet transplantation in humans. Four patients who developed diabetes after total pancreatectomy were candidates for the autologous transplantation of pancreatic islet. Because the patients had contraindications for intraportal infusion, islets were infused in the BM. In all recipients, islets engrafted successfully as shown by measurable posttransplantation C-peptide levels and histopathological evidence of insulin-producing cells or molecular markers of endocrine tissue in BM biopsy samples analyzed during follow-up. Thus far, we have recorded no adverse events related to the infusion procedure or the presence of islets in the BM. Islet function was sustained for the maximum follow-up of 944 days. The encouraging results of this pilot study provide new perspectives in identifying alternative sites for islet infusion in patients with type 1 diabetes. Moreover, this is the first unequivocal example of successful engraftment of endocrine tissue in the BM in humans. PMID:23733196

  13. Apoptosis induction of human endometriotic epithelial and stromal cells by noscapine

    Directory of Open Access Journals (Sweden)

    Mohammad Rasoul Khazaei

    2016-09-01

    Full Text Available Objective(s: Endometriosis is a complex gynecologic disease with unknown etiology. Noscapine has been introduced as a cancer cell suppressor. Endometriosis was considered as a cancer like disorder, The aim of present study was to investigate noscapine apoptotic effect on human endometriotic epithelial and stromal cells in vitro. Materials and Methods:In this in vitro study, endometrial biopsies from endometriosis patients (n=9 were prepared and digested by an enzymatic method (collagenase I, 2 mg/ml. Stromal and epithelial cells were separated by sequential filtration through a cell strainer and ficoll layering. The cells of each sample were divided into five groups: control (0, 10, 25, 50 and 100 micromole/liter (µM concentration of noscapine and were cultured for three different periods of times; 24, 48 and 72 hr. Cell viability was assessed by colorimetric assay. Nitric oxide (NO concentration was measured by Griess reagent. Cell death was analyzed by Acridine Orange (AO–Ethidium Bromide (EB double staining and Terminal deoxynucleotidyl transferase (TdT dUTP Nick-End Labeling (TUNEL assay. Data were analyzed by one-way ANOVA. Results: Viability of endometrial epithelial and stromal cells significantly decreased in 10, 25, 50 and 100 µM noscapine concentration in 24, 48, 72 hr (P

  14. Good Preservation of Stromal Cells and No Apoptosis in Human Ovarian Tissue after Vitrification

    Directory of Open Access Journals (Sweden)

    Raffaella Fabbri

    2014-01-01

    Full Text Available The aim of this study was to develop a vitrification procedure for human ovarian tissue cryopreservation in order to better preserve the ovarian tissue. Large size samples of ovarian tissue retrieved from 15 female-to-male transgender subjects (18–38 years were vitrified using two solutions (containing propylene glycol, ethylene glycol, and sucrose at different concentrations in an open system. Light microscopy, transmission electron microscopy, and TUNEL assay were applied to evaluate the efficiency of the vitrification protocol. After vitrification/warming, light microscopy showed oocyte nucleus with slightly thickened chromatin and irregular shape, while granulosa and stromal cells appeared well preserved. Transmission electron microscopy showed oocytes with slightly irregular nuclear shape and finely dispersed chromatin. Clear vacuoles and alterations in cellular organelles were seen in the oocyte cytoplasm. Stromal cells had a moderately dispersed chromatin and homogeneous cytoplasm with slight vacuolization. TUNEL assay revealed the lack of apoptosis induction by vitrification in all ovarian cell types. In conclusion after vitrification/warming the stromal compartment maintained morphological and ultrastructural features similar to fresh tissue, while the oocyte cytoplasm was slightly damaged. Although these data are encouraging, further studies are necessary and essential to optimize vitrification procedure.

  15. Up-regulation of bone marrow stromal protein 2 (BST2) in breast cancer with bone metastasis

    International Nuclear Information System (INIS)

    Cai, Dongqing; Cao, Jie; Li, Zhen; Zheng, Xin; Yao, Yao; Li, Wanglin; Yuan, Ziqiang

    2009-01-01

    Bone metastases are frequent complications of breast cancer. Recent literature implicates multiple chemokines in the formation of bone metastases in breast cancer. However, the molecular mechanism of metastatic bone disease in breast cancer remains unknown. We have recently made the novel observation of the BST2 protein expression in human breast cancer cell lines. The purpose of our present study is to investigate the expression and the role of BST2 in bone metastatic breast cancer. cDNA microarray analysis was used to compare the BST2 gene expression between a metastatic to bone human breast cancer cell line (MDA-231BO) and a primary human breast cancer cell line (MDA-231). The BST2 expression in one bone metastatic breast cancer and seven non-bone metastatic breast cancer cell lines were also determined using real-time RT-PCR and Western blot assays. We then employed tissue array to further study the BST2 expression in human breast cancer using array slides containing 20 independent breast cancer tumors that formed metastatic bone lesions, 30 non-metastasis-forming breast cancer tumors, and 8 normal breast tissues. In order to test the feasibility of utilizing BST2 as a serum marker for the presence of bone metastasis in breast cancer, we had measured the BST2 expression levels in human serums by using ELISA on 43 breast cancer patients with bone metastasis, 43 breast cancer patients without bone metastasis, and 14 normal healthy controls. The relationship between cell migration and proliferation and BST2 expression was also studied in a human breast recombinant model system using migration and FACS analysis. The microarray demonstrated over expression of the BST2 gene in the bone metastatic breast cancer cell line (MDA-231BO) compared to the primary human breast cancer cell line (MDA-231). The expression of the BST2 gene was significantly increased in the bone metastatic breast cancer cell lines and tumor tissues compared to non-bone metastatic breast cancer

  16. Human bone marrow mesenchymal stem cells for retinal vascular injury.

    Science.gov (United States)

    Wang, Jin-Da; An, Ying; Zhang, Jing-Shang; Wan, Xiu-Hua; Jonas, Jost B; Xu, Liang; Zhang, Wei

    2017-09-01

    To examine the potential of intravitreally implanted human bone marrow-derived mesenchymal stem cells (BMSCs) to affect vascular repair and the blood-retina barrier in mice and rats with oxygen-induced retinopathy, diabetic retinopathy or retinal ischaemia-reperfusion damage. Three study groups (oxygen-induced retinopathy group: 18 C57BL/6J mice; diabetic retinopathy group: 15 rats; retinal ischaemia-reperfusion model: 18 rats) received BMSCs injected intravitreally. Control groups (oxygen-induced retinopathy group: 12 C57BL/6J mice; diabetic retinopathy group: 15 rats; retinal ischaemia-reperfusion model: 18 rats) received an intravitreal injection of phosphate-buffered saline. We applied immunohistological techniques to measure retinal vascularization, spectroscopic measurements of intraretinally extravasated fluorescein-conjugated dextran to quantify the blood-retina barrier breakdown, and histomorphometry to assess retinal thickness and retinal ganglion cell count. In the oxygen-induced retinopathy model, the study group with intravitreally injected BMSCs as compared with the control group showed a significantly (p = 0.001) smaller area of retinal neovascularization. In the diabetic retinopathy model, study group and control group did not differ significantly in the amount of intraretinally extravasated dextran. In the retinal ischaemia-reperfusion model, on the 7th day after retina injury, the retina was significantly thicker in the study group than in the control group (p = 0.02), with no significant difference in the retinal ganglion cell count (p = 0.36). Intravitreally implanted human BMSCs were associated with a reduced retinal neovascularization in the oxygen-induced retinopathy model and with a potentially cell preserving effect in the retinal ischaemia-reperfusion model. Intravitreal BMSCs may be of potential interest for the therapy of retinal vascular disorders. © 2016 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley

  17. [Effect of the compound of poly lactic-co-glycolic acid and bone marrow stromal cells modified by osteoprotegerin gene on the periodontal regeneration in Beagle dog periodontal defects].

    Science.gov (United States)

    Zhou, Wei; Zhao, Chun-Hui; Mei, Ling-Xuan

    2010-06-01

    To evaluate the effect of the osteoprotegerin (OPG) gene-modified autologous bone marrow stromal cells (BMSCs) on regeneration of periodontal defects, and to provide new experimental evidence to explore the gene therapy for periodontal disease. pSecTag2/B-opg was transduced into BMSCs by lipofectamine 2000. The expression of OPG protein in the BMSCs was detected by immunocytochemistry and Western blot. Inverted phase contrast microscope and scanning electron microscopy (SEM) were used to observe the morphology and proliferation of the BMSCs(OPG) on on the surface of the poly lactic-co-glycolic (PLGA). Horizontal alveolar bone defect (4 mmx4 mmx 3 mm) were surgically created in the buccal aspect of the mandibular premolar, and were randomly assigned to receive BMSCs(OPG)-PLGA (cells/material/OPG), BMSCs-PLGA (cells/material), PLGA (material), or root planning only (blank control). The animals were euthanized at 6 weeks post surgery for histological analysis. The height of new alveolar bone and cementum and the formation of new connective tissue were analyzed and compared. All data were statistically analyzed using the q test. The BMSCs transfected by human OPG gene can highly express OPG protein. SEM observations demonstrated that BMSCs(OPG) were able to proliferate and massively colonize on the scaffolds structure. After 6 weeks, the height of new alveolar bone and cementum and the formation of new connective tissue were significantly greater in the experimental group than in the control groups (P < 0.05). BMSCs(OPG)-PLGA can significantly promote the regeneration of dog's periodontal bone defects. Gene therapy utilizing OPG may offer the potential for periodontal tissue engineering applications.

  18. Migration of bone marrow and cord blood mesenchymal stem cells in vitro is regulated by stromal-derived factor-1-CXCR4 and hepatocyte growth factor-c-met axes and involves matrix metalloproteinases.

    Science.gov (United States)

    Son, Bo-Ra; Marquez-Curtis, Leah A; Kucia, Magda; Wysoczynski, Marcin; Turner, A Robert; Ratajczak, Janina; Ratajczak, Mariusz Z; Janowska-Wieczorek, Anna

    2006-05-01

    Human mesenchymal stem cells (MSCs) are increasingly being considered in cell-based therapeutic strategies for regeneration of various organs/tissues. However, the signals required for their homing and recruitment to injured sites are not yet fully understood. Because stromal-derived factor (SDF)-1 and hepatocyte growth factor (HGF) become up-regulated during tissue/organ damage, in this study we examined whether these factors chemoattract ex vivo-expanded MSCs derived from bone marrow (BM) and umbilical cord blood (CB). Specifically, we investigated the expression by MSCs of CXCR4 and c-met, the cognate receptors of SDF-1 and HGF, and their functionality after early and late passages of MSCs. We also determined whether MSCs express matrix metalloproteinases (MMPs), including membrane type 1 (MT1)-MMP, matrix-degrading enzymes that facilitate the trafficking of hematopoietic stem cells. We maintained expanded BM- or CB-derived MSCs for up to 15-18 passages with monitoring of the expression of 1) various tissue markers (cardiac and skeletal muscle, neural, liver, and endothelial cells), 2) functional CXCR4 and c-met, and 3) MMPs. We found that for up to 15-18 passages, both BM- and CB-derived MSCs 1) express mRNA for cardiac, muscle, neural, and liver markers, as well as the vascular endothelial (VE) marker VE-cadherin; 2) express CXCR4 and c-met receptors and are strongly attracted by SDF-1 and HGF gradients; 3) express MMP-2 and MT1-MMP transcripts and proteins; and 4) are chemo-invasive across the reconstituted basement membrane Matrigel. These in vitro results suggest that the SDF-1-CXCR4 and HGF-c-met axes, along with MMPs, may be involved in recruitment of expanded MSCs to damaged tissues.

  19. MicroRNA-138 regulates osteogenic differentiation of human stromal (mesenchymal) stem cells in vivo

    DEFF Research Database (Denmark)

    Eskildsen, Tilde; Taipaleenmäki, Hanna; Stenvang, Jan

    2011-01-01

    Elucidating the molecular mechanisms that regulate human stromal (mesenchymal) stem cell (hMSC) differentiation into osteogenic lineage is important for the development of anabolic therapies for treatment of osteoporosis. MicroRNAs (miRNAs) are short, noncoding RNAs that act as key regulators......-regulated during osteoblast differentiation of hMSCs. Overexpression of miR-138 inhibited osteoblast differentiation of hMSCs in vitro, whereas inhibition of miR-138 function by antimiR-138 promoted expression of osteoblast-specific genes, alkaline phosphatase (ALP) activity, and matrix mineralization. Furthermore...

  20. Cloning, identification, and functional analysis of bone marrow stromal cell antigen-2 from sika deer (Cervus nippon).

    Science.gov (United States)

    Wang, Jiawen; Bian, Shuai; Liu, Meichun; Zhang, Xin; Wang, Siming; Bai, Xueyuan; Zhao, Daqing; Zhao, Yu

    2018-06-30

    BST-2(tetherin/CD317/HM1.24) has been identified as a cellular antiviral factor that inhibits the release of a wide range of enveloped viruses from infected cells. Orthologs of BST-2 have been identified in several species including humans, monkeys, cows, sheep, pigs, and mice. In this study, we cloned the gene and characterized the protein of the BST-2 homolog from sika deer (Cervus nippon). cnBST-2 shares 37.8% and 74.2% identity with the BST-2 homologs from Homo sapiens and Ovis aries, respectively. The extracellular domain of cnBST-2 has two putative N-linked glycosylation sites and three potential dimerization sites. cnBST-2 was shown to be expressed on the cell surface, like human BST-2. Exogenous expression of cnBST-2 resulted in potent inhibition of HIV-1 particle release in 293T cells; however, this activity resisted antagonism by HIV-1 Vpu. Moreover, cnBST-2 was not able to activate nuclear factor-κB, in contrast to human BST-2. This study is the first report of the isolation and characterization of BST-2 from C. nippon. Copyright © 2018 Elsevier B.V. All rights reserved.

  1. Cytogenetic adaptive response induced by pre-exposure in human lymphocytes and marrow cells of mice

    International Nuclear Information System (INIS)

    Zhang Lianzhen; Deng Zhicheng

    1993-01-01

    The cytogenetic adaptive response induced by pre-exposure in human lymphocytes and marrow cells of mice were studied. The results of this study showed that human lymphocytes in vitro and mouse marrow cells in vivo can become adapted to low-level irradiation from 3 H-TdR or exposure to a low dose of X-or γ-irradiation, so that they become less sensitive to the chromosomal damage effects of subsequent exposures. (4 tabs.)

  2. Expression of Siglec-11 by human and chimpanzee ovarian stromal cells, with uniquely human ligands: implications for human ovarian physiology and pathology

    Science.gov (United States)

    Wang, Xiaoxia; Chow, Renee; Deng, Liwen; Anderson, Dan; Weidner, Noel; Godwin, Andrew K; Bewtra, Chanda; Zlotnik, Albert; Bui, Jack; Varki, Ajit; Varki, Nissi

    2011-01-01

    Siglecs (Sialic acid-binding Immunoglobulin Superfamily Lectins) are cell surface signaling receptors of the I-type lectin group that recognize sialic acid-bearing glycans. CD33-related-Siglecs are a subset with expression primarily in cells of hematopoietic origin and functional relevance to immune reactions. Earlier we reported a human-specific gene conversion event that markedly changed the coding region for the extracellular domain of Siglec-11, associated with human-specific expression in microglia (Hayakawa T, Angata T, Lewis AL, Mikkelsen TS, Varki NM, Varki A. 2005. A human-specific gene in microglia. Science. 309:1693). Analyzing human gene microarrays to define new patterns of expression, we observed high levels of SIGLEC11 transcript in the ovary and adrenal cortex. Thus, we examined human and chimpanzee tissues using a well-characterized anti-Siglec-11 mouse monoclonal antibody. Although adrenal expression was variable and confined to infiltrating macrophages in capillaries, ovarian expression of Siglec-11 in both humans and chimpanzees was on fibroblasts, the first example of Siglec expression on mesenchyme-derived stromal cells. Cytokines from such ovarian stromal fibroblasts play important roles in follicle development and ovulation. Stable transfection of SIGLEC11 into a primary human ovarian stromal fibroblast cell line altered the secretion of growth-regulated oncogene α, interleukin (IL)-10, IL-7, transforming growth factor β1 and tumor necrosis factor-α, cytokines involved in ovarian physiology. Probing for Siglec-11 ligands revealed distinct and strong mast cell expression in human ovaries, contrasting to diffuse stromal ligands in chimpanzee ovaries. Interestingly, there was a trend of increased Siglec-11 expression in post-menopausal ovaries compared with pre-menopausal ones. Siglec-11 expression was also found on human ovarian stromal tumors and in polycystic ovarian syndrome, a human-specific disease. These results indicate potential

  3. Persistence of human parvovirus B19 in multipotent mesenchymal stromal cells expressing the erythrocyte P antigen: implications for transplantation

    Science.gov (United States)

    Sundin, Mikael; Lindblom, Anna; Örvell, Claes; Barrett, A.John; Sundberg, Berit; Watz, Emma; Wikman, Agneta; Broliden, Kristina; Le Blanc, Katarina

    2014-01-01

    Multipotent mesenchymal stromal cells (MSC) are used to improve the outcome of hematopoietic stem cell transplantation and in regenerative medicine. However, MSC may harbor persistent viruses that may compromise their clinical benefit. Retrospectively screened, 1 of 20 MSC from healthy donors contained parvovirus B19 (B19) DNA. We found that MSC express the B19 receptor (the globoside P antigen) and a co-receptor (Ku 80), and can transmit B19 to bone marrow cells in vitro, suggesting that the virus can persist in the marrow stroma of healthy individuals. Two stem cell transplant patients received the B19 positive MSC as treatment for graft-versus-host disease. Neither developed viremia nor symptomatic B19 infection. These results demonstrate for the first time that persistent B19 in MSC can infect hematopoietic cells and underscore the importance of monitoring B19 transmission by MSC products. PMID:18804048

  4. Wnt-dependent osteogenic commitment of bone marrow stromal cells using a novel GSK3β inhibitor

    Directory of Open Access Journals (Sweden)

    David A. Cook

    2014-03-01

    Using human BMSCs grown in adipogenic medium, we confirmed that AR28-mediated Wnt signalling caused a significant (p < 0.05 dose-dependent reduction of adipogenic markers. In osteogenic media, including dexamethasone, AR28 caused significant (p < 0.05 decreases in alkaline phosphatase (ALP activity compared to vehicle controls, indicative of a reduced osteogenic response. However, when excluding dexamethasone from the osteogenic media, increases in both ALP and mineralisation were identified following AR28 treatment, which was blocked by mitomycin C. Pre-treatment of BMSCs with AR28 for 7 days before osteogenic induction also increased ALP activity and mineralisation. Furthermore, BMP2-induced osteogenic differentiation was strongly enhanced by AR28 addition within 3 days, but without concomitant changes in cell number, therefore revealing BMP-dependent and independent mechanisms for Wnt-induced osteogenesis.

  5. Participation of bone marrow stromal cells in hemopoietic recovery of rats irradiated and then parabiosed with a non-irradiated litter mate, 2. Scanning and transmission electron microscopic observations

    Energy Technology Data Exchange (ETDEWEB)

    Kagawa, Koichi; Hayashi, Keiki; Awai, Michiyasu

    1986-07-01

    A light microscopical study on the recovery process after lethal irradiation and parabiosis has been made. Electron microscopically, in the bone marrow of lethally irradiated rats, hemorrhage occurred due to detachment of sinus endothelial cells. Afterwards, reticulum cells with small intracytoplasmic lipid droplets appeared. On day 3, these cells were rapidly replaced by the reticulum cells with large lipid droplets, and resulted in fatty marrow within 7 days. Spindle-shaped fibroblastoid reticulum cells were also observed. In the bone marrow of lethally irradiated rats parabiosed with non-treated litter mates, hemopoiesis was initiated by adhesion of nucleated blood cells to intricated fine cytoplasmic pseudopods of fat-storage cells. On days 3 to 5, in parallel with progressive hemopoietic recovery, fibroblastoid and reticulum cells with large lipid droplets decreased whereas those with small droplets increased. On day 8, reticulum cells with lipid droplets were seldom seen, and hemopoietic distribution became the same as normal. These results suggested that bone marrow stromal cells, namely reticulum, fat-storage, and fibroblastoid cells share a common cellular origin, and also that they regain their structure and function when fat-storage cells were placed in contact with hemopoietic precursor cells.

  6. Poly(γ-Glutamic Acid) as an Exogenous Promoter of Chondrogenic Differentiation of Human Mesenchymal Stem/Stromal Cells.

    Science.gov (United States)

    Antunes, Joana C; Tsaryk, Roman; Gonçalves, Raquel M; Pereira, Catarina Leite; Landes, Constantin; Brochhausen, Christoph; Ghanaati, Shahram; Barbosa, Mário A; Kirkpatrick, C James

    2015-06-01

    Cartilage damage and/or aging effects can cause constant pain, which limits the patient's quality of life. Although different strategies have been proposed to enhance the limited regenerative capacity of cartilage tissue, the full production of native and functional cartilaginous extracellular matrix (ECM) has not yet been achieved. Poly(γ-glutamic acid) (γ-PGA), a naturally occurring polyamino acid, biodegradable into glutamate residues, has been explored for tissue regeneration. In this work, γ-PGA's ability to support the production of cartilaginous ECM by human bone marrow mesenchymal stem/stromal cells (MSCs) and nasal chondrocytes (NCs) was investigated. MSC and NC pellets were cultured in basal medium (BM), chondrogenic medium (CM), and CM-γ-PGA-supplemented medium (CM+γ-PGA) over a period of 21 days. Pellet size/shape was monitored with time. At 14 and 21 days of culture, the presence of sulfated glycosaminoglycans (sGAGs), type II collagen (Col II), Sox-9, aggrecan, type XI collagen (Col XI), type X collagen (Col X), calcium deposits, and type I collagen (Col I) was analyzed. After excluding γ-PGA's cytotoxicity, earlier cell condensation, higher sGAG content, Col II, Sox-9 (day 14), aggrecan, and Col X (day 14) production was observed in γ-PGA-supplemented MSC cultures, with no signs of mineralization or Col I. These effects were not evident with NCs. However, Sox-9 (at day 14) and Col X (at days 14 and 21) were increased, decreased, or absent, respectively. Overall, γ-PGA improved chondrogenic differentiation of MSCs, increasing ECM production earlier in culture. It is proposed that γ-PGA incorporation in novel biomaterials has a beneficial impact on future approaches for cartilage regeneration.

  7. Potential role of herbal remedies in stem cell therapy: proliferation and differentiation of human mesenchymal stromal cells.

    Science.gov (United States)

    Udalamaththa, Vindya Lankika; Jayasinghe, Chanika Dilumi; Udagama, Preethi Vidya

    2016-08-11

    Stem cell therapy has revolutionized modern clinical therapy with the potential of stem cells to differentiate into many different cell types which may help to replace different cell lines of an organism. Innumerous trials are carried out to merge new scientific knowledge and techniques with traditional herbal extracts that may result in less toxic, affordable, and highly available natural alternative therapeutics. Currently, mesenchyamal stromal cell (MSC) lines are treated with individual and mixtures of crude herbal extracts, as well as with purified compounds from herbal extracts, to investigate the mechanisms and effects of these on stem cell growth and differentiation. Human MSCs (hMSCs) possess multilineage, i.e., osteogenic, neurogenic, adipogenic, chondrogenic, and myogenic, differentiation abilities. The proliferative and differentiation properties of hMSCs treated with herbal extracts have shown promise in diseases such as osteoporosis, neurodegenerative disorders, and other tissue degenerative disorders. Well characterized herbal extracts that result in increased rates of tissue regeneration may be used in both stem cell therapy and tissue engineering for replacement therapy, where the use of scaffolds and vesicles with enhanced attaching and proliferative properties could be highly advantageous in the latter. Although the clinical application of herbal extracts is still in progress due to the variability and complexity of bioactive constituents, standardized herbal preparations will strengthen their application in the clinical context. We have critically reviewed the proliferative and differentiation effects of individual herbal extracts on hMSCs mainly derived from bone marrow and elaborated on the plausible underlying mechanisms of action. To be fruitfully used in reparative and regenerative therapy, future directions in this area of study should (i) make use of hMSCs derived from different non-traditional sources, including medical waste material

  8. Effects of Bone Marrow Mesenchymal Stromal Cell Therapy in Experimental Cutaneous Leishmaniasis in BALB/c Mice Induced by Leishmania amazonensis

    Science.gov (United States)

    Pereira, Joyce Carvalho; Ramos, Tadeu Diniz; Silva, Johnatas Dutra; de Mello, Mirian França; Pratti, Juliana Elena Silveira; da Fonseca-Martins, Alessandra Marcia; Firmino-Cruz, Luan; Kitoko, Jamil Zola; Chaves, Suzana Passos; Gomes, Daniel Claudio De Oliveira; Diaz, Bruno Lourenço; Rocco, Patricia R. M.; de Matos Guedes, Herbert Leonel

    2017-01-01

    Cutaneous leishmaniasis remains both a public health and a therapeutic challenge. To date, no ideal therapy for cutaneous leishmaniasis has been identified, and no universally accepted therapeutic regimen and approved vaccines are available. Due to the mesenchymal stromal cell (MSC) immunomodulatory capacity, they have been applied in a wide variety of disorders, including infectious, inflammatory, and allergic diseases. We evaluated the potential effects of bone marrow MSC therapy in a murine model of cutaneous leishmaniasis. In vitro, coculture of infected macrophages with MSC increased parasite load on macrophages in comparison with controls (macrophages without MSCs). In vivo, BALB/c mice were infected with 2 × 106 Leishmania amazonensis (Josefa strain) promastigotes in the footpad. 7 and 37 days after infection, animals were treated with 1 × 105 MSCs, either intralesional (i.l.), i.e., in the same site of infection, or intravenously (i.v.), through the external jugular vein. Control animals received the same volume (50 µL) of phosphate-buffered saline by i.l. or i.v. routes. The lesion progression was assessed by its thickness measured by pachymetry. Forty-two days after infection, animals were euthanized and parasite burden in the footpad and in the draining lymph nodes was quantified by the limiting dilution assay (LDA), and spleen cells were phenotyped by flow cytometry. No significant difference was observed in lesion progression, regardless of the MSC route of administration. However, animals treated with i.v. MSCs presented a significant increase in parasite load in comparison with controls. On the other hand, no harmful effect due to MSCs i.l. administered was observed. The spleen cellular profile analysis showed an increase of IL-10 producing T CD4+ and TCD8+ cells in the spleen only in mice treated with i.v. MSC. The excessive production of IL-10 could be associated with the disease-aggravating effects of MSC therapy when intravenously

  9. Design and validation of a consistent and reproducible manufacture process for the production of clinical-grade bone marrow-derived multipotent mesenchymal stromal cells.

    Science.gov (United States)

    Codinach, Margarita; Blanco, Margarita; Ortega, Isabel; Lloret, Mireia; Reales, Laura; Coca, Maria Isabel; Torrents, Sílvia; Doral, Manel; Oliver-Vila, Irene; Requena-Montero, Miriam; Vives, Joaquim; Garcia-López, Joan

    2016-09-01

    Multipotent mesenchymal stromal cells (MSC) have achieved a notable prominence in the field of regenerative medicine, despite the lack of common standards in the production processes and suitable quality controls compatible with Good Manufacturing Practice (GMP). Herein we describe the design of a bioprocess for bone marrow (BM)-derived MSC isolation and expansion, its validation and production of 48 consecutive batches for clinical use. BM samples were collected from the iliac crest of patients for autologous therapy. Manufacturing procedures included: (i) isolation of nucleated cells (NC) by automated density-gradient centrifugation and plating; (ii) trypsinization and expansion of secondary cultures; and (iii) harvest and formulation of a suspension containing 40 ± 10 × 10(6) viable cells. Quality controls were defined as: (i) cell count and viability assessment; (ii) immunophenotype; and (iii) sterility tests, Mycoplasma detection, endotoxin test and Gram staining. A 3-week manufacturing bioprocess was first designed and then validated in 3 consecutive mock productions, prior to producing 48 batches of BM-MSC for clinical use. Validation included the assessment of MSC identity and genetic stability. Regarding production, 139.0 ± 17.8 mL of BM containing 2.53 ± 0.92 × 10(9) viable NC were used as starting material, yielding 38.8 ± 5.3 × 10(6) viable cells in the final product. Surface antigen expression was consistent with the expected phenotype for MSC, displaying high levels of CD73, CD90 and CD105, lack of expression of CD31 and CD45 and low levels of HLA-DR. Tests for sterility, Mycoplasma, Gram staining and endotoxin had negative results in all cases. Herein we demonstrated the establishment of a feasible, consistent and reproducible bioprocess for the production of safe BM-derived MSC for clinical use. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  10. Osteoinduction and proliferation of bone-marrow stromal cells in three-dimensional poly (ε-caprolactone)/ hydroxyapatite/collagen scaffolds.

    Science.gov (United States)

    Wang, Ting; Yang, Xiaoyan; Qi, Xin; Jiang, Chaoyin

    2015-05-08

    Osteoinduction and proliferation of bone-marrow stromal cells (BMSCs) in three-dimensional (3D) poly(ε-caprolactone) (PCL) scaffolds have not been studied throughly and are technically challenging. This study aimed to optimize nanocomposites of 3D PCL scaffolds to provide superior adhesion, proliferation and differentiation environment for BMSCs in this scenario. BMSCs were isolated and cultured in a novel 3D tissue culture poly(ε-caprolactone) (PCL) scaffold coated with poly-lysine, hydroxyapatite (HAp), collagen and HAp/collagen. Cell morphology was observed and BMSC biomarkers for osteogenesis, osteoblast differentiation and activation were analyzed. Scanning Electron Microscope (SEM) micrographs showed that coating materials were uniformly deposited on the surface of PCL scaffolds and BMSCs grew and aggregated to form clusters during 3D culture. Both mRNA and protein levels of the key players of osteogenesis and osteoblast differentiation and activation, including runt-related transcription factor 2 (Runx2), alkaline phosphates (ALP), osterix, osteocalcin, and RANKL, were significantly higher in BMSCs seeded in PCL scaffolds coated with HAp or HAp/collagen than those seeded in uncoated PCL scaffolds, whereas the expression levels were not significantly different in collagen or poly-lysine coated PCL scaffolds. In addition, poly-lysine, collagen, HAp/collagen, and HAp coated PCL scaffolds had significantly more viable cells than uncoated PCL scaffolds, especially scaffolds with HAp/collagen and collagen-alone coatings. That BMSCs in HAp or HAp/collagen PCL scaffolds had remarkably higher ALP activities than those in collagen-coated alone or uncoated PCL scaffolds indicating higher osteogenic differentiation levels of BMSCs in HAp or HAp/collagen PCL scaffolds. Moreover, morphological changes of BMSCs after four-week of 3D culture confirmed that BMSCs successfully differentiated into osteoblast with spread-out phenotype in HAp/collagen coated PCL scaffolds

  11. PHD-2 Suppression in Mesenchymal Stromal Cells Enhances Wound Healing.

    Science.gov (United States)

    Ko, Sae Hee; Nauta, Allison C; Morrison, Shane D; Hu, Michael S; Zimmermann, Andrew S; Chung, Michael T; Glotzbach, Jason P; Wong, Victor W; Walmsley, Graham G; Peter Lorenz, H; Chan, Denise A; Gurtner, Geoffrey C; Giaccia, Amato J; Longaker, Michael T

    2018-01-01

    Cell therapy with mesenchymal stromal cells is a promising strategy for tissue repair. Restoration of blood flow to ischemic tissues is a key step in wound repair, and mesenchymal stromal cells have been shown to be proangiogenic. Angiogenesis is critically regulated by the hypoxia-inducible factor (HIF) superfamily, consisting of transcription factors targeted for degradation by prolyl hydroxylase domain (PHD)-2. The aim of this study was to enhance the proangiogenic capability of mesenchymal stromal cells and to use these modified cells to promote wound healing. Mesenchymal stromal cells harvested from mouse bone marrow were transduced with short hairpin RNA (shRNA) against PHD-2; control cells were transduced with scrambled shRNA (shScramble) construct. Gene expression quantification, human umbilical vein endothelial cell tube formation assays, and wound healing assays were used to assess the effect of PHD knockdown mesenchymal stromal cells on wound healing dynamics. PHD-2 knockdown mesenchymal stromal cells overexpressed HIF-1α and multiple angiogenic factors compared to control (p cells treated with conditioned medium from PHD-2 knockdown mesenchymal stromal cells exhibited increased formation of capillary-like structures and enhanced migration compared with human umbilical vein endothelial cells treated with conditioned medium from shScramble-transduced mesenchymal stromal cells (p cells healed at a significantly accelerated rate compared with wounds treated with shScramble mesenchymal stromal cells (p cells (p cells augments their proangiogenic potential in wound healing therapy. This effect appears to be mediated by overexpression of HIF family transcription factors and up-regulation of multiple downstream angiogenic factors.

  12. Discrepancy of biologic behavior influenced by bone marrow derived cells in lung cancer.

    Science.gov (United States)

    Zhang, Jie; Niu, Xiao-Min; Liao, Mei-Lin; Liu, Yun; Sha, Hui-Fang; Zhao, Yi; Yu, Yong-Feng; Tan, Qiang; Xiang, Jia-Qing; Fang, Jing; Lv, Dan-Dan; Li, Xue-Bing; Lu, Shun; Chen, Hai-Quan

    2010-11-01

    Disseminated cancer cells may initially require local nutrients and growth factors to thrive and survive in bone marrow. However, data on the influence of bone marrow derived cells (BMDC, also called bone stromal cells in some publications) on lung cancer cells is largely unexplored. This study explored the mechanism of how bone stromal factors contribute to the bone tropism in lung cancer. The difference among lung cancer cell lines in their abilities to metastasize to bone was found using the SCID animal model. Supernatant of bone marrow aspiration (BM) and condition medium from human bone stromal cells (BSC) were used to study the activity of bone stromal factors. We found bone stromal factors significantly increased the proliferation, invasion, adhesion and expression of angiogenosis-related factors, and inhibited the apoptosis for high bone metastasis H460 lung cancer cells. These biologic effects were not seen in SPC-A1 or A549 cells, which are low bone metastasis lung cancer cells. Adhesion of H460 cells to surface coated with bone stromal cells can activate some signal transduction pathways, and alter the expression of adhesion associated factors, including integrin β 3 and ADAMTS-1, two potential targets related with bone metastasis. We concluded that bone marrow derived cells had a profound effect on biological behavior of lung cancers, therefore favoring the growth of lung cancer cells in bone.

  13. Regulation of cholesterol 25-hydroxylase expression by vitamin D3 metabolites in human prostate stromal cells

    International Nuclear Information System (INIS)

    Wang, J.-H.; Tuohimaa, Pentti

    2006-01-01

    Vitamin D 3 plays an important role in the control of cell proliferation and differentiation. Cholesterol 25-hydroxylase (CH25H) is an enzyme converting cholesterol into 25-hydroxycholesterol. Vitamin D 3 as well as 25-hydroxycholesterol has been shown to inhibit cell growth and induce cell apoptosis. Here we show that 10 nM 1α,25(OH) 2 D 3 and 500 nM 25OHD 3 upregulate CH25H mRNA expression in human primary prostate stromal cells (P29SN). Protein synthesis inhibitor cycloheximide does not block 1α,25(OH) 2 D 3 mediated upregulation of CH25H mRNA. Transcription inhibitor actinomycin D blocks basal level as well as 1α,25(OH) 2 D 3 induced CH25H mRNA expression. 1α,25(OH) 2 D 3 has no effect on CH25H mRNA stability. 25-Hydroxycholesterol significantly decreased the P29SN cell number. A CH25H enzyme inhibitor, desmosterol, increases basal cell number but has no significant effect on vitamin D 3 treated cells. Our data suggest that ch25h could be a vitamin D 3 target gene and may partly mediate anti-proliferative action of vitamin D 3 in human primary prostate stromal cells

  14. Transcriptomic comparisons between cultured human adipose tissue-derived pericytes and mesenchymal stromal cells

    Directory of Open Access Journals (Sweden)

    Lindolfo da Silva Meirelles

    2016-03-01

    Full Text Available Mesenchymal stromal cells (MSCs, sometimes called mesenchymal stem cells, are cultured cells able to give rise to mature mesenchymal cells such as adipocytes, osteoblasts, and chondrocytes, and to secrete a wide range of trophic and immunomodulatory molecules. Evidence indicates that pericytes, cells that surround and maintain physical connections with endothelial cells in blood vessels, can give rise to MSCs (da Silva Meirelles et al., 2008 [1]; Caplan and Correa, 2011 [2]. We have compared the transcriptomes of highly purified, human adipose tissue pericytes subjected to culture-expansion in pericyte medium or MSC medium, with that of human adipose tissue MSCs isolated with traditional methods to test the hypothesis that their transcriptomes are similar (da Silva Meirelles et al., 2015 [3]. Here, we provide further information and analyses of microarray data from three pericyte populations cultured in pericyte medium, three pericyte populations cultured in MSC medium, and three adipose tissue MSC populations deposited in the Gene Expression Omnibus under accession number GSE67747. Keywords: Mesenchymal stromal cells, Mesenchymal stem cells, Pericytes, Microarrays

  15. Clinical-grade production of human mesenchymal stromal cells: occurrence of aneuploidy without transformation.

    Science.gov (United States)

    Tarte, Karin; Gaillard, Julien; Lataillade, Jean-Jacques; Fouillard, Loic; Becker, Martine; Mossafa, Hossein; Tchirkov, Andrei; Rouard, Hélène; Henry, Catherine; Splingard, Marie; Dulong, Joelle; Monnier, Delphine; Gourmelon, Patrick; Gorin, Norbert-Claude; Sensebé, Luc

    2010-02-25

    Clinical-grade human mesenchymal stromal cells (MSCs) have been expanded in vitro for tissue engineering or immunoregulatory purposes without standardized culture conditions or release criteria. Although human MSCs show poor susceptibility for oncogenic transformation, 2 recent studies described their capacity to accumulate chromosomal instability and to give rise to carcinoma in immunocompromised mice after long-term culture. We thus investigated the immunologic and genetic features of MSCs expanded with fetal calf serum and fibroblast growth factor or with platelet lysate in 4 cell-therapy facilities during 2 multicenter clinical trials. Cultured MSCs showed a moderate expression of human leukocyte antigen-DR without alteration of their low immunogenicity or their immunomodulatory capacity. Moreover, some transient and donor-dependent recurring aneuploidy was detected in vitro, independently of the culture process. However, MSCs with or without chromosomal alterations showed progressive growth arrest and entered senescence without evidence of transformation either in vitro or in vivo.

  16. Cyclic hydrostatic pressure promotes a stable cartilage phenotype and enhances the functional development of cartilaginous grafts engineered using multipotent stromal cells isolated from bone marrow and infrapatellar fat pad.

    Science.gov (United States)

    Carroll, S F; Buckley, C T; Kelly, D J

    2014-06-27

    The objective of this study was to investigate how joint specific biomechanical loading influences the functional development and phenotypic stability of cartilage grafts engineered in vitro using stem/progenitor cells isolated from different source tissues. Porcine bone marrow derived multipotent stromal cells (BMSCs) and infrapatellar fat pad derived multipotent stromal cells (FPSCs) were seeded in agarose hydrogels and cultured in chondrogenic medium, while simultaneously subjected to 10MPa of cyclic hydrostatic pressure (HP). To mimic the endochondral phenotype observed in vivo with cartilaginous tissues engineered using BMSCs, the culture media was additionally supplemented with hypertrophic factors, while the loss of phenotype observed in vivo with FPSCs was induced by withdrawing transforming growth factor (TGF)-β3 from the media. The application of HP was found to enhance the functional development of cartilaginous tissues engineered using both BMSCs and FPSCs. In addition, HP was found to suppress calcification of tissues engineered using BMSCs cultured in chondrogenic conditions and acted to maintain a chondrogenic phenotype in cartilaginous grafts engineered using FPSCs. The results of this study point to the importance of in vivo specific mechanical cues for determining the terminal phenotype of chondrogenically primed multipotent stromal cells. Furthermore, demonstrating that stem or progenitor cells will appropriately differentiate in response to such biophysical cues might also be considered as an additional functional assay for evaluating their therapeutic potential. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. Stromal cells expressing hedgehog-interacting protein regulate the proliferation of myeloid neoplasms

    International Nuclear Information System (INIS)

    Kobune, M; Iyama, S; Kikuchi, S; Horiguchi, H; Sato, T; Murase, K; Kawano, Y; Takada, K; Ono, K; Kamihara, Y; Hayashi, T; Miyanishi, K; Sato, Y; Takimoto, R; Kato, J

    2012-01-01

    Aberrant reactivation of hedgehog (Hh) signaling has been described in a wide variety of human cancers including cancer stem cells. However, involvement of the Hh-signaling system in the bone marrow (BM) microenvironment during the development of myeloid neoplasms is unknown. In this study, we assessed the expression of Hh-related genes in primary human CD34 + cells, CD34 + blastic cells and BM stromal cells. Both Indian Hh (Ihh) and its signal transducer, smoothened (SMO), were expressed in CD34 + acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS)-derived cells. However, Ihh expression was relatively low in BM stromal cells. Remarkably, expression of the intrinsic Hh-signaling inhibitor, human Hh-interacting protein (HHIP) in AML/MDS-derived stromal cells was markedly lower than in healthy donor-derived stromal cells. Moreover, HHIP expression levels in BM stromal cells highly correlated with their supporting activity for SMO + leukemic cells. Knockdown of HHIP gene in stromal cells increased their supporting activity although control cells marginally supported SMO + leukemic cell proliferation. The demethylating agent, 5-aza-2′-deoxycytidine rescued HHIP expression via demethylation of HHIP gene and reduced the leukemic cell-supporting activity of AML/MDS-derived stromal cells. This indicates that suppression of stromal HHIP could be associated with the proliferation of AML/MDS cells

  18. Is fatty acid composition of human bone marrow significant to bone health?

    Science.gov (United States)

    Pino, Ana María; Rodríguez, J Pablo

    2017-12-16

    The bone marrow adipose tissue (BMAT) is a conserved component of the marrow microenvironment, providing storage and release of energy and stabilizing the marrow extent. Also, it is recognized both the amount and quality of BMAT are relevant to preserve the functional relationships between BMAT, bone, and blood cell production. In this article we ponder the information supporting the tenet that the quality of BMAT is relevant to bone health. In the human adult the distribution of BMAT is heterogeneous over the entire skeleton, and both BMAT accumulation and bone loss come about with aging in healthy populations. But some pathological conditions which increase BMAT formation lead to bone impairment and fragility. Analysis in vivo of the relative content of saturated and unsaturated fatty acids (FA) in BMAT indicates site-related bone marrow fat composition and an association between increased unsaturation index (UI) and bone health. With aging some impairment ensues in the regulation of bone marrow cells and systemic signals leading to local chronic inflammation. Most of the bone loss diseases which evolve altered BMAT composition have as common factors aging and/or chronic inflammation. Both saturated and unsaturated FAs originate lipid species which are active mediators in the inflammation process. Increased free saturated FAs may lead to lipotoxicity of bone marrow cells. The pro-inflammatory, anti-inflammatory or resolving actions of compounds derived from long chain poly unsaturated FAs (PUFA) on bone cells is varied, and depending on the metabolism of the parent n:3 or n:6 PUFAs series. Taking together the evidence substantiate that marrow adipocyte function is fundamental for an efficient link between systemic and marrow fatty acids to accomplish specific energy or regulatory needs of skeletal and marrow cells. Further, they reveal marrow requirements of PUFAs. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Properties and growth of human bone marrow mesenchymal stromal cells cultivated in different media

    Czech Academy of Sciences Publication Activity Database

    Turnovcová, Karolína; Růžičková, Kateřina; Vaněček, Václav; Syková, Eva; Jendelová, Pavla

    2009-01-01

    Roč. 11, č. 7 (2009), s. 874-875 ISSN 1465-3249 R&D Projects: GA MŠk(CZ) LC554 Grant - others:GA MŠk(CZ) 1M0538; GA ČR(CZ) GD309/08/H079; EC FP6 Rescue(XE) LSHB-CT-2005-518233; EC FP6 Enimet(XE) LSHM-CT-2005-019063 Program:1M Institutional research plan: CEZ:AV0Z50390703 Keywords : cell cycle * cell differentiation * antigens Subject RIV: FH - Neurology Impact factor: 2.204, year: 2009

  20. Human bone-marrow-derived mesenchymal stem cells

    DEFF Research Database (Denmark)

    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...

  1. CD54-Mediated Interaction with Pro-inflammatory Macrophages Increases the Immunosuppressive Function of Human Mesenchymal Stromal Cells

    Directory of Open Access Journals (Sweden)

    Nicolas Espagnolle

    2017-04-01

    Full Text Available Summary: Mesenchymal stromal cells (MSCs sense and modulate inflammation and represent potential clinical treatment for immune disorders. However, many details of the bidirectional interaction of MSCs and the innate immune compartment are still unsolved. Here we describe an unconventional but functional interaction between pro-inflammatory classically activated macrophages (M1MΦ and MSCs, with CD54 playing a central role. CD54 was upregulated and enriched specifically at the contact area between M1MФ and MSCs. Moreover, the specific interaction induced calcium signaling and increased the immunosuppressive capacities of MSCs dependent on CD54 mediation. Our data demonstrate that MSCs can detect an inflammatory microenvironment via a direct and physical interaction with innate immune cells. This finding opens different perspectives for MSC-based cell therapy. : Mesenchymal stromal cells (MSCs are promising for cell-based therapy in inflammatory disorders by switching off the immune response. Varin and colleagues demonstrate that MSCs and inflammatory macrophages communicate via an unconventional but functional interaction that strongly increases the immunosuppressive capacities of MSCs. This new communication between the innate immune system and MSCs opens new perspectives for MSC-based cell therapy. Keywords: macrophages, bone marrow mesenchymal stromal cells, functional interaction, CD54, immunosuppression, indoleamine 2,3-dioxygenase, cell therapy

  2. Origin of hemopoietic stromal progenitor cells in chimeras

    International Nuclear Information System (INIS)

    Chertkov, J.L.; Drize, N.J.; Gurevitch, O.A.; Samoylova, R.S.

    1985-01-01

    Intravenously injected bone marrow cells do not participate in the regeneration of hemopoietic stromal progenitors in irradiated mice, nor in the curetted parts of the recipient's marrow. The hemopoietic stromal progenitors in allogeneic chimeras are of recipient origin. The adherent cell layer (ACL) of long-term cultures of allogeneic chimera bone marrow contains only recipient hemopoietic stromal progenitors. However, in ectopic hemopoietic foci produced by marrow implantation under the renal capsule and repopulated by the recipient hemopoietic cells after irradiation and reconstitution by syngeneic hemopoietic cells, the stromal progenitors were of implant donor origin, as were stromal progenitors of the ACL in long-term cultures of hemopoietic cells from ectopic foci. Our results confirm that the stromal and hemopoietic progenitors differ in origin and that hemopoietic stromal progenitors are not transplantable by the intravenous route in mice

  3. Transplantation of human neonatal foreskin stromal cells in ex vivo organotypic cultures of embryonic chick femurs

    DEFF Research Database (Denmark)

    Aldahmash, Abdullah; Vishnubalaji, Radhakrishnan

    2017-01-01

    NSSCs in ex vivo organotypic cultures of embryonic chick femurs. Isolated embryonic chick femurs (E10 and E11) were cultured for 10 days together with micro-mass cell pellets of hNSSCs, human umbilical vein endothelial cells (HUVEC) or a combination of the two cell types. Changes in femurs gross morphology......We have previously reported that human neonatal foreskin stromal cells (hNSSCs) promote angiogenesis in vitro and in chick embryo chorioallantoic membrane (CAM) assay in vivo. To examine the in vivo relevance of this observation, we examined in the present study the differentiation potential of h......NSSC + HUVEC cultures. Our data suggest that organotypic cultures can be employed to test the differentiation potential of stem cells and demonstrate the importance of stem cell interaction with 3D-intact tissue microenvironment for their differentiation....

  4. Derivation of Stromal (Skeletal, Mesenchymal) Stem-like cells from Human Embryonic Stem Cells

    DEFF Research Database (Denmark)

    Mahmood, Amer; Harkness, Linda; Abdallah, Basem

    2012-01-01

    EBs using BMP2 (bone morphogenic protein 2) combined with standard osteoblast induction medium led to weak osteoblastic induction. Conversely, subcutaneous in vivo implantation of day 20 hEBs in immune deficient mice, mixed with hydroxyapatite/tricalcium phosphate (HA/TCP) as an osteoconductive scaffold......Derivation of bone forming cells (osteoblasts) from human embryonic stem cells (hESC) is a pre-requisite for their use in clinical applications. However, there is no standard protocol for differentiating hESC into osteoblastic cells. The aim of this study was to identify the emergence of a human...... stromal (mesenchymal, skeletal) stem cell (hMSC)-like population, known to be osteoblastic cell precursors and to test their osteoblastic differentiation capacity in ex vivo cultures and in vivo. We cultured hESC in a feeder-free environment using serum replacement and as suspension aggregates (embryoid...

  5. Genetically-modified pig mesenchymal stromal cells: xenoantigenicity and effect on human T-cell xenoresponses.

    Science.gov (United States)

    Ezzelarab, Mohamed; Ezzelarab, Corin; Wilhite, Tyler; Kumar, Goutham; Hara, Hidetaka; Ayares, David; Cooper, David K C

    2011-01-01

    Mesenchymal stromal cells (MSC) are being investigated as immunomodulatory therapy in the field of transplantation, particularly islet transplantation. While MSC can regenerate across species barriers, the immunoregulatory influence of genetically modified pig MSC (pMSC) on the human and non-human primate T-cell responses has not been studied. Mesenchymal stromal cells from wild-type (WT), α1,3-galactosyltransferase gene knockout (GTKO) and GTKO pigs transgenic for the human complement-regulatory protein CD46 (GTKO/CD46) were isolated and tested for differentiation. Antibody binding and T-cell responses to WT and GTKO pMSC in comparison with GTKO pig aortic endothelial cells (pAEC) were investigated. The expression of swine leukocyte antigen (SLA) class II (SLA II) was tested. Costimulatory molecules CD80 and CD86 mRNA levels were measured. Human T-cell proliferation and the production of pro-inflammatory cytokines in response to GTKO and GTKO/CD46 pMSC in comparison with human MSC (hMSC) were evaluated. α1,3-galactosyltransferase gene knockout and GTKO/CD46 pMSC isolation and differentiation were achieved in vitro. Binding of human antibodies and T-cell responses were lower to GTKO than those to WT pMSC. Human and baboon (naïve and sensitized) antibody binding were significantly lower to GTKO pMSC than to GTKO pAEC. Before activation, human CD4(+) T-cell response to GTKO pMSC was significantly weaker than that to GTKO pAEC, even after pIFN-γ activation. More than 99% of GTKO/CD46 pMSC expressed hCD46. Human peripheral blood mononuclear cells and CD4(+) T-cell responses to GTKO and GTKO/CD46 pMSC were comparable with those to hMSC, and all were significantly lower than to GTKO pAEC. GTKO/CD46 pMSC downregulated human T-cell proliferation as efficiently as hMSC. The level of proinflammatory cytokines IL-2, IFN-γ, TNF-α, and sCD40L correlated with the downregulation of T-cell proliferation by all types of MSC. Genetically modified pMSC is significantly less

  6. miRNA signature and Dicer requirement during human endometrial stromal decidualization in vitro.

    Directory of Open Access Journals (Sweden)

    Carlos Estella

    Full Text Available Decidualization is a morphological and biochemical transformation of endometrial stromal fibroblast into differentiated decidual cells, which is critical for embryo implantation and pregnancy establishment. The complex regulatory networks have been elucidated at both the transcriptome and the proteome levels, however very little is known about the post-transcriptional regulation of this process. miRNAs regulate multiple physiological pathways and their de-regulation is associated with human disorders including gynaecological conditions such as endometriosis and preeclampsia. In this study we profile the miRNAs expression throughout human endometrial stromal (hESCs decidualization and analyze the requirement of the miRNA biogenesis enzyme Dicer during this process. A total of 26 miRNAs were upregulated and 17 miRNAs downregulated in decidualized hESCs compared to non-decidualized hESCs. Three miRNAs families, miR-181, miR-183 and miR-200, are down-regulated during the decidualization process. Using miRNAs target prediction algorithms we have identified the potential targets and pathways regulated by these miRNAs. The knockdown of Dicer has a minor effect on hESCs during in vitro decidualization. We have analyzed a battery of decidualization markers such as cell morphology, Prolactin, IGFBP-1, MPIF-1 and TIMP-3 secretion as well as HOXA10, COX2, SP1, C/EBPß and FOXO1 expression in decidualized hESCs with decreased Dicer function. We found decreased levels of HOXA10 and altered intracellular organization of actin filaments in Dicer knockdown decidualized hESCs compared to control. Our results provide the miRNA signature of hESC during the decidualization process in vitro. We also provide the first functional characterization of Dicer during human endometrial decidualization although surprisingly we found that Dicer plays a minor role regulating this process suggesting that alternative biogenesis miRNAs pathways must be involved in human

  7. Serial in vivo imaging of the porcine heart after percutaneous, intramyocardially injected 111In-labeled human mesenchymal stromal cells

    DEFF Research Database (Denmark)

    Lyngbaek, Stig; Ripa, Rasmus S; Haack-Sørensen, Mandana

    2010-01-01

    This pilot trial aimed to investigate the utilization of (111)In-labeling of mesenchymal stromal cells (MSC) for in vivo tracking after intramyocardial transplantation in a xenotransplantation model with gender mismatched cells. Human male MSC were expanded ex vivo and labeled with (111)In...

  8. Serial in vivo imaging of the porcine heart after percutaneous, intramyocardially injected (111)In-labeled human mesenchymal stromal cells

    DEFF Research Database (Denmark)

    Lyngbæk, Stig; Ripa, Rasmus Sejersten; Haack-Sørensen, Mandana

    2009-01-01

    This pilot trial aimed to investigate the utilization of (111)In-labeling of mesenchymal stromal cells (MSC) for in vivo tracking after intramyocardial transplantation in a xenotransplantation model with gender mismatched cells. Human male MSC were expanded ex vivo and labeled with (111)In...

  9. MicroRNA Levels as Prognostic Markers for the Differentiation Potential of Human Mesenchymal Stromal Cell Donors

    NARCIS (Netherlands)

    Georgi, Nicole; Taipaleenmaeki, H.; Raiss, C.C.; Groen, N.; Portalska, K.K.; van Blitterswijk, Clemens; de Boer, Jan; Post, Janine Nicole; van Wijnen, A.; Karperien, Hermanus Bernardus Johannes

    2015-01-01

    The ability of human mesenchymal stromal/stem cells (hMSCs) to differentiate into various mesenchymal cell lineages makes them a promising cell source for the use in tissue repair strategies. Because the differentiation potential of hMSCs differs between donors, it is necessary to establish

  10. Human mesenchymal stromal cells reduce influenza A H5N1-associated acute lung injury in vitro and in vivo.

    Science.gov (United States)

    Chan, Michael C W; Kuok, Denise I T; Leung, Connie Y H; Hui, Kenrie P Y; Valkenburg, Sophie A; Lau, Eric H Y; Nicholls, John M; Fang, Xiaohui; Guan, Yi; Lee, Jae W; Chan, Renee W Y; Webster, Robert G; Matthay, Michael A; Peiris, J S Malik

    2016-03-29

    Influenza can cause acute lung injury. Because immune responses often play a role, antivirals may not ensure a successful outcome. To identify pathogenic mechanisms and potential adjunctive therapeutic options, we compared the extent to which avian influenza A/H5N1 virus and seasonal influenza A/H1N1 virus impair alveolar fluid clearance and protein permeability in an in vitro model of acute lung injury, defined the role of virus-induced soluble mediators in these injury effects, and demonstrated that the effects are prevented or reduced by bone marrow-derived multipotent mesenchymal stromal cells. We verified the in vivo relevance of these findings in mice experimentally infected with influenza A/H5N1. We found that, in vitro, the alveolar epithelium's protein permeability and fluid clearance were dysregulated by soluble immune mediators released upon infection with avian (A/Hong Kong/483/97, H5N1) but not seasonal (A/Hong Kong/54/98, H1N1) influenza virus. The reduced alveolar fluid transport associated with down-regulation of sodium and chloride transporters was prevented or reduced by coculture with mesenchymal stromal cells. In vivo, treatment of aged H5N1-infected mice with mesenchymal stromal cells increased their likelihood of survival. We conclude that mesenchymal stromal cells significantly reduce the impairment of alveolar fluid clearance induced by A/H5N1 infection in vitro and prevent or reduce A/H5N1-associated acute lung injury in vivo. This potential adjunctive therapy for severe influenza-induced lung disease warrants rapid clinical investigation.

  11. Total lymphatic irradiation and bone marrow in human heart transplantation

    International Nuclear Information System (INIS)

    Kahn, D.R.; Hong, R.; Greenberg, A.J.; Gilbert, E.F.; Dacumos, G.C.; Dufek, J.H.

    1984-01-01

    Six patients, aged 36 to 59 years, had heart transplants for terminal myocardial disease using total lymphatic irradiation (TLI) and donor bone marrow in addition to conventional therapy. All patients were poor candidates for transplantation because of marked pulmonary hypertension, unacceptable tissue matching, or age. Two patients are living and well more than four years after the transplants. Two patients died of infection at six and seven weeks with normal hearts. One patient, whose preoperative pulmonary hypertension was too great for an orthotopic heart transplant, died at 10 days after such a procedure. The other patient died of chronic rejection seven months postoperatively. Donor-specific tolerance developed in 2 patients. TLI and donor bone marrow can produce specific tolerance to donor antigens and allow easy control of rejection, but infection is still a major problem. We describe a new technique of administering TLI with early reduction of prednisone that may help this problem

  12. Quality Control Assays for Clinical-Grade Human Mesenchymal Stromal Cells: Methods for ATMP Release.

    Science.gov (United States)

    Radrizzani, Marina; Soncin, Sabrina; Lo Cicero, Viviana; Andriolo, Gabriella; Bolis, Sara; Turchetto, Lucia

    2016-01-01

    Mesenchymal stromal/stem cells (MSC) are promising candidates for the development of cell-based therapies for various diseases and are currently being evaluated in a number of clinical trials (Sharma et al., Transfusion 54:1418-1437, 2014; Ikebe and Suzuki, Biomed Res Int 2014:951512, 2014). MSC for therapeutic applications are classified as advanced therapy medicinal products (ATMP) (Regulation (EC) No 1394/2007 of the European Parliament and of the Council of 13 November 2007 on advanced therapy medicinal products and amending Directive 2001/83/EC and Regulation (EC) No 726/2004) and must be prepared according to good manufacturing practices ( http://ec.europa.eu/health/documents/eudralex/vol-4 ). They may be derived from different starting materials (mainly bone marrow (BM), adipose tissue, or cord blood) and applied as fresh or cryopreserved products, in the autologous as well as an allogeneic context (Sharma et al., Transfusion 54:1418-1437, 2014; Ikebe and Suzuki, Biomed Res Int 2014:951512, 2014; Sensebé and Bourin, Transplantation 87(9 Suppl):S49-S53, 2009). In any case, they require an approved and well-defined panel of assays in order to be released for clinical use.This chapter describes analytical methods implemented and performed in our cell factory as part of the release strategy for an ATMP consisting of frozen autologous BM-derived MSC. Such methods are designed to assess the safety (sterility, endotoxin, and mycoplasma assays) and identity/potency (cell count and viability, immunophenotype and clonogenic assay) of the final product. Some assays are also applied to the biological starting material (sterility) or carried out as in-process controls (sterility, cell count and viability, immunophenotype, clonogenic assay).The validation strategy for each analytical method is described in the accompanying Chapter 20 .

  13. Inhibiting actin depolymerization enhances osteoblast differentiation and bone formation in human stromal stem cells

    DEFF Research Database (Denmark)

    Chen, Li; Shi, Kaikai; Frary, Charles

    2015-01-01

    Remodeling of the actin cytoskeleton through actin dynamics is involved in a number of biological processes, but its role in human stromal (skeletal) stem cells (hMSCs) differentiation is poorly understood. In the present study, we demonstrated that stabilizing actin filaments by inhibiting gene...... expression of the two main actin depolymerizing factors (ADFs): Cofilin 1 (CFL1) and Destrin (DSTN) in hMSCs, enhanced cell viability and differentiation into osteoblastic cells (OB) in vitro, as well as heterotopic bone formation in vivo. Similarly, treating hMSC with Phalloidin, which is known to stabilize...... polymerized actin filaments, increased hMSCs viability and OB differentiation. Conversely, Cytocholasin D, an inhibitor of actin polymerization, reduced cell viability and inhibited OB differentiation of hMSC. At a molecular level, preventing Cofilin phosphorylation through inhibition of LIM domain kinase 1...

  14. Human adipose-derived stromal/stem cell isolation, culture, and osteogenic differentiation.

    Science.gov (United States)

    Qureshi, Ammar T; Chen, Cong; Shah, Forum; Thomas-Porch, Caasy; Gimble, Jeffrey M; Hayes, Daniel J

    2014-01-01

    Annually, more than 200,000 elective liposuction procedures are performed in the United States and over a million worldwide. The ease of harvest and abundance make human adipose-derived stromal/stem cells (hASCs) isolated from lipoaspirates an attractive, readily available source of adult stem cells that have become increasingly popular for use in many studies. Here, we describe common methods for hASC culture, preservation, and osteogenic differentiation. We introduce methods of ceramic, polymer, and composite scaffold synthesis with a description of morphological, chemical, and mechanical characterization techniques. Techniques for scaffold loading are compared, and methods for determining cell loading efficiency and proliferation are described. Finally, we provide both qualitative and quantitative techniques for in vitro assessment of hASC osteogenic differentiation. © 2014 Elsevier Inc. All rights reserved.

  15. [Progesterone Promotes Human Bone Marrow Mesenchymal Stem Cells to Synthesize Fibronectin via ERK Pathway].

    Science.gov (United States)

    Wu, Zhen-Yong; Chen, Jing-Li; Huang, Shu; Zhang, Hui; Wang, Fang; Wang, Yan; Bi, Xiao-Yun; Guo, Zi-Kuan

    2015-12-01

    To investigate whether the progesterone can promote fibronection (FN) synthesis by human bone marrow mesenchymal stem cells (MSCs) and to explore the potential underlying mechanism. The human bone marrow MSCs were cultured in a serum-free medium with progesterone for 72 hours, the MTT test was performed to observe the proliferation status and adhension ability of the treated cells. Western blot was used to detect the content of FN in MSDs with GAPDH as the internal reference, the phosphorylation of ERK1/2, as well as the FN content in MSC treated by PD98059, a specific inhibitor of ERK1/2. The progesterone at a range of certain doses not effect on the proliferation of human bone marrow MSCs. Progesterone (25 µg/L) treatment enhanced the FN expression and adherent ability of marrow MSCs. Progesterone could induce prompt phosphorylation of ERK 1/2 and its promoting effects on FN synthesis was reversed by PD98059. The progesterone can promote FN synthesis by human bone marrow MSCs via ERK 1/2 pathway, and it might be used to culture MSCs in serum-free medium.

  16. In vitro differentiation of neural cells from human adipose tissue derived stromal cells.

    Science.gov (United States)

    Dave, Shruti D; Patel, Chetan N; Vanikar, Aruna V; Trivedi, Hargovind L

    2018-01-01

    Stem cells, including neural stem cells (NSCs), are endowed with self-renewal capability and hence hold great opportunity for the institution of replacement/protective therapy. We propose a method for in vitro generation of stromal cells from human adipose tissue and their differentiation into neural cells. Ten grams of donor adipose tissue was surgically resected from the abdominal wall of the human donor after the participants' informed consents. The resected adipose tissue was minced and incubated for 1 hour in the presence of an enzyme (collagenase-type I) at 37 0 C followed by its centrifugation. After centrifugation, the supernatant and pellets were separated and cultured in a medium for proliferation at 37 0 C with 5% CO2 for 9-10 days in separate tissue culture dishes for generation of mesenchymal stromal cells (MSC). At the end of the culture, MSC were harvested and analyzed. The harvested MSC were subjected for further culture for their differentiation into neural cells for 5-7 days using differentiation medium mainly comprising of neurobasal medium. At the end of the procedure, culture cells were isolated and studied for expression of transcriptional factor proteins: orthodenticle homolog-2 (OTX-2), beta-III-tubulin (β3-Tubulin), glial-fibrillary acid protein (GFAP) and synaptophysin-β2. In total, 50 neural cells-lines were generated. In vitro generated MSC differentiated neural cells' mean quantum was 5.4 ± 6.9 ml with the mean cell count being, 5.27 ± 2.65 × 10 3/ μl. All of them showed the presence of OTX-2, β3-Tubulin, GFAP, synaptophysin-β2. Neural cells can be differentiated in vitro from MSC safely and effectively. In vitro generated neural cells represent a potential therapy for recovery from spinal cord injuries and neurodegenerative disease.

  17. Mesenchymal stromal cells improve human islet function through released products and extracellular matrix.

    Science.gov (United States)

    Arzouni, Ahmed A; Vargas-Seymour, Andreia; Rackham, Chloe L; Dhadda, Paramjeet; Huang, Guo-Cai; Choudhary, Pratik; Nardi, Nance; King, Aileen J F; Jones, Peter M

    2017-12-01

    The aims of the present study were (i) to determine whether the reported beneficial effects of mesenchymal stromal cells (MSCs) on mouse islet function extend to clinically relevant human tissues (islets and MSCs), enabling translation into improved protocols for clinical human islet transplantation; and (ii) to identify possible mechanisms through which human MSCs influence human islet function. Human islets were co-cultured with human adipose tissue-derived MSCs (hASCs) or pre-treated with its products - extracellular matrix (ECM) and annexin A1 (ANXA1). Mouse islets were pre-treated with mouse MSC-derived ECM. Islet insulin secretory function was assessed in vitro by radioimmunoassay. Quantitative RT-PCR was used to screen human adipMSCs for potential ligands of human islet G-protein-coupled receptors. We show that co-culture with hASCs improves human islet secretory function in vitro , as measured by glucose-stimulated insulin secretion, confirming previous reports using rodent tissues. Furthermore, we demonstrate that these beneficial effects on islet function can be partly attributed to the MSC-derived products ECM and ANXA1. Our results suggest that hASCs have the potential to improve the quality of human islets isolated for transplantation therapy of Type 1 diabetes. Furthermore, it may be possible to achieve improvements in human islet quality in a cell-free culture system by using the MSC-derived products ANXA1 and ECM. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  18. The role of human umbilical cord tissue-derived mesenchymal stromal cells (UCX® in the treatment of inflammatory arthritis

    Directory of Open Access Journals (Sweden)

    Santos Jorge M

    2013-01-01

    Full Text Available Abstract Background ECBio has developed proprietary technology to consistently isolate, expand and cryopreserve a well-characterized population of stromal cells from human umbilical cord tissue (UCX® cells. The technology has recently been optimized in order to become compliant with Advanced Medicine Therapeutic Products. In this work we report the immunosuppressive capacity of UCX® cells for treating induced autoimmune inflammatory arthritis. Methods UCX® cells were isolated using a proprietary method (PCT/IB2008/054067 that yields a well-defined number of cells using a precise proportion between tissue digestion enzyme activity units, tissue mass, digestion solution volume and void volume. The procedure includes three recovery steps to avoid non-conformities related to cell recovery. UCX® surface markers were characterized by flow cytometry and UCX® capacity to expand in vitro and to differentiate into adipocyte, chondrocyte and osteoblast-like cells was evaluated. Mixed Lymphocyte Reaction (MLR assays were performed to evaluate the effect of UCX® cells on T-cell activation and Treg conversion assays were also performed in vitro. Furthermore, UCX® cells were administered in vivo in both a rat acute carrageenan-induced arthritis model and rat chronic adjuvant induced arthritis model for arthritic inflammation. UCX® anti-inflammatory activity was then monitored over time. Results UCX® cells stained positive for CD44, CD73, CD90 and CD105; and negative for CD14, CD19 CD31, CD34, CD45 and HLA-DR; and were capable to differentiate into adipocyte, chondrocyte and osteoblast-like cells. UCX® cells were shown to repress T-cell activation and promote the expansion of Tregs better than bone marrow mesenchymal stem cells (BM-MSCs. Accordingly, xenogeneic UCX® administration in an acute carrageenan-induced arthritis model showed that human UCX® cells can reduce paw edema in vivo more efficiently than BM-MSCs. Finally, in a chronic adjuvant

  19. Human endometrial stromal stem cells differentiate into megakaryocytes with the ability to produce functional platelets.

    Directory of Open Access Journals (Sweden)

    Jinju Wang

    Full Text Available Human endometrium is a high dynamic tissue that contains endometrial stromal stem cells (hESSCs. The hESSCs have been differentiated into a number of cell lineages. However, differentiation of hESSCs into megakaryocytes (MKs has not yet been investigated. The aim of this study was to investigate the feasibility of MK generation from hESSCs and subsequent production of functional platelets (PLTs. In our study, hESSCs were cultured from endometrial stromal cells as confirmed by positive stromal cell specific markers (CD90 and CD29 and negative hematopoietic stem cell markers (CD45 and CD34 expression. Then, hESSCs were differentiated in a medium supplemented with thrombopoietin (TPO for 18 days. The MK differentiation was analyzed by flow cytometry and confocal microscopy. The differentiation medium was collected for PLT production analysis by flow cytometry, transmission electron microscopy and functional measurements. Our results show: 1 MKs were successfully generated from hESSCs as identified by expression of specific markers (CD41a: 1 ± 0.09% and 39 ± 3.0%; CD42b: 1.2 ± 0.06% and 28 ± 2.0%, control vs. differentiation accompanied with reduction of pluripotent transcription factors (Oct4 and Sox2 expression; 2 The level of PLTs in the differentiation medium was 16 ± 1 number/µl as determined by size (2-4 µm and CD41a expression (CD41a: 1 ± 0.4% and 90±2.0%, control vs. differentiation; 3 Generated PLTs were functional as evidenced by the up-regulation of CD62p expression and fibrinogen binding following thrombin stimulation; 4 Released PLTs showed similar ultra-structure characteristics (alpha granules, vacuoles and dense tubular system as PLTs from peripheral blood determined by electron microscopic analysis. Data demonstrate the feasibility of generating MKs from hESSCs, and that the generated MKs release functional PLTs. Therefore, hESSCs could be a potential new stem cell source for in vitro MK/PLT production.

  20. Hypoxic stress simultaneously stimulates vascular endothelial growth factor via hypoxia-inducible factor-1α and inhibits stromal cell-derived factor-1 in human endometrial stromal cells.

    Science.gov (United States)

    Tsuzuki, Tomoko; Okada, Hidetaka; Cho, Hisayuu; Tsuji, Shoko; Nishigaki, Akemi; Yasuda, Katsuhiko; Kanzaki, Hideharu

    2012-02-01

    Hypoxia of the human endometrium is a physiologic event occurring during the perimenstrual period and the local stimulus for angiogenesis. The aim of this study was to investigate the effects of hypoxic stress on the regulation of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1/CXCL12), and the potential role of hypoxia-inducible factor-1α (HIF-1α) in the endometrium. Human endometrial stromal cells (ESCs, n= 22 samples) were studied in vitro. ESCs were cultured under hypoxic and normoxic conditions and treated with cobalt chloride (CoCl₂; a hypoxia-mimicking agent) and/or echinomycin, a small-molecule inhibitor of HIF-1α activity. The mRNA levels and production of VEGF and SDF-1 were assessed by real-time PCR and ELISA, respectively. The HIF-1α protein levels were measured using western blot analysis. Hypoxia simultaneously induced the expression of mRNA and production of VEGF and attenuated the expression and production of SDF-1 from ESCs in a time-dependent manner. Similar changes were observed in the ESCs after stimulation with CoCl₂ in a dose-dependent manner. CoCl₂ significantly induced the expression of HIF-1α protein, and its highest expression was observed at 6 h. Echinomycin inhibited hypoxia-induced VEGF production without affecting the HIF-1α protein level and cell toxicity and had no effect on SDF-1 secretion (P hypoxic conditions that could influence angiogenesis in the human endometrium.

  1. Survival of Free and Encapsulated Human and Rat Islet Xenografts Transplanted into the Mouse Bone Marrow

    Science.gov (United States)

    Meier, Raphael P. H.; Seebach, Jörg D.; Morel, Philippe; Mahou, Redouan; Borot, Sophie; Giovannoni, Laurianne; Parnaud, Geraldine; Montanari, Elisa; Bosco, Domenico; Wandrey, Christine; Berney, Thierry; Bühler, Leo H.; Muller, Yannick D.

    2014-01-01

    Bone marrow was recently proposed as an alternative and potentially immune-privileged site for pancreatic islet transplantation. The aim of the present study was to assess the survival and rejection mechanisms of free and encapsulated xenogeneic islets transplanted into the medullary cavity of the femur, or under the kidney capsule of streptozotocin-induced diabetic C57BL/6 mice. The median survival of free rat islets transplanted into the bone marrow or under the kidney capsule was 9 and 14 days, respectively, whereas that of free human islets was shorter, 7 days (bone marrow) and 10 days (kidney capsule). Infiltrating CD8+ T cells and redistributed CD4+ T cells, and macrophages were detected around the transplanted islets in bone sections. Recipient mouse splenocytes proliferated in response to donor rat stimulator cells. One month after transplantation under both kidney capsule or into bone marrow, encapsulated rat islets had induced a similar degree of fibrotic reaction and still contained insulin positive cells. In conclusion, we successfully established a small animal model for xenogeneic islet transplantation into the bone marrow. The rejection of xenogeneic islets was associated with local and systemic T cell responses and macrophage recruitment. Although there was no evidence for immune-privilege, the bone marrow may represent a feasible site for encapsulated xenogeneic islet transplantation. PMID:24625569

  2. Survival of free and encapsulated human and rat islet xenografts transplanted into the mouse bone marrow.

    Directory of Open Access Journals (Sweden)

    Raphael P H Meier

    Full Text Available Bone marrow was recently proposed as an alternative and potentially immune-privileged site for pancreatic islet transplantation. The aim of the present study was to assess the survival and rejection mechanisms of free and encapsulated xenogeneic islets transplanted into the medullary cavity of the femur, or under the kidney capsule of streptozotocin-induced diabetic C57BL/6 mice. The median survival of free rat islets transplanted into the bone marrow or under the kidney capsule was 9 and 14 days, respectively, whereas that of free human islets was shorter, 7 days (bone marrow and 10 days (kidney capsule. Infiltrating CD8+ T cells and redistributed CD4+ T cells, and macrophages were detected around the transplanted islets in bone sections. Recipient mouse splenocytes proliferated in response to donor rat stimulator cells. One month after transplantation under both kidney capsule or into bone marrow, encapsulated rat islets had induced a similar degree of fibrotic reaction and still contained insulin positive cells. In conclusion, we successfully established a small animal model for xenogeneic islet transplantation into the bone marrow. The rejection of xenogeneic islets was associated with local and systemic T cell responses and macrophage recruitment. Although there was no evidence for immune-privilege, the bone marrow may represent a feasible site for encapsulated xenogeneic islet transplantation.

  3. Evaluation of umbilical cord blood CD34+ hematopoietic stem cells expansion with inhibition of TGF-β receptorII in co-culture with bone marrow mesenchymal stromal cells.

    Science.gov (United States)

    Sohrabi Akhkand, Saman; Amirizadeh, Naser; Nikougoftar, Mahin; Alizadeh, Javad; Zaker, Farhad; Sarveazad, Arash; Joghataei, Mohammad Taghi; Faramarzi, Mahmood

    2016-08-01

    Umbilical cord blood (UCB) is an important source of hematopoietic stem cells (HSCs). However, low number of HSCs in UCB has been an obstacle for adult hematopoietic stem cell transplantation. The expansion of HSCs in culture is one approach to overcome this problem. In this study, we investigated the expansion of UCB-HSCs by using human bone marrow mesenchymal stromal cells (MSCs) as feeder layer as well as inhibiting the TGF-β signaling pathway through reduction of TGF-βRII expression. CD34(+) cells were isolated from UCB and transfected by SiRNA targeting TGF-βRII mRNA. CD34(+) cells were expanded in four culture media with different conditions, including 1) expansion of CD34(+) cells in serum free medium containing growth factors, 2) expansion of cells transfected with SiRNA targeting TGF-βRII in medium containing growth factors, 3) expansion of cells in presence of growth factors and MSCs, 4) expansion of cells transfected with SiRNA targeting TGF-βRII on MSCs feeder layer in medium containing growth factors. These culture conditions were evaluated for the number of total nucleated cells (TNCs), CD34 surface marker as well as using CFU assay on 8th day after culture. The fold increase in CD34(+) cells, TNCs, and colony numbers (71.8±6.9, 93.2±10.2 and 128±10, respectively) was observed to be highest in fourth culture medium compared to other culture conditions. The difference between number of cells in four culture media in 8th day compared to unexpanded cells (0day) before expansion was statistically significant (P<0.05). The results showed that transfection of CD34(+) cells with SiRNA targeting TGF-βRII and their co-culture with MSCs could considerably increase the number of progenitors. Therefore, this method could be useful for UCB-HSCs expansion. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Automated processing of human bone marrow grafts for transplantation.

    Science.gov (United States)

    Zingsem, J; Zeiler, T; Zimmermanm, R; Weisbach, V; Mitschulat, H; Schmid, H; Beyer, J; Siegert, W; Eckstein, R

    1993-01-01

    Prior to purging or cryopreservation, we concentrated 21 bone marrow (BM) harvests using a modification of the 'grancollect-protocol' of the Fresenius AS 104 cell separator with the P1-Y set. Within 40-70 min, the initial marrow volume of 1,265 ml (+/- 537 ml) was processed two to three times. A mean of 47% (+/- 21%) of the initial mononuclear cells was recovered in a mean volume of 128 ml (+36 ml). The recovery of clonogenic cells, measured by CFU-GM assays, was 68% (+/- 47%). Red blood cells in the BM concentrates were reduced to 7% (+/- 4%) of the initial number. The procedure was efficient and yielded a BM cell fraction suitable for purging, cryopreservation and transplantation. At this time, 10 of the 21 patients whose BM was processed using this technique have been transplanted. Seven of these 10 patients have been grafted using the BM alone. Three of the 10 patients showed reduced cell viability and colony growth in the thawed BM samples, and therefore obtained BM and peripheral blood-derived stem cells. All transplanted patients showed an evaluable engraftment, achieving 1,000 granulocytes per microliter of peripheral blood in a mean of 18 days.

  5. Human and Autologous Adipose-derived Stromal Cells Increase Flap Survival in Rats Independently of Host Immune Response

    DEFF Research Database (Denmark)

    Toyserkani, Navid Mohamadpour; Jensen, Charlotte Harken; Andersen, Ditte Caroline

    2018-01-01

    evaluated after 7 days. RESULTS: The mean survival rates for SVF treatment regardless of human or autologous origin were significantly increased as compared with the control group. Adipose stem/stromal cell and SVF lysate injection did not increase flap survival. Vessel density was increased for human...... injections lead to increased vessel density, but it did not necessarily lead to increased flap survival. Further research should elaborate which molecular events make SVF treatment more efficacious than ASC....

  6. Impaired Angiogenic Potential of Human Placental Mesenchymal Stromal Cells in Intrauterine Growth Restriction.

    Science.gov (United States)

    Mandò, Chiara; Razini, Paola; Novielli, Chiara; Anelli, Gaia Maria; Belicchi, Marzia; Erratico, Silvia; Banfi, Stefania; Meregalli, Mirella; Tavelli, Alessandro; Baccarin, Marco; Rolfo, Alessandro; Motta, Silvia; Torrente, Yvan; Cetin, Irene

    2016-04-01

    Human placental mesenchymal stromal cells (pMSCs) have never been investigated in intrauterine growth restriction (IUGR). We characterized cells isolated from placental membranes and the basal disc of six IUGR and five physiological placentas. Cell viability and proliferation were assessed every 7 days during a 6-week culture. Expression of hematopoietic, stem, endothelial, and mesenchymal markers was evaluated by flow cytometry. We characterized the multipotency of pMSCs and the expression of genes involved in mitochondrial content and function. Cell viability was high in all samples, and proliferation rate was lower in IUGR compared with control cells. All samples presented a starting heterogeneous population, shifting during culture toward homogeneity for mesenchymal markers and occurring earlier in IUGR than in controls. In vitro multipotency of IUGR-derived pMSCs was restricted because their capacity for adipocyte differentiation was increased, whereas their ability to differentiate toward endothelial cell lineage was decreased. Mitochondrial content and function were higher in IUGR pMSCs than controls, possibly indicating a shift from anaerobic to aerobic metabolism, with the loss of the metabolic characteristics that are typical of undifferentiated multipotent cells. This study demonstrates that the loss of endothelial differentiation potential and the increase of adipogenic ability are likely to play a significant role in the vicious cycle of abnormal placental development in intrauterine growth restriction (IUGR). This is the first observation of a potential role for placental mesenchymal stromal cells in intrauterine growth restriction, thus leading to new perspectives for the treatment of IUGR. ©AlphaMed Press.

  7. Regulating Chondrogenesis of Human Mesenchymal Stromal Cells with a Retinoic Acid Receptor-Beta Inhibitor: Differential Sensitivity of Chondral Versus Osteochondral Development

    Directory of Open Access Journals (Sweden)

    Solvig Diederichs

    2014-05-01

    Full Text Available Aim: Main objective was to investigate whether the synthetic retinoic acid receptor (RAR-β antagonist LE135 is able to drive in vitro chondrogenesis of human mesenchymal stromal cells (MSCs or improve differentiation by suppressing hypertrophic chondrocyte development. Methods: Chondrogenesis of human bone marrow and adipose tissue-derived MSCs was induced in micromass pellet culture for six weeks. Effects of LE135 alone and in combinatorial treatment with TGF-β on deposition of cartilaginous matrix including collagen type II and glycosaminoglycans, on deposition of non-hyaline cartilage collagens type I and X, and on hypertrophy markers including alkaline phosphatase (ALP, indian hedghehog (IHH and matrix metalloproteinase (MMP-13 were assessed. Results: LE135 was no inducer of chondrogenesis and failed to stimulate deposition of collagen type II and glycosaminoglycans. Moreover, addition of LE135 to TGF-β-treated pellets inhibited cartilaginous matrix deposition and gene expression of COL2A1. In contrast, non-hyaline cartilage collagens were less sensitive to LE135 and hypertrophy markers remained unaffected. Conclusion: This demonstrates a differential sensitivity of chondral versus endochondral differentiation pathways to RARβ signaling; however, opposite to the desired direction. The relevance of trans-activating versus trans-repressing RAR signaling, including effects on activator protein (AP-1 is discussed and implications for overcoming current limits of hMSC chondrogenesis are considered.

  8. Regulating chondrogenesis of human mesenchymal stromal cells with a retinoic Acid receptor-Beta inhibitor: differential sensitivity of chondral versus osteochondral development.

    Science.gov (United States)

    Diederichs, Solvig; Zachert, Kerstin; Raiss, Patric; Richter, Wiltrud

    2014-01-01

    Main objective was to investigate whether the synthetic retinoic acid receptor (RAR)-β antagonist LE135 is able to drive in vitro chondrogenesis of human mesenchymal stromal cells (MSCs) or improve differentiation by suppressing hypertrophic chondrocyte development. Chondrogenesis of human bone marrow and adipose tissue-derived MSCs was induced in micromass pellet culture for six weeks. Effects of LE135 alone and in combinatorial treatment with TGF-β on deposition of cartilaginous matrix including collagen type II and glycosaminoglycans, on deposition of non-hyaline cartilage collagens type I and X, and on hypertrophy markers including alkaline phosphatase (ALP), indian hedghehog (IHH) and matrix metalloproteinase (MMP)-13 were assessed. LE135 was no inducer of chondrogenesis and failed to stimulate deposition of collagen type II and glycosaminoglycans. Moreover, addition of LE135 to TGF-β-treated pellets inhibited cartilaginous matrix deposition and gene expression of COL2A1. In contrast, non-hyaline cartilage collagens were less sensitive to LE135 and hypertrophy markers remained unaffected. This demonstrates a differential sensitivity of chondral versus endochondral differentiation pathways to RARβ signaling; however, opposite to the desired direction. The relevance of trans-activating versus trans-repressing RAR signaling, including effects on activator protein (AP)-1 is discussed and implications for overcoming current limits of hMSC chondrogenesis are considered. © 2014 S. Karger AG, Basel.

  9. Progestins inhibit estradiol-induced vascular endothelial growth factor and stromal cell-derived factor 1 in human endometrial stromal cells.

    Science.gov (United States)

    Okada, Hidetaka; Okamoto, Rika; Tsuzuki, Tomoko; Tsuji, Shoko; Yasuda, Katsuhiko; Kanzaki, Hideharu

    2011-09-01

    To investigate whether 17β-estradiol (E(2)) and progestins exert direct effects on vascular endothelial growth factor (VEGF) and stromal cell-derived factor 1 (SDF-1/CXCL12) in human endometrial stromal cells (ESCs) and thereby to clarify the regulatory function of these local angiogenic factors in the endometrium. In vitro experiment. Research laboratory at Kansai Medical University. Fourteen patients undergoing hysterectomy for benign reasons. ESCs were cultured with E(2) and/or various clinically relevant progestins (medroxyprogesterone acetate [MPA], norethisterone [NET], levonorgestrel [LNG], dienogest [DNG], and progesterone [P]). The mRNA levels and production of VEGF and SDF-1 were assessed by real-time reverse-transcription polymerase chain reaction and ELISA, respectively. E(2) significantly induced the mRNA levels and protein production of VEGF and SDF-1 in ESCs. MPA could antagonize the E(2)-stimulated effects in a time- and dose-dependent manner, and this effect could be reversed by RU-486 (P receptor antagonist). All of the progestins (MPA, NET, LNG, and DNG; 10(-9) to 10(-7) mol/L) attenuated E(2)-induced VEGF and SDF-1 production, whereas P showed these inhibitory effects only when present in a high concentration (10(-7) mol/L). Progestins have inhibitory effects on E(2)-induced VEGF and SDF-1 in ESCs. These results may indicate a potential mechanism for action of the female sex steroids in the human endometrium that can be helpful for various clinical applications. Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  10. Cell shape and spreading of stromal (mesenchymal) stem cells cultured on fibronectin coated gold and hydroxyapatite surfaces

    DEFF Research Database (Denmark)

    Dolatshahi-Pirouz, A; Jensen, Thomas Hartvig Lindkjær; Kolind, Kristian

    2011-01-01

    In order to identify the cellular mechanisms leading to the biocompatibility of hydroxyapatite implants, we studied the interaction of human bone marrow derived stromal (mesenchymal) stem cells (hMSCs) with fibronectin-coated gold (Au) and hydroxyapatite (HA) surfaces. The adsorption of fibronectin...

  11. Cannabidiol Activates Neuronal Precursor Genes in Human Gingival Mesenchymal Stromal Cells.

    Science.gov (United States)

    Soundara Rajan, Thangavelu; Giacoppo, Sabrina; Scionti, Domenico; Diomede, Francesca; Grassi, Gianpaolo; Pollastro, Federica; Piattelli, Adriano; Bramanti, Placido; Mazzon, Emanuela; Trubiani, Oriana

    2017-06-01

    In the last years, mesenchymal stromal cells (MSCs) from oral tissues have received considerable interest in regenerative medicine since they can be obtained with minimal invasive procedure and exhibit immunomodulatory properties. This study was aimed to investigate whether in vitro pre-treatment of MSCs obtained from human gingiva (hGMSCs) with Cannabidiol (CBD), a cannabinoid component produced by the plant Cannabis sativa, may promote human gingiva derived MSCs to differentiate toward neuronal precursor cells. Specifically, we have treated the hGMSCs with CBD (5 µM) for 24 h in order to evaluate the expression of genes involved in cannabidiol signaling, cell proliferation, self-renewal and multipotency, and neural progenitor cells differentiation. Next generation sequencing (NGS) demonstrated that CBD activates genes associated with G protein coupled receptor signaling in hGMSCs. Genes involved in DNA replication, cell cycle, proliferation, and apoptosis were regulated. Moreover, genes associated with the biological process of neuronal progenitor cells (NCPs) proliferation, neuron differentiation, neurogenesis, and nervous system development were significantly modulated. From our results, we hypothesize that human gingiva-derived MSCs conditioned with CBD could represent a valid method for improving the hGMSCs phenotype and thus might be a potential therapeutic tool in the treatment of neurodegenerative diseases. J. Cell. Biochem. 118: 1531-1546, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  12. Data on isolating mesenchymal stromal cells from human adipose tissue using a collagenase-free method

    Directory of Open Access Journals (Sweden)

    Wassim Shebaby

    2016-03-01

    Full Text Available The present dataset describes a detailed protocol to isolate mesenchymal cells from human fat without the use of collagenase. Human fat specimen, surgically cleaned from non-fat tissues (e.g., blood vessels and reduced into smaller fat pieces of around 1–3 mm size, is incubated in complete culture media for five to seven days. Then, cells started to spread out from the fat explants and to grow in cultures according to an exponential pattern. Our data showed that primary mesenchymal cells presenting heterogeneous morphology start to acquire more homogenous fibroblastic-like shape when cultured for longer duration or when subcultured into new flasks. Cell isolation efficiency as well as cell doubling time were also calculated throughout the culturing experimentations and illustrated in a separate figure thereafter. This paper contains data previously considered as an alternative protocol to isolate adipose-derived mesenchymal stem cell published in “Proliferation and differentiation of human adipose-derived mesenchymal stem cells (ASCs into osteoblastic lineage are passage dependent” [1]. Keywords: Adipose tissue, mesenchymal stromal cell, cell culture, doubling time

  13. Immune Humanization of Immunodeficient Mice Using Diagnostic Bone Marrow Aspirates from Carcinoma Patients

    Science.gov (United States)

    Werner-Klein, Melanie; Proske, Judith; Werno, Christian; Schneider, Katharina; Hofmann, Hans-Stefan; Rack, Brigitte; Buchholz, Stefan; Ganzer, Roman; Blana, Andreas; Seelbach-Göbel, Birgit; Nitsche, Ulrich

    2014-01-01

    Tumor xenografts in immunodeficient mice, while routinely used in cancer research, preclude studying interactions of immune and cancer cells or, if humanized by allogeneic immune cells, are of limited use for tumor-immunological questions. Here, we explore a novel way to generate cancer models with an autologous humanized immune system. We demonstrate that hematopoietic stem and progenitor cells (HSPCs) from bone marrow aspirates of non-metastasized carcinoma patients, which are taken at specialized centers for diagnostic purposes, can be used to generate a human immune system in NOD-scid IL2rγ(null) (NSG) and HLA-I expressing NSG mice (NSG-HLA-A2/HHD) comprising both, lymphoid and myeloid cell lineages. Using NSG-HLA-A2/HHD mice, we show that responsive and self-tolerant human T cells develop and human antigen presenting cells can activate human T cells. As critical factors we identified the low potential of bone marrow HSPCs to engraft, generally low HSPC numbers in patient-derived bone marrow samples, cryopreservation and routes of cell administration. We provide here an optimized protocol that uses a minimum number of HSPCs, preselects high-quality bone marrow samples defined by the number of initially isolated leukocytes and intra-femoral or intra-venous injection. In conclusion, the use of diagnostic bone marrow aspirates from non-metastasized carcinoma patients for the immunological humanization of immunodeficient mice is feasible and opens the chance for individualized analyses of anti-tumoral T cell responses. PMID:24830425

  14. Immune humanization of immunodeficient mice using diagnostic bone marrow aspirates from carcinoma patients.

    Directory of Open Access Journals (Sweden)

    Melanie Werner-Klein

    Full Text Available Tumor xenografts in immunodeficient mice, while routinely used in cancer research, preclude studying interactions of immune and cancer cells or, if humanized by allogeneic immune cells, are of limited use for tumor-immunological questions. Here, we explore a novel way to generate cancer models with an autologous humanized immune system. We demonstrate that hematopoietic stem and progenitor cells (HSPCs from bone marrow aspirates of non-metastasized carcinoma patients, which are taken at specialized centers for diagnostic purposes, can be used to generate a human immune system in NOD-scid IL2rγ(null (NSG and HLA-I expressing NSG mice (NSG-HLA-A2/HHD comprising both, lymphoid and myeloid cell lineages. Using NSG-HLA-A2/HHD mice, we show that responsive and self-tolerant human T cells develop and human antigen presenting cells can activate human T cells. As critical factors we identified the low potential of bone marrow HSPCs to engraft, generally low HSPC numbers in patient-derived bone marrow samples, cryopreservation and routes of cell administration. We provide here an optimized protocol that uses a minimum number of HSPCs, preselects high-quality bone marrow samples defined by the number of initially isolated leukocytes and intra-femoral or intra-venous injection. In conclusion, the use of diagnostic bone marrow aspirates from non-metastasized carcinoma patients for the immunological humanization of immunodeficient mice is feasible and opens the chance for individualized analyses of anti-tumoral T cell responses.

  15. Selective inhibition of B lymphocytes in TBTC-treated human bone marrow long-term culture.

    NARCIS (Netherlands)

    Carfi', M.; Bowe, G.; Pieters, R.; Gribaldo, L.

    2010-01-01

    Tributyltin chloride (TBTC) is well known for its immunotoxic effect, in particular towards immature thymocytes. TBTC is also known to induce adipocyte differentiation in primary human bone marrow cultures, which is reflected in the decrease in a number of adipocyte-derived cytokines, chemokines and

  16. The Human Figure Drawing with Donor and Nondonor Siblings of Pediatric Bone Marrow Transplant Patients.

    Science.gov (United States)

    Packman, Wendy L.; Beck, Vanessa L.; VanZutphen, Kelly H.; Long, Janet K.; Spengler, Gisele

    2003-01-01

    There is little research on the psychological impact of bone marrow transplantation (BMT) on family members. This study uses the Human Figure Drawing (HFD) to measure siblings' emotional distress toward BMT. Among the siblings, feelings of isolation, anger, depression, anxiety, and low self-esteem emerged as major themes. Findings indicate the…

  17. Mesenchymal Stromal Cells from Osteoarthritic Synovium Are a Distinct Population Compared to Their Bone-Marrow Counterparts regarding Surface Marker Distribution and Immunomodulation of Allogeneic CD4+ T-Cell Cultures

    Directory of Open Access Journals (Sweden)

    Sebastien Hagmann

    2016-01-01

    Full Text Available Introduction. The participation of an inflammatory joint milieu has been described in osteoarthritis (OA pathogenesis. Mesenchymal stromal cells (MSCs play an important role in modulating inflammatory processes. Based on previous studies in an allogeneic T-cell coculture model, we aimed at further determining the role of synovial MSCs in OA pathogenesis. Methods. Bone-marrow (BM and synovial membrane (SM MSCs from hip joints of late stage OA patients and CD4+ T-cells from healthy donors were analysed regarding surface marker expression before and after coculture. Proliferation upon CD3/CD28 stimulation and cytokine analyses were compared between MSCs. Results. SM-MSCs differed from BM-MSCs in several surface markers and their osteogenic differentiation potential. Cocultures of both MSCs with CD4+ T-cells resulted in recruitment of CD45RA+ FoxP3+ regulatory T-cells. Upon stimulation, only SM-MSCs suppressed CD4+ T-cell proliferation, while both SM-MSCs and BM-MSCs modified cytokine profiles through suppressing IL-2 and TNF-α as well as increasing IL-6 secretion. Conclusions. Synovial MSCs from OA joints are a unique fraction that can be distinguished from their bone-marrow derived counterparts. Their unique ability to suppress CD3/CD28 induced CD4+ T-cell proliferation makes them a potential target for future therapeutic approaches.

  18. Octanoate in Human Albumin Preparations Is Detrimental to Mesenchymal Stromal Cell Culture

    Directory of Open Access Journals (Sweden)

    Way-Wua Wong

    2015-01-01

    Full Text Available Cell therapies hold great promise as the next major advance in medical treatment. To enable safe, effective ex vivo culture whilst maintaining cell phenotype, growth media constituents must be carefully controlled. We have used a chemically defined mesenchymal stromal cell culture medium to investigate the influence of different preparations of human serum albumin. We examined two aspects of cell culture, growth rate as measured by population doubling time and colony forming ability which is a representative measure of the stemness of the cell population. Albumin preparations showed comparative differences in both of these criteria. Analysis of the albumin bound fatty acids also showed differences depending on the manufacturing procedure used. We demonstrated that octanoate, an additive used to stabilize albumin during pasteurization, slows growth and lowers colony forming ability during ex vivo culture. Further to this we also found the level of Na+/K+ ATPase, a membrane bound cation pump inhibited by octanoate, is increased in cells exposed to this compound. We conclude that the inclusion of human serum albumin in ex vivo growth media requires careful consideration of not only the source of albumin, but also the associated molecular cargo, for optimal cell growth and behavior.

  19. Chick embryo xenograft model reveals a novel perineural niche for human adipose-derived stromal cells

    Directory of Open Access Journals (Sweden)

    Ingrid R. Cordeiro

    2015-09-01

    Full Text Available Human adipose-derived stromal cells (hADSC are a heterogeneous cell population that contains adult multipotent stem cells. Although it is well established that hADSC have skeletal potential in vivo in adult organisms, in vitro assays suggest further differentiation capacity, such as into glia. Thus, we propose that grafting hADSC into the embryo can provide them with a much more instructive microenvironment, allowing the human cells to adopt diverse fates or niches. Here, hADSC spheroids were grafted into either the presumptive presomitic mesoderm or the first branchial arch (BA1 regions of chick embryos. Cells were identified without previous manipulations via human-specific Alu probes, which allows efficient long-term tracing of heterogeneous primary cultures. When grafted into the trunk, in contrast to previous studies, hADSC were not found in chondrogenic or osteogenic territories up to E8. Surprisingly, 82.5% of the hADSC were associated with HNK1+ tissues, such as peripheral nerves. Human skin fibroblasts showed a smaller tropism for nerves. In line with other studies, hADSC also adopted perivascular locations. When grafted into the presumptive BA1, 74.6% of the cells were in the outflow tract, the final goal of cardiac neural crest cells, and were also associated with peripheral nerves. This is the first study showing that hADSC could adopt a perineural niche in vivo and were able to recognize cues for neural crest cell migration of the host. Therefore, we propose that xenografts of human cells into chick embryos can reveal novel behaviors of heterogeneous cell populations, such as response to migration cues.

  20. Derivation of Insulin Producing Cells From Human Endometrial Stromal Stem Cells and Use in the Treatment of Murine Diabetes

    OpenAIRE

    Santamaria, Xavier; Massasa, Efi E; Feng, Yuzhe; Wolff, Erin; Taylor, Hugh S

    2011-01-01

    Pancreatic islet cell transplantation is an effective approach to treat type 1 diabetes, however the shortage of cadaveric donors and limitations due to rejection require alternative solutions. Multipotent cells derived from the uterine endometrium have the ability to differentiate into mesodermal and ectodermal cellular lineages, suggesting the existence of mesenchymal stem cells in this tissue. We differentiated human endometrial stromal stem cells (ESSC) into insulin secreting cells using ...

  1. Leukemia inhibitory factor increases the proliferation of human endometrial stromal cells and expression of genes related to pluripotency

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    Mojdeh Salehnia

    2017-08-01

    Full Text Available Background: Concerning the low population of human endometrial mesenchymal cells within the tissue and their potential application in the clinic and tissue engineering, some researches have been focused on their in vitro expansion. Objective: The aim of this study was to evaluate the effect of leukemia inhibitory factor (LIF as a proliferative factor on the expansion and proliferation of human endometrial stromal cells. Materials and Methods: In this experimental study, the isolated and cultured human endometrial stromal cells from women at ovulatory phase aged 20-35 years, after fourth passage were divided into control and LIF-treated groups. In the experimental group, the endometrial cells were treated by 10 ng/ml LIF in culture media and the cultured cells without adding LIF considered as control group. Both groups were evaluated and compared for proliferation rate using MTT assay, for CD90 marker by flow cytometric analysis and for the expression of Oct4, Nanog, PCNA and LIFr genes using real-time RT-PCR. Results: The proliferation rate of control and LIF-treated groups were 1.17±0.17 and 1.61±0.06 respectively and there was a significant increase in endometrial stromal cell proliferation following in vitro treatment by LIF compared to control group (p=0.049. The rate of CD90 positive cells was significantly increased in LIFtreated group (98.96±0.37% compared to control group (94.26±0.08% (p=0.0498. Also, the expression ratio of all studied genes was significantly increased in the LIFtreated group compared to control group (p=0.0479. Conclusion: The present study showed that LIF has a great impact on proliferation, survival, and maintenance of pluripotency of human endometrial stromal cells and it could be applicable in cell therapies.

  2. Relative biological effectiveness of tritiated water on human chromosomes of lymphocytes and bone marrow cells

    International Nuclear Information System (INIS)

    Tanaka, Kimio; Sawada, Shozo; Kamada, Nanao

    1992-01-01

    One of the major toxic effluent from nuclear power industries is tritiated water (HTO), which is released into the environment in large quantities. Low dose radiation effects and dose rate effects of HTO on human lymphocytes and bone marrow cells are not well studied. The present study was performed to investigate dose-response relationship for chromosome aberration frequencies in the human lymphocytes and bone marrow cells, by HTO in-vitro exposure at low dose ranges of 0.1 to 1 Gy. Go lymphocytes and bone marrow cells were incubated for 10 - 150 minutes with HTO at 2 cGy/min. Also 60 Co γ and 137 Cs γ rays were used as controls. Dicentric chromosomes were scored in 1,000 to 2,000 cells of each experimental series. The RBE values of HTO at low dose range for the induction of dicentric chromosomes and chromatid type aberrations were 2.7 in lymphocytes and approximately 3.8 in bone marrow cells with respect to 60 Co γ ray, respectively. Also lymphocytes were chronically exposed to HTO for 24 to 72 hrs at lower dose rates (0.2 and 0.05 cGy/min). The yields of dicentrics and rings decreased with the reduction in the dose rate of HTO, presenting a clear dose rate effects of HTO. These results provide an useful information for the assessment for health risk in humans exposed to low concentration level to HTO. (author)

  3. Plasticity of human dental pulp stromal cells with bioengineering platforms: a versatile tool for regenerative medicine.

    Science.gov (United States)

    Barachini, Serena; Danti, Serena; Pacini, Simone; D'Alessandro, Delfo; Carnicelli, Vittoria; Trombi, Luisa; Moscato, Stefania; Mannari, Claudio; Cei, Silvia; Petrini, Mario

    2014-12-01

    In recent years, human dental pulp stromal cells (DPSCs) have received growing attention due to their characteristics in common with other mesenchymal stem cells, in addition to the ease with which they can be harvested. In this study, we demonstrated that the isolation of DPSCs from third molar teeth of healthy individuals allowed the recovery of dental mesenchymal stem cells that showed self-renewal and multipotent differentiation capability. DPSCs resulted positive for CD73, CD90, CD105, STRO-1, negative for CD34, CD45, CD14 and were able to differentiate into osteogenic and chondrogenic cells. We also assayed the angiogenic potential of DPSCs, their capillary tube-like formation was assessed using an in vitro angiogenesis assay and the uptake of acetylated low-density lipoprotein was measured as a marker of endothelial function. Based on these results, DPSCs were capable of differentiating into cells with phenotypic and functional features of endothelial cells. Furthermore, this study investigated the growth and differentiation of human DPSCs under a variety of bioengineering platforms, such as low frequency ultrasounds, tissue engineering and nanomaterials. DPSCs showed an enhanced chondrogenic differentiation under ultrasound application. Moreover, DPSCs were tested on different scaffolds, poly(vinyl alcohol)/gelatin (PVA/G) sponges and human plasma clots. We showed that both PVA/G and human plasma clot are suitable scaffolds for adhesion, growth and differentiation of DPSCs toward osteoblastic lineages. Finally, we evaluated the interactions of DPSCs with a novel class of nanomaterials, namely boron nitride nanotubes (BNNTs). From our investigation, DPSCs have appeared as a highly versatile cellular tool to be employed in regenerative medicine. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Human mesenchymal stromal cell-secreted lactate induces M2-macrophage differentiation by metabolic reprogramming

    Science.gov (United States)

    Civini, Sara; Pacelli, Consiglia; Dieng, Mame Massar; Lemieux, William; Jin, Ping; Bazin, Renée; Patey, Natacha; Marincola, Francesco M.; Moldovan, Florina; Zaouter, Charlotte; Trudeau, Louis-Eric; Benabdhalla, Basma; Louis, Isabelle; Beauséjour, Christian; Stroncek, David; Le Deist, Françoise; Haddad, Elie

    2016-01-01

    Human mesenchymal stromal cells (MSC) have been shown to dampen immune response and promote tissue repair, but the underlying mechanisms are still under investigation. Herein, we demonstrate that umbilical cord-derived MSC (UC-MSC) alter the phenotype and function of monocyte-derived dendritic cells (DC) through lactate-mediated metabolic reprogramming. UC-MSC can secrete large quantities of lactate and, when present during monocyte-to-DC differentiation, induce instead the acquisition of M2-macrophage features in terms of morphology, surface markers, migratory properties and antigen presentation capacity. Microarray expression profiling indicates that UC-MSC modify the expression of metabolic-related genes and induce a M2-macrophage expression signature. Importantly, monocyte-derived DC obtained in presence of UC-MSC, polarize naïve allogeneic CD4+ T-cells into Th2 cells. Treatment of UC-MSC with an inhibitor of lactate dehydrogenase strongly decreases lactate concentration in culture supernatant and abrogates the effect on monocyte-to-DC differentiation. Metabolic analysis further revealed that UC-MSC decrease oxidative phosphorylation in differentiating monocytes while strongly increasing the spare respiratory capacity proportional to the amount of secreted lactate. Because both MSC and monocytes are recruited in vivo at the site of tissue damage and inflammation, we propose the local increase of lactate concentration induced by UC-MSC and the consequent enrichment in M2-macrophage generation as a mechanism to achieve immunomodulation. PMID:27070086

  5. Characterization of Human Knee and Chin Adipose-Derived Stromal Cells

    Directory of Open Access Journals (Sweden)

    Magali Kouidhi

    2015-01-01

    Full Text Available Animal study findings have revealed that individual fat depots are not functionally equivalent and have different embryonic origins depending on the anatomic location. Mouse bone regeneration studies have also shown that it is essential to match the Hox code of transplanted cells and host tissues to achieve correct repair. However, subcutaneous fat depots from any donor site are often used in autologous fat grafting. Our study was thus carried out to determine the embryonic origins of human facial (chin and limb (knee fat depots and whether they had similar features and molecular matching patterns. Paired chin and knee fat depots were harvested from 11 subjects and gene expression profiles were determined by DNA microarray analyses. Adipose-derived stromal cells (ASCs from both sites were isolated and analyzed for their capacity to proliferate, form clones, and differentiate. Chin and knee fat depots expressed a different HOX code and could have different embryonic origins. ASCs displayed a different phenotype, with chin-ASCs having the potential to differentiate into brown-like adipocytes, whereas knee-ASCs differentiated into white adipocytes. These results highlighted different features for these two fat sites and indicated that donor site selection might be an important factor to be considered when applying adipose tissue in cell-based therapies.

  6. miR-141-3p inhibits human stromal (mesenchymal) stem cell proliferation and differentiation

    DEFF Research Database (Denmark)

    Qiu, Weimin; Kassem, Moustapha

    2014-01-01

    Wnt signaling determines human stromal (mesenchymal) stem cell (hMSC) differentiation fate into the osteoblast or adipocyte lineage. microRNAs (miRNAs) are small RNA molecules of 21-25 nucleotides that regulate many aspects of osteoblast biology. Thus, we examined miRNAs regulated by Wnt signaling...... in hMSC. We identified miRNA (miR)-141-3p as a Wnt target which in turn inhibited Wnt signaling. Moreover, miR-141-3p inhibited hMSC proliferation by arresting cells at the G1 phase of the cell cycle. miR-141-3p inhibited osteoblast differentiation of hMSC as evidenced by reduced alkaline phosphatase...... activity, gene expression and in vitro mineralized matrix formation. Bioinformatic studies, Western blot analysis and 3'UTR reporter assay demonstrated that cell division cycle 25A (CDC25A) is a direct target of miR-141-3p. siRNA-mediated knock-down of CDC25A inhibited hMSC proliferation and osteoblast...

  7. Xeno-Free Strategies for Safe Human Mesenchymal Stem/Stromal Cell Expansion: Supplements and Coatings

    Directory of Open Access Journals (Sweden)

    M. Cimino

    2017-01-01

    Full Text Available Human mesenchymal stem/stromal cells (hMSCs have generated great interest in regenerative medicine mainly due to their multidifferentiation potential and immunomodulatory role. Although hMSC can be obtained from different tissues, the number of available cells is always low for clinical applications, thus requiring in vitro expansion. Most of the current protocols for hMSC expansion make use of fetal bovine serum (FBS as a nutrient-rich supplement. However, regulatory guidelines encourage novel xeno-free alternatives to define safer and standardized protocols for hMSC expansion that preserve their intrinsic therapeutic potential. Since hMSCs are adherent cells, the attachment surface and cell-adhesive components also play a crucial role on their successful expansion. This review focuses on the advantages/disadvantages of FBS-free media and surfaces/coatings that avoid the use of animal serum, overcoming ethical issues and improving the expansion of hMSC for clinical applications in a safe and reproducible way.

  8. Human platelet lysate supplementation of mesenchymal stromal cell delivery: issues of xenogenicity and species variability.

    Science.gov (United States)

    Allen, Ashley B; Butts, Emily B; Copland, Ian B; Stevens, Hazel Y; Guldberg, Robert E

    2017-10-01

    Immunogenicity of fetal bovine serum (FBS) poses a problem for its use in the propagation of autologous mesenchymal stromal cells (MSCs) for cell therapy. Human platelet lysate (hPL), an enriched growth factor solution containing mitogenic and angiogenic cues, has potential utility in replacing FBS for human MSC (hMSC) delivery strategies. Despite its potentiation of hMSC number in vitro, little is known concerning its capacity to supplement implanted hMSC-seeded constructs and promote tissue regeneration in vivo. In this study, we tested the effects of incorporating hPL in cell-seeded constructs implanted subcutaneously into immunocompromised rats, investigated in vitro interactions between hPL and rat MSCs (rMSCs) and determined interspecies variability in the PL product [hPL vs rat PL (rPL)] and its effect on cultured MSCs (hPL/hMSCs vs rPL/rMSCs). The overarching aim was to determine the utility of hPL to foster MSC survival in preclinical rodent models. Exposure to hPL-supplemented media resulted in rMSC death, by a process attributable to heat-labile proteins, but not membrane attack complex formation. In the in vitro syngeneic model, the rodent product proved fundamentally distinct from the human product, with rPL having substantially lower growth factor content than hPL. Moreover, contrary to the positive effects of hPL on hMSC expansion, rPL did not reduce rMSC doubling time for the serum concentrations examined. When tested in vivo, hPL did not improve cell survival within hydrogel constructs through 2 weeks postimplantation. In summary, this study highlights the many facets of xenogenicity and interspecies variability that must be considered in the preclinical evaluation of hPL. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  9. Evaluation of human platelet lysate versus fetal bovine serum for culture of mesenchymal stromal cells.

    Science.gov (United States)

    Hemeda, Hatim; Giebel, Bernd; Wagner, Wolfgang

    2014-02-01

    Culture media for therapeutic cell preparations-such as mesenchymal stromal cells (MSCs)-usually comprise serum additives. Traditionally, fetal bovine serum is supplemented in basic research and in most clinical trials. Within the past years, many laboratories adapted their culture conditions to human platelet lysate (hPL), which further stimulates proliferation and expansion of MSCs. Particularly with regard to clinical application, human alternatives for fetal bovine serum are clearly to be preferred. hPL is generated from human platelet units by disruption of the platelet membrane, which is commonly performed by repeated freeze and thaw cycles. Such culture supplements are notoriously ill-defined, and many parameters contribute to batch-to-batch variation in hPL such as different amounts of plasma, a broad range of growth factors and donor-specific effects. The plasma components of hPL necessitate addition of anticoagulants such as heparins to prevent gelatinization of hPL medium, and their concentration must be standardized. Labels for description of hPL-such as "xenogen-free," "animal-free" and "serum free"-are not used consistently in the literature and may be misleading if not critically assessed. Further analysis of the precise composition of relevant growth factors, attachment factors, microRNAs and exosomes will pave the way for optimized and defined culture conditions. The use of hPL has several advantages and disadvantages: they must be taken into account because the choice of cell culture additive has major impact on cell preparations. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  10. Bone marrow blood vessel ossification and "microvascular dead space" in rat and human long bone.

    Science.gov (United States)

    Prisby, Rhonda D

    2014-07-01

    Severe calcification of the bone microvascular network was observed in rats, whereby the bone marrow blood vessels appeared ossified. This study sought to characterize the magnitude of ossification in relation to patent blood vessels and adipocyte content in femoral diaphyses. Additionally, this study confirmed the presence of ossified vessels in patients with arteriosclerotic vascular disease and peripheral vascular disease and cellulitis. Young (4-6 month; n=8) and old (22-24 month; n=8) male Fischer-344 rats were perfused with barium sulfate to visualize patent bone marrow blood vessels. Femoral shafts were processed for bone histomorphometry to quantify ossified (Goldner's Trichrome) and calcified (Alizarin Red) vessels. Adipocyte content was also determined. Additional femora (n=5/age group) were scanned via μCT to quantify microvascular ossification. Bone marrow blood vessels from the rats and the human patients were also isolated and examined via microscopy. Ossified vessels (rats and humans) had osteocyte lacunae on the vessel surfaces and "normal" vessels were transitioning into bone. The volume of ossified vessels was 4800% higher (pnecrosis. Progression of bone microvascular ossification may provide the common link associated with age-related changes in bone and bone marrow. The clinical implications may be evident in the difficulties treating bone disease in the elderly. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Identification and Characterization of Plasma Cells in Normal Human Bone Marrow by High-Resolution Flow Cytometry

    NARCIS (Netherlands)

    Terstappen, Leonardus Wendelinus Mathias Marie; Johnsen, Steen; Segers-Nolten, Gezina M.J.; Loken, Michael R.

    1990-01-01

    The low frequency of plasma cells and the lack of specific cell surface markers has been a major obstacle for a detailed characterization of plasma cells in normal human bone marrow. Multiparameter flow cytometry enabled the identification of plasma cells in normal bone marrow aspirates. The plasma

  12. Storage effect on viability and biofunctionality of human adipose tissue-derived stromal cells.

    Science.gov (United States)

    Falah, Mizied; Rayan, Anwar; Srouji, Samer

    2015-09-01

    In our recent studies, the transplantation of human adipose tissue-derived stromal cells (ASCs) has shown promise for treatment of diseases related to bone and joint disorders. For the current clinical applications, ASCs were formulated and suspended in PlasmaLyte A supplemented with heparin, glucose and human serum albumin, balanced to pH 7.4 with sodium bicarbonate. This cell solution constitutes 20% of the overall transplanted mixture and is supplemented with hyaluronic acid (60%) and OraGraft particles (20%). We intended to investigate the effect of this transplantation mixture on the viability and biofunctionality of ASCs in bone formation. Freshly harvested cells were resuspended and incubated in the indicated mixture for up to 48 h at 4°C. Cell viability was assessed using trypan blue and AlamarBlue, and cell functionality was determined by quantifying their adhesion rate in vitro and bone formation in an ectopic mouse model. More than 80% of the ASCs stored in the transplantation mixture were viable for up to 24 h. Cell viability beyond 24 h in storage decreased to approximately 50%. In addition, an equal degree of bone formation was observed between the cells transplanted following incubation in transplantation mixture for up to 24 h and zero-time non-incubated cells (control). The viability and functionality of ASCs stored in the presented formulation will make such cell therapy accessible to larger and more remote populations. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  13. Clonal chromosomal and genomic instability during human multipotent mesenchymal stromal cells long-term culture.

    Directory of Open Access Journals (Sweden)

    Victoria Nikitina

    Full Text Available Spontaneous mutagenesis often leads to appearance of genetic changes in cells. Although human multipotent mesenchymal stromal cells (hMSC are considered as genetically stable, there is a risk of genomic and structural chromosome instability and, therefore, side effects of cell therapy associated with long-term effects. In this study, the karyotype, genetic variability and clone formation analyses have been carried out in the long-term culture MSC from human gingival mucosa.The immunophenotype of MSC has been examined using flow cytofluorometry and short tandem repeat (STR analysis has been carried out for authentication. The karyotype has been examined using GTG staining and mFISH, while the assessment of the aneuploidy 8 frequency has been performed using centromere specific chromosome FISH probes in interphase cells.The immunophenotype and STR loci combination did not change during the process of cultivation. From passage 23 the proliferative activity of cultured MSCs was significantly reduced. From passage 12 of cultivation, clones of cells with stable chromosome aberrations have been identified and the biggest of these (12% are tetrasomy of chromosome 8. The random genetic and structural chromosomal aberrations and the spontaneous level of chromosomal aberrations in the hMSC long-term cultures were also described.The spectrum of spontaneous chromosomal aberrations in MSC long-term cultivation has been described. Clonal chromosomal aberrations have been identified. A clone of cells with tetrasomy 8 has been detected in passage 12 and has reached the maximum size by passage 18 before and decreased along with the reduction of proliferative activity of cell line by passage 26. At later passages, the MSC line exhibited a set of cells with structural variants of the karyotype with a preponderance of normal diploid cells. The results of our study strongly suggest a need for rigorous genetic analyses of the clone formation in cultured MSCs before

  14. Human amniotic mesenchymal stromal cell transplantation improves endometrial regeneration in rodent models of intrauterine adhesions.

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    Gan, Lu; Duan, Hua; Xu, Qian; Tang, Yi-Qun; Li, Jin-Jiao; Sun, Fu-Qing; Wang, Sha

    2017-05-01

    Intrauterine adhesion (IUA) is a common uterine cavity disease characterized by the unsatisfactory regeneration of damaged endometria. Recently, stem cell transplantation has been proposed to promote the recovery process. Here we investigated whether human amniotic mesenchymal stromal cells (hAMSCs), a valuable resource for transplantation therapy, could improve endometrial regeneration in rodent IUA models. Forty female Sprague-Dawley rats were randomly assigned to five groups: normal, sham-operated, mechanical injury, hAMSC transplantation, and negative control group. One week after intervention and transplantation, histological analyses were performed, and immunofluorescent and immunohistochemical expression of cell-specific markers and messenger RNA expression of cytokines were measured. Thicker endometria, increased gland numbers and fewer fibrotic areas were found in the hAMSC transplantation group compared with the mechanical injury group. Engraftment of hAMSCs was detected by the presence of anti-human nuclear antigen-positive cells in the endometrial glands of the transplantation uteri. Transplantation of hAMSCs significantly decreased messenger RNA levels of pro-inflammatory cytokines (tumor necrosis factor-α and interleukin-1β), and increased those of anti-inflammatory cytokines (basic fibroblast growth factor, and interleukin-6) compared with the injured uterine horns. Immunohistochemical expression of endometrial epithelial cells was revealed in specimens after hAMSC transplantation, whereas it was absent in the mechanically injured uteri. hAMSC transplantation promotes endometrial regeneration after injury in IUA rat models, possibly due to immunomodulatory properties. These cells provide a more easily accessible source of stem cells for future research into the impact of cell transplantation on damaged endometria. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  15. Direct Head-To-Head Comparison of Cationic Liposome-Mediated Gene Delivery to Mesenchymal Stem/Stromal Cells of Different Human Sources: A Comprehensive Study

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    Boura, Joana S.; dos Santos, Francisco; Gimble, Jeffrey M.; Cardoso, Carla M.P.; Madeira, Catarina; Cabral, Joaquim M.S.

    2013-01-01

    Abstract Nonviral gene delivery to human mesenchymal stem/stromal cells (MSC) can be considered a very promising strategy to improve their intrinsic features, amplifying the therapeutic potential of these cells for clinical applications. In this work, we performed a comprehensive comparison of liposome-mediated gene transfer efficiencies to MSC derived from different human sources—bone marrow (BM MSC), adipose tissue-derived cells (ASC), and umbilical cord matrix (UCM MSC). The results obtained using a green fluorescent protein (GFP)-encoding plasmid indicated that MSC isolated from BM and UCM are more amenable to genetic modification when compared to ASC as they exhibited superior levels of viable, GFP+ cells 48 hr post-transfection, 58±7.1% and 54±3.8%, respectively, versus 33±4.7%. For all cell sources, high cell recoveries (≈50%) and viabilities (>85%) were achieved, and the transgene expression was maintained for 10 days. Levels of plasmid DNA uptake, as well as kinetics of transgene expression and cellular division, were also determined. Importantly, modified cells were found to retain their characteristic immunophenotypic profile and multilineage differentiation capacity. By using the lipofection protocol optimized herein, we were able to maximize transfection efficiencies to human MSC (maximum of 74% total GFP+ cells) and show that lipofection is a promising transfection strategy for MSC genetic modification, especially when a transient expression of a therapeutic gene is required. Importantly, we also clearly demonstrated that intrinsic features of MSC from different sources should be taken into consideration when developing and optimizing strategies for MSC engineering with a therapeutic gene. PMID:23360350

  16. Canine and human gastrointestinal stromal tumors display similar mutations in c-KIT exon 11

    International Nuclear Information System (INIS)

    Gregory-Bryson, Emmalena; Bartlett, Elizabeth; Kiupel, Matti; Hayes, Schantel; Yuzbasiyan-Gurkan, Vilma

    2010-01-01

    Gastrointestinal stromal tumors (GISTs) are common mesenchymal neoplasms in the gastrointestinal tract of humans and dogs. Little is known about the pathogenesis of these tumors. This study evaluated the role of c-KIT in canine GISTs; specifically, we investigated activating mutations in exons 8, 9, 11, 13, and 17 of c-KIT and exons 12, 14, and 18 of platelet-derived growth factor receptor, alpha polypeptide (PDGFRA), all of which have been implicated in human GISTs. Seventeen canine GISTs all confirmed to be positive for KIT immunostaining were studied. Exons 8, 9, 11, 13 and 17 of c-KIT and exons 12, 14, and 18 of PDGFRA, were amplified from DNA isolated from formalin-fixed paraffin-embedded samples. Of these seventeen cases, six amplicons of exon 11 of c-KIT showed aberrant bands on gel electrophoresis. Sequencing of these amplicons revealed heterozygous in-frame deletions in six cases. The mutations include two different but overlapping six base pair deletions. Exons 8, 9, 13, and 17 of c-KIT and exons 12, 14, and 18 of PDGFRA had no abnormalities detected by electrophoresis and sequencing did not reveal any mutations, other than synonymous single nucleotide polymorphisms (SNPs) found in exon 11 of c-KIT and exons 12 and 14 of PDGFRA. The deletion mutations detected in canine GISTs are similar to those previously found in the juxtamembrane domain of c-KIT in canine cutaneous mast cell tumors in our laboratory as well as to those reported in human GISTs. Interestingly, none of the other c-KIT or PDGFRA exons showed any abnormalities in our cases. This finding underlines the critical importance of c-KIT in the pathophysiology of canine GISTs. The expression of KIT and the identification of these activating mutations in c-KIT implicate KIT in the pathogenesis of these tumors. Our results indicate that mutations in c-KIT may be of prognostic significance and that targeting KIT may be a rational approach to treatment of these malignant tumors. This study further

  17. Characterization of human erythroid burst-promoting activity derived from bone marrow conditioned media

    International Nuclear Information System (INIS)

    Porter, P.N.; Ogawa, M.

    1982-01-01

    Bone marrow conditioned media (BMCM) increases burst number and the incorporation of 59 Fe into heme by bursts when peripheral blood or bone marrow cells are cultured at limiting serum concentrations. Burst-promoting activity (BPA) has now been purified approximately 300-fold from this source by ion-exchange chromatography on DEAE-Sephadex and absorption chromatography on hydroxyapatite agarose gel. Marrow BPA increased burst number and hemoglobin (Hb) synthesis in a dose-dependent manner. A larger increase in Hb synthesis than in burst number was consistently observed, which was probably a consequence of the increase in the number of cells per burst that occurs in the presence of BPA. The role of BPA in culture could be distinguished from erythropoietin (Ep), since no bursts grew in the absence of Ep, whether or not BPA was present, and since it had no effect on the growth of erythroid colonies scored at day 5 of culture. Our purified fraction did not support the growth of CFU-C in culture. Activity was stable at temperatures of 70 degrees C or lower for 10 min; exposure to 80 degrees C resulted in approximately 50% loss of activity. BPA was completely inactivated by treatment at 100 degrees C for 10 min. Thus, human bone marrow cells produce a heat-sensitive factor that specifically promotes the growth of early erythroid progenitors in culture

  18. Changes of the proliferation kinetics of human bone marrow in vivo through hydroxyurea

    International Nuclear Information System (INIS)

    Ertl, E.

    1982-01-01

    A 10-hour oral continuous infusion with hydroxyurea (HU) at a non-toxic concentration was performed in 20 malignoma patients with undisturbed bone marrow. Bone marrow taken before, during and after HU-administration was examined for 3H-TdR incorporation by means of autoradiography and liquid scintimetry, for cell phase distribution by means of flow cytophotometry, morphologically and by means of CFUc. 3H-TdR incorporation into bone marrow cells dropped to 16% of the initial value under HU and rose to 156% 10 h after HU-effect terminated. Cytophotometry did not furnish any proof of a decrease of S-phase cells or increase of cells in G 1 -to-S-transition during HU. S-cells rise to 129% of the initial value 10 h after having fallen below minimum inhibition concentration. Under HU, there is an equal number of cells in S which incorporate much less 3H-thymidine; after HU more S-cells incorporate more 3H-thymidine than before HU. During HU action, DNA synthesis activity is reduced to 17% and reaches the initial value with 105% afterwards. In human bone marrow, hydroxyurea in non-toxic concentration causes a temporary DNA synthesis inhibition in terms of activity reduction and partial arrest in S. A stop-and-go of the cell cycle effected by HU does not occur; the effect is rather a slow-down of DNA synthesis. (orig./MG) [de

  19. Early Intravenous Delivery of Human Brain Stromal Cells Modulates Systemic Inflammation and Leads to Vasoprotection in Traumatic Spinal Cord Injury.

    Science.gov (United States)

    Badner, Anna; Vawda, Reaz; Laliberte, Alex; Hong, James; Mikhail, Mirriam; Jose, Alejandro; Dragas, Rachel; Fehlings, Michael

    2016-08-01

    : Spinal cord injury (SCI) is a life-threatening condition with multifaceted complications and limited treatment options. In SCI, the initial physical trauma is closely followed by a series of secondary events, including inflammation and blood spinal cord barrier (BSCB) disruption, which further exacerbate injury. This secondary pathology is partially mediated by the systemic immune response to trauma, in which cytokine production leads to the recruitment/activation of inflammatory cells. Because early intravenous delivery of mesenchymal stromal cells (MSCs) has been shown to mitigate inflammation in various models of neurologic disease, this study aimed to assess these effects in a rat model of SCI (C7-T1, 35-gram clip compression) using human brain-derived stromal cells. Quantitative polymerase chain reaction for a human-specific DNA sequence was used to assess cell biodistribution/clearance and confirmed that only a small proportion (approximately 0.001%-0.002%) of cells are delivered to the spinal cord, with the majority residing in the lung, liver, and spleen. Intriguingly, although cell populations drastically declined in all aforementioned organs, there remained a persistent population in the spleen at 7 days. Furthermore, the cell infusion significantly increased splenic and circulating levels of interleukin-10-a potent anti-inflammatory cytokine. Through this suppression of the systemic inflammatory response, the cells also reduced acute spinal cord BSCB permeability, hemorrhage, and lesion volume. These early effects further translated into enhanced functional recovery and tissue sparing 10 weeks after SCI. This work demonstrates an exciting therapeutic approach whereby a minimally invasive cell-transplantation procedure can effectively reduce secondary damage after SCI through systemic immunomodulation. Central nervous system pericytes (perivascular stromal cells) have recently gained significant attention within the scientific community. In addition to

  20. Greater sparing of stromal progenitor cells than of haemopoietic stem cells in γ-irradiated mouse marrow using low dose-rates

    International Nuclear Information System (INIS)

    Hendry, J.H.; Wang, S.B.; Testa, N.G.

    1984-01-01

    The Do value fibroblastoid colony-forming units in mouse bone-marrow increased from 1.7 Gy using γ-rays at 4.2 Gy/minute, to 2.6 Gy at 4.5 cGy/minute. In contrast, the sensitivity of bone-marrow stem cells was very little changed (Do approximately 0.9 Gy). At 7.5 Gy acute single dose, the dose sparing achieved for CFU-F using 4.5 cGy/minute was a factor of 1.4, inbetween the values reported for lung of 1.8 and for haemopoiesis of 1.2. Although the role of CFU-F in the haemopoietic environment has not been established, the content of CFU-F can predict the ability of irradiated marrow to sustain haemopoiesis in the long term. Hence the data imply that the haemopoiesis environment, as well as the dose-limiting lung, benefits from the use of low dose-rates for haemopoietic ablations in the treatment of leukaemia. No significant further sparing of CFU-F was achieved using a lower dose-rate of 1.4 cGy per minute

  1. Multipotent Mesenchymal Stromal Stem Cell Expansion by Plating Whole Bone Marrow at a Low Cellular Density: A More Advantageous Method for Clinical Use

    Directory of Open Access Journals (Sweden)

    Katia Mareschi

    2012-01-01

    Full Text Available Mesenchymal stem cells (MSCs are a promising source for cell therapy due to their pluripotency and immunomodulant proprieties. As the identification of “optimal” conditions is important to identify a standard procedure for clinical use. Percoll, Ficoll and whole bone marrow directly plated were tested from the same sample as separation methods. The cells were seeded at the following densities: 100 000, 10 000, 1000, 100, 10 cells/cm2. After reaching confluence, the cells were detached, pooled and re-plated at 1000, 500, 100, and 10 cells/cm2. Statistical analyses were performed. Cumulative Population Doublings (PD did not show significant differences for the separation methods and seeding densities but only for the plating density. Some small quantity samples plated in T25 flasks at plating densities of 10 and 100 cells/cm2 did not produce any expansion. However, directly plated whole bone marrow resulted in a more advantageous method in terms of CFU-F number, cellular growth and minimal manipulation. No differences were observed in terms of gross morphology, differentiation potential or immunophenotype. These data suggest that plating whole bone marrow at a low cellular density may represent a good procedure for MSC expansion for clinical use.

  2. Multipotent Mesenchymal Stromal Stem Cell Expansion by Plating Whole Bone Marrow at a Low Cellular Density: A More Advantageous Method for Clinical Use

    Science.gov (United States)

    Mareschi, Katia; Rustichelli, Deborah; Calabrese, Roberto; Gunetti, Monica; Sanavio, Fiorella; Castiglia, Sara; Risso, Alessandra; Ferrero, Ivana; Tarella, Corrado; Fagioli, Franca

    2012-01-01

    Mesenchymal stem cells (MSCs) are a promising source for cell therapy due to their pluripotency and immunomodulant proprieties. As the identification of “optimal” conditions is important to identify a standard procedure for clinical use. Percoll, Ficoll and whole bone marrow directly plated were tested from the same sample as separation methods. The cells were seeded at the following densities: 100 000, 10 000, 1000, 100, 10 cells/cm2. After reaching confluence, the cells were detached, pooled and re-plated at 1000, 500, 100, and 10 cells/cm2. Statistical analyses were performed. Cumulative Population Doublings (PD) did not show significant differences for the separation methods and seeding densities but only for the plating density. Some small quantity samples plated in T25 flasks at plating densities of 10 and 100 cells/cm2 did not produce any expansion. However, directly plated whole bone marrow resulted in a more advantageous method in terms of CFU-F number, cellular growth and minimal manipulation. No differences were observed in terms of gross morphology, differentiation potential or immunophenotype. These data suggest that plating whole bone marrow at a low cellular density may represent a good procedure for MSC expansion for clinical use. PMID:23715383

  3. Effect of low oxygen tension on the biological characteristics of human